Laboratory Standards Guideline for Primary
Health Care Clinics in Iraq
March 2013
Table of contents
Table of contents ....................................................................................................................... 1
Figure 25: A flow diagram for the preliminary identification of staphylococci
Bookmark not defined.
125..... Error!
Acronyms ................................................................................................................................. 6
1. Introduction ........................................................................................................................... 8
2. Setting up a primary health care laboratory........................................................................... 8
3. Safety in the Laboratory ...................................................................................................... 11
3.1 Physical Hazards ........................................................................................................... 11
3.2 Chemical Hazards.......................................................................................................... 12
3.3 Biological Hazards ........................................................................................................ 12
3.4 Safety Measures ............................................................................................................ 13
23.5 Cleaning, disinfection, sterilization and disposal of biohazard waste. ........................ 16
3.6 Disposal of Biohazard Waste ........................................................................................ 19
4. Hematology ......................................................................................................................... 22
4.1 Collection & Handling of blood .................................................................................... 22
4.2 Part One: Automated Blood Count Techniques ............................................................ 26
4.2.1 Hemoglobin Concentration .................................................................................... 27
4.2.2 Red Blood CELL Count ......................................................................................... 27
4.2.3 Counting Systems ................................................................................................... 27
4.2.4 Reliability of Electronic Counters .......................................................................... 28
4.2.6 Packed Cell Volume and Mean Cell Volume ......................................................... 31
4.2.7 Variation in Red Cell Volumes: ............................................................................. 32
4.2.8 Total White Blood Cell Count ................................................................................ 33
4.2.9 Automated Differential Count ................................................................................ 33
4.2.10 New White Cell Parameters ................................................................................. 34
4.2.11 Platelet Count ....................................................................................................... 35
4.2.12 Mean Platelet Volume .......................................................................................... 35
4.2.13 Reticulocytes Count.............................................................................................. 36
4.2.14 Immature Reticulocyte Fraction ........................................................................... 36
4.2.15 Calibration of Automated Blood Cell Counters ................................................... 36
4.2.16 Flagging of Automated Blood Counts .................................................................. 37
4.3 Part Two: Manual Techniques....................................................................................... 38
4.3.1 Haemoglobin Estimation ........................................................................................ 38
4.3.2 Packed Cell Volume or Haematocrit ...................................................................... 41
4.3.3 Total White Blood Cell Count ................................................................................ 44
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4.3.4 Platelet Count ......................................................................................................... 47
4.3.5 Erythrocyte Sedimentation Rate ............................................................................. 50
4.3.6 Reticulocyte Count ................................................................................................. 53
4.2.7 PREPARATION OF BLOOD FILMS ON SLIDES.......................................... 55
4.3.11 Antiglobulin or Coombs' Test .............................................................................. 58
4.3.12 ABO & Rh Group................................................................................................. 61
5. Serology............................................................................................................................... 61
5.1Rapid Plasma Reagin (RPR) TEST ................................................................................ 61
5.2 TPHA(Treponema pallidum Haemagglutination Assay ) TEST ................................... 64
5.3 Widal Test ((agglutination test)) ................................................................................... 66
5.4 Rose Bengal Test for Brucellosis .................................................................................. 70
Principle........................................................................................................................... 70
5.5 C-Reactive Protein (CRP) Test ..................................................................................... 72
5.6 ASO latex slide agglutination test ................................................................................. 74
5.6 Rheumatoid Factor (RF) ................................................................................................ 75
5.7 Toxoplasmosis Test ........................................................................................................... 77
5.8 Infectious Mononucleosis test ....................................................................................... 80
5.9 Rota Virus Latex Test.................................................................................................... 81
5.10 Pregnancy Test ............................................................................................................ 82
5.10.1 Rapid test for detection of human Chorionic Gonadotrophin in Urine ................ 82
5.10.2 Direct HCG Latex Pregnancy Kit............................................................................. 84
6. Bacteriology ........................................................................................................................ 85
6.1 General urine examination ............................................................................................ 85
6.1.1 Macroscopical Examination of Urine ................................................................... 86
6.1.2 Microscopical Examination of Urine ..................................................................... 92
6.2 General Stool Examination.......................................................................................... 100
6.2.1 Macroscopical examination .................................................................................. 101
6.2.2 Microscopic Examination ..................................................................................... 102
6.3 Urine Culture ............................................................................................................... 106
6.4 Throat Swab ................................................................................................................ 115
6.5 Nasal Swab .................................................................................................................. 121
6.6 Smart Tests .................................................................................................................. 125
6.6.1 Helicobacter pylori Serum/Whole Blood ............................................................. 125
6.6.2 Salmonella Typhi Antigen .................................................................................... 128
6.6.3 Vibrio Cholerae Rapid Visual Antigen Detection Test ........................................ 131
6.7 Preparation of Media ................................................................................................... 135
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6.7.1 Blood agar: TSA with 5% sheep blood ................................................................ 136
6.7.2 Chocolate agar with TSA base and growth supplement ....................................... 136
6.7.3 MacConkey agar (MAC) ...................................................................................... 137
6.7.4 Mueller-Hinton agar ............................................................................................. 138
6.7.5 Cary-Blair medium, Amies medium, and Stuarts medium ................................... 139
6.8 Staining Smears ........................................................................................................... 139
6.8.1 Gram’s Stain ......................................................................................................... 139
6.8.2 Ziehl-Neelsen’s stain (for acid fast bacilli) .......................................................... 140
7 Parasitology ........................................................................................................................ 141
7.1 Malaria parasite diagnosis ........................................................................................... 141
8. Biochemistry ..................................................................................................................... 148
8.1 Blood glucose .............................................................................................................. 148
8.2 Liver Function Test ..................................................................................................... 150
8.2.1 Alanine Aminotransferase (ALT) ......................................................................... 150
8.2.2 Aspartate Aminotransferase (AST) ...................................................................... 152
8.2.3 Alkaline Phosphatase............................................................................................ 153
8.2.4 Total Serum Bilirubin ........................................................................................... 155
8.3 Renal Function test ...................................................................................................... 157
8.3.1 Blood Urea............................................................................................................ 157
8.3.2 Serum Creatinine .................................................................................................. 160
8.3.3 Uric acid ............................................................................................................... 161
8.4 Lipid Profile ................................................................................................................ 163
8.4.1 Total Cholesterol .................................................................................................. 163
8.4.2 Triglycerides ......................................................................................................... 165
8.4.3 HDL Cholesterol .................................................................................................. 167
8.4.4 LDL Cholesterol ................................................................................................... 169
Annex 1 ................................................................................................................................. 172
References: ............................................................................................................................ 180
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Table of Figures
Figure 1: Plan for a one room laboratory .................................................................................. 9
Figure 2: Indicates another possible arrangement of a peripheral laboratory ..... 10
Figure 3: Common symbols of hazards ................................................................................... 14
Figure 4:Anterior surface of the left arm showing veins most suitable for venepuncture ....... 24
Figure 5: Venepuncture technique using needle and syringe. ................................................. 24
Figure 6: Venepuncture technique using an evacuated container ........................................... 25
Figure 7:The areas of the foot of a baby or infant which are suitable for obtaining capillary
blood. ....................................................................................................................................... 25
Figure 8: Effect of threshold discrimination............................................................................ 28
Figure 9: Setting Discrimination Thresholds .......................................................................... 31
Figure 10: Counting chamber .................................................................................................. 46
Figure11: Counting Chamber .................................................................................................. 49
Figure 12: Coombs Test .......................................................................................................... 60
Figure 13: Appearance of Positive and Negative RPR Results ............................................... 64
Figure 14: Results of PT .......................................................................................................... 84
Fig15: common urine cells ...................................................................................................... 95
Fig16: different casts seen in urine .......................................................................................... 96
Fig17: common urine crystals ................................................................................................. 99
Fig18: Infectious agents ........................................................................................................ 100
Figure19: Ova in GSE ........................................................................................................... 105
Figure 20: Entamoeba histolytica & Giardia lamblia (HPF) ................................................. 105
Fig 21: Identification of Streptococci and Enterococci Flowchart ........................................ 111
Fig 22: Presumptive Identification of Staphylococcus Species Flowchart ........................... 112
Figure 25: A flow diagram for the preliminary identification of staphylococci.................... 124
Figure 26: Interpretation of Smart Test Results .................................................................... 127
Figure 27: Interpretation of Salmonella Smart Test .............................................................. 130
Figure28: Rapid Test Instruction Sheet, for a graphic representation of the test procedure . 133
Figure 29:Visual Interpretation of Rapid Dipstick Test Results ........................................... 135
Figure 30: P. Falciparum ....................................................................................................... 145
Figure 31: P. Ovale ................................................................................................................ 146
Figure 32: P. Vivax ............................................................................................................... 147
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Table of Tables
Table1: TPHA results .............................................................................................................. 66
Table 2:Record agglutination .................................................................................................. 68
Table 3: Determination of antibody titer ................................................................................. 68
Table 4: Example of slide test are shown in the following table ............................................. 68
Table 5: indication of significant serum titers ......................................................................... 69
Table 6: Determination of antibody titer ................................................................................. 71
Table 7: Examples of Rose Bengal results .............................................................................. 72
Table 8: Reagents of Infectious Mononucleosis Test .............................................................. 80
Table 9: Degree of Precipitation.............................................................................................. 89
Table 10: Differentiation of b-haemolytic streptococci ........................................................ 119
Table 11: Culture media, conditions and organisms for all specimens ................................. 121
Table 12: Differentiation of medically important species of Staphylococcus .................... 123
Table 13: Expected Test Result Outcomes for the Dipstick and .......................................... 134
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Acronyms
AAFB
AIDS
ALP
ALT
AST
BHSP
CRP
CSF
CV
DNA
EDTA
ELISA
ESR
GIT
Hb
HCG
Hct
Hi
HiCN
HIV
IDDM
Ig
IHA
IU
KIA
LDH
LPS
MAC
MCH
MCV
MIU
MoH
MPV
MPXI
MRSA
NIDDM
NRBS
PCV
PDW
PHC
PPE
R
RBCs
Acid and Alcohol Fast Bacilli
Acquired Immune Deficiency Syndrome
Alkaline Phosphatase
Alanine Aminotransferase
Aspartate Aminotransferase
Basic Health Services Package
C- Reactive Protein
Cerebro-Spinal Fluid
Coefficient of Variation
Deoxyribonucleic Acid
Ethylenediaminetetraacetic Acid
Enzyme-Linked Immunosorbent Assay
Erythrocyte Sedimentation Rate
Gastro Intestinal Tract
Hemoglobin
Human Chorionic Gonadotropin
Hematocrit
Meth hemoglobin
Hemiglobincyanide
Human Immune Virus
Insulin Dependent Diabetes Miletus
Immunoglobulin
Indirect Haemogglutination
International Unit
Klinger Iron Agar
Low Density Lipids
Lipopolysaccharides
MacConky
Mean Corpuscular Hemoglobin
Mean Corpuscular Volume
Motility-Indole-Urase
Ministry of Health
Mean Platelet Volume
Mean Peroxidase Activity Index
Methicillin Resistant Staph Aurous
Non-Insulin Dependent Diabetes Mellitus
Nucleated Red Blood Cells
Packed Cell Volume
Platelet Distribution Width
Primary Health Care
Personal Protective Equipment
Reactive
Red Blood Cells
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RDW
RF
RNA
RPM
RPR
SD
SHb
SOPs
SPA
SR
TCBS
TNCC
TPHA
TSA
URC
USAID
UTI
VCA
VDRL
WBC
XLD
Red cell Distribution Width
Rheumatoid Factor
Ribonucleic Acid
Round Per Minute
Rapid Plasma Reagin
Standards Deviation
Sulphemoglobin
Standard Operating Procedures
Suprapubic Aspirate
Strong Reactive
Thiosulfate Citrate Bile Salts
Total Nucleated Cell Count
Treponema Pallidum Particle Agglutination
Tryptic Soya Agar
University Research Company
United States Agency of International Development
Urinary Tract Infection
Viral Structural Antigens
Venereal Disease Research Laboratory
White Blood Cells
Xylose Lysine Desoxycholate
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1. Introduction
The Iraqi Ministry of Health (MoH) is undergoing health reform and moving forward with
reorganizing and stabilizing its health services. Ensuring access to routine, high-quality, and
equitable healthcare has emerged as a critical need, and the Government of Iraq is responding
with vigor and commitment to improving the quality of primary health care (PHC) services.
To assist with these efforts, USAID awarded University Research Co. LLC (URC) the fouryear Primary Health Care Project in Iraq (USAID/PHCPI) to support the MoH in achieving
its goal of improving the quality of care for all Iraqis. The project aims to strengthen health
sector capacity to provide essential PHC services and expand availability of such services
throughout the country, with increasing coverage for vulnerable and at-risk populations, such
as children under five, women, and internally displaced persons.
The "Laboratory Standard Operating Procedures (SOPs)," often called the "Methods,"
discusses how the experiment occurred. Documenting the procedures of the laboratory
experiment is important not only so that others can repeat the results but also so that you can
replicate the work later, if the need arises. Historically, laboratory procedures have been
written as first-person narratives as opposed to second-person sets of instructions.
Achieving a proper depth in laboratory procedures is challenging. In general, adequate
information should be given in order for staff to be able to carry out the procedure. For that
reason, details that will affect the outcome are included.
The Basic Health Services Package (BHSP) for Iraq was considered in developing this
guideline as all the SOPs for the investigations are listed in the BHSP.
2. Setting up a primary health care laboratory

A one room laboratory: Figure 1 sets out the possible arrangement of a peripheral
medical laboratory attached to a health center. It shows a laboratory suitable for
carrying out some or all of the techniques described in the manual. The plan is
limited to one room, since often this is all the space that is available for the
laboratory. The room should measure at least 5m X 6m.
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Figure 1: Plan for a one room laboratory
Alternative plan for a one-room laboratory ( check figure2) 1: outpatient’s table; 2: handoperated centrifuge; 3: microscopes; 4: hematology area; 5: colorimeter; 6: water bath ; 7:
electric centrifuge; 8: syphilis serology and biochemistry area; 9: reagent refrigerator; 10:
reagent shelf; 11: glassware shelf; 12: balance; 13: staining box; 14: area for examination of
sputum specimens; 15: Bunsen burner; 16: sinks; 17: waste sink; 18: bed for patients; 19:
record-keeping area; 20: area for examination of stool specimens; 21: area for examination of
urine specimens; 22: area for reception of specimens; 23: gas bottle.
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Figure 2: Indicates another possible arrangement of a peripheral
laboratory.
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3. Safety in the Laboratory
Quite common hazards and accidents can occur in the laboratory. The need and importance of
laboratory safety should be the real concern of the laboratory. Laboratory accidents and
hazards are controlled by the use of:
 Simple precautions
 Foresight (prudence)
 Safety devices
Above all a ‘real concern’ or a ‘built in concern’ or ‘safety mindedness’ for oneself and the
other fellow working at the next bench is very essential.
Laboratory accidents range from minor injury, illness or loss of body parts to death. There are
many factors that contribute to laboratory accidents. These include:
 Poorly designed laboratory
 Overcrowding of materials
 Poor training
 Lack of concentration
 Noisy and untidy working environment
 Carelessness and neglect
 Overwork and fatigue
 Hot and humid climatic conditions
 Hurrying to finish work on time
 Emergency condition (especially during night hours)
Source of Laboratory Hazards
 Physical
 Chemical
 Biological
3.1 Physical Hazards
A. Poorly Designed Laboratory Buildings
Due attention should be given in the design of laboratories.
The management, the laboratory personnel and the architecture should be involved in the
standard design of the lab.
B. Burns
Burns may be caused by:
 Flammable chemicals and stains, or by reagents catching alight.
 Fires from spirit lamps, Bunsen burners, lighted tapers (e.g. when heating ZiehlNeelsen stain), or from faulty electrical equipment or overloaded circuits. Spirit
burners should not be used in direct sunlight because in bright light the flame can be
difficult to see.
 Corrosive chemicals being spilt on the skin or ingested when mouth –pipetting.
C. Electric shock
Electric shock can be caused by:
 Faulty electrical circuits.
 Incorrect installation of equipment.
 Touching exposed live wires.
D. Cuts
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Cuts may be caused by:
 Breakages.
 Using glassware that is cracked or has damaged edges.
 Walking on glass chippings.
3.2 Chemical Hazards
A. Toxic harmful chemicals
Inhaling fumes from toxic chemicals
Ingesting toxic chemicals by mouth pipetting
Skin contact
B. Explosive chemicals
Injury from explosions can be caused by:
Incompatible chemical exploding
Leaking gas
C. Flammable chemicals causing fire
Burns
D. Kinds of chemicals
a. Corrosive (strong acids & alkalis)
 Concentrated sulphuric acid
 Nitric acid
 Sodium hydroxide
 Potassium Hydroxide
b. Toxic irritating chemicals-cause death or serious ill
health if swallowed, inhaled, and by skin contact.
 Potassium cyanide – Chloroform
 Barium chloride - Sodium azide
c. Flammable chemicals
 Ether – Acetone
 Romanowsky stains - Methanol
d. Explosive chemicals
 Picric acid
e. Carcinogens
 Chemicals that cause cancer through ingestion, inhalation, skin contact
 Proven carcinogen chemicals include benzidin, otoulidine, and Nitrophenol.
 The risk is proportional to the length of exposure, frequency of exposure, and
concentration of the chemical.
3.3 Biological Hazards
Laboratory acquired infections
Infection can be caused by:
 Pathogens being inhaled in aerosols (airborne droplets) when snap-closing specimen
containers, dispensing or pipetting infectious fluids, or centrifuging infectious
material in open buckets. Aerosols may also be formed and inhaled following
breakages or after spilling infectious fluids. Breakages in centrifuges can be
particularly hazardous if the centrifuge is opened before the aerosols have settled.
 Pathogen being ingested from contaminated fingers, or in food that has been
contaminated, e.g. by being stored in a laboratory refrigerator. Care should be taken
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


to avoid the fingers or other parts of the body touching infected material. Mouthpipetting specimens and cultures is one of the commonest ways of ingesting
pathogens.
Pathogens entering the skin through needle punctures, cuts, scratches, insect bites,
sores or other open skin lesions. Laboratory workers must always handle infected
needles with great care.
Pathogens can also be acquired from unclean or nondisinfected room floors and
walls, water taps and laboratory benches.
Pathogens are acquired directly through careless contacts with patients or carrier staff
through breathing, hand contact, etc.
3.4 Safety Measures
Safely Designed and Organized Laboratory
It is clear that a poorly designed laboratory and overcrowding can increase the risk of
laboratory accidents. It is, therefore, important to know how the laboratory should be
designed with regard to safety considerations. The following are some of the features.
A. Adequate floor, bench and storage space
B. A floor that is well constructed with a surface that is nonslip, impermeable to liquids,
and resistant to those chemicals used in the laboratory.
C. Walls those are smooth, free from cracks, impermeable to liquids and easily
washable.
D. A door at each end of the lab so that lab staff will not be trapped should a firebreak
out.
E. Adequate ventilation with windows that can be opened.
F. Sectioning of the laboratory into separate rooms with places for patients, visitors, and
reception of specimens.
G. Bench surface that are without cracks, impervious, washable, and resistant to
disinfectants and chemicals.
H. Suitable storage facilities that include a well-ventilated, fire proof, locked store, for
the storage of flammable chemicals.
I. A good supply of gas, water, electric power & wall electric points
J. Provision of protective safety cabinets, fire extinguishers at accessible points, and
adequate waste disposal area, etc.
Safe use of Laboratory Equipment
A. Positioning
 Suitable and ideal place for operation
 Avoid overcrowding of a bench with equipment
 Position equipment correctly that requires special facilities like ventilation, shield
from sunlight, and great care.
B. Installation


Should be carried out by the supplier or by the health unit electrician or trained lab
equipment technician.
Important points to consider for safe installation
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


Ensure that the voltage of the new equipment is the same as that of the electricity
supply.
Check that the power required by the instrument does not exceed the power supply
circuit of the lab.
Make sure that the equipment is wired correctly, and the wiring system have
grounded conductor.
Safe use of Electrical Equipment
The supplier should demonstrate the use of an apparatus. If this is not possible, the operation
and service manual should be carefully studied before the equipment is operated.
Points to consider with regard to the safe use of electrical equipment:
1. Hands should be dry completely, and also the floor on which the operator is standing.
2. The electric supply must be disconnected when performing any maintenance and at
the end of the day’s work.
3. If a fuse should blow, do not automatically put in a new one until the circuit is
checked.
Safe use and Storage of Chemicals and Reagents
Even in the smallest lab, dangerous chemicals are used directly or incorporated into stains and
reagents. These include highly flammable chemicals such as ether or methanol, highly
corrosive chemicals such as phenol or sulphuric acid, or toxic and harmful chemicals such as
formaldehyde solution.
The correct handling and storage of hazardous chemicals is essential to prevent injury and
damage. It is particularly important to keep chemicals out of direct sunlight and avoid
overheating in chemical stores and the laboratory.
Overheating can decompose many chemicals, cause explosions, or the formation of toxic
fumes.
Labeling of dangerous chemicals and reagents
To reduce accidents caused by chemicals, many countries have introduced legislation,
requiring manufacturers to label dangerous chemicals with hazard symbols and to provide
simple safety instructions.
The six accepted danger symbols currently in use are toxic, corrosive, explosive, oxidizing,
highly flammable, and harmful or irritant. The safe use and storage of these hazardous
chemicals is presented in detail in the lecture note introduction to med lab.
Figure 3: Common symbols of hazards
Preventing Laboratory Infection
All specimens received in the lab should be regarded as potentially pathogenic. For example,
a blood specimen sent for measuring hemoglobin may contain highly infectious organisms.
Laboratory acquired infections can be prevented by:
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





Practicing personal hygiene
Wearing of laboratory coat and protective gloves
Safe handling of specimens and infectious materials.
Avoiding mouth-pipetting
Disposing safely of specimens and contaminated material.
Being immunized against highly infectious pathogens
Practice of Personal Hygiene
This includes:
 Washing of hands and arms with soap and water before and after work
 Wearing protective clothing and gloves whenever possible.

Not wearing protective clothing outside of the lab area.

Changing gloves when cut or infected.Covering any cuts, insect bites open sores, or
wounds with a water proof adhesive dressing.
Wearing closed shoes and not walking barefoot.
Not eating, drinking, chewing gum, smoking or applying cosmetics in any part of the
lab.


Safe Handling and Disposal of Specimens
Special precautions should be taken when collecting specimens, especially blood specimens,
and when testing specimens and handling infected material.
Safety measures involved are:
 Careful handling and disposal of materials used for collecting specimens.
 Making contaminated materials non-infectious by using appropriate decontamination
methods.
 Wearing gloves and a plastic apron when collecting blood suspected of having a
highly infectious disease like AIDS.
 Avoid contamination of fingers, other body parts, and working surface.
 Specimens suspected of having hepatitis, and AIDS must be labeled ‘HIGH RISK’.
Strict Prohibition of Mouth Pipetting
Pathogens may be ingested during mouth pipetting, either by direct aspiration or from the
mouth ends of pipettes which have been contaminated from fingers or benches. Accidents
caused by mouth pipetting include infection, poisoning, chemical burns, and other injuries
from chemicals. There are many inexpensive and simple ways to measure and dispense safely
without mouth pipetting.
Miscellaneous
 Immunization- protective inoculations against certain pathogenic organisms are
necessary.
 The use of signs- display suitable safety signs both prohibitive (don’t) and commands
(do).
 First aid- basic practical training in first aid helps to reduce suffering and
consequences of serious accidents.
Elements of a Laboratory Safety Program
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A successful laboratory safety program requires the participation of persons at every level of
the laboratory staff.
 Safety does not occur only with the appointment of safety officer or safety
committee.
 Safety does not occur by having employees solely perform their assigned duties in
manner which they feel is most efficient.
 Laboratory safety requires the full participation of every member of the staff.
Management Responsibility
 Establish a policy relative to the design and implementation of his safety program.
 Delegate authority for implementing the program
 Provide a safe and healthful work place
 Provide fund for the implementation of the program
 Assess the program- establish a mechanism to ensure safety
 Establish safety committee- regular report, recommendations regarding need for
modification of the program.
Safety Officer Responsibility
 Technical advisor to the program
 Assist in the development of safe work method
 Advise management on safety issues
 Assist safety committee
 Provide a variety of communication, e.g. hazard notice, safety data
 Review a variety of plans which include facility designs, special equipment purchase
in relation to safety.
Supervisor Responsibility
 Train the staff in lab practices required for safe conduct of work.
 Evaluate regularity of the laboratory facilities, equipment, personnel and work place.
 Correct unsafe condition as fire hazards, physical hazards, and defective equipment.
Employees’ Responsibility
 Use of safe equipment
 Report of malfunctioning of equipment
 Report injuries or exposure
 Report hazard or unsafe condition to supervisors
 Receiving proper safety training.
 Ask questions if uncertain about safety regulations
23.5 Cleaning, disinfection, sterilization and disposal of biohazard
waste.
Cleaning glassware
 Instructions for cleaning: Wear protective clothing and gloves when cleaning lab
equipmentglass containers (Erlenmeyer flasks, beakers, test-tubes)
 pipettes
Glass containers
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New glassware
Glassware that has never been used may be slightly alkaline.
In order to neutralize it:




Prepare a bowl containing 3 liters of water and 60 ml of concentrated
hydrochloricacid (i.e. a 2% solution of acid).
Leave the new glassware completely immersed in this solution for 24 hours.
Rinse twice with ordinary water and once with demineralized water.
Dry.
Dirty glassware
 Preliminary rinsing: Rinse twice in cold or lukewarm water (never rinse bloodstained
tubes in hot water). If the glassware has been used for fluids containing protein, it
should be rinsed immediately and then washed (never allow it to dry before rinsing).
 Soaking in detergent solution: Prepare a bowl of water mixed with washing powder
or liquid detergent. Put the rinsed glassware in the bowl and brush the inside of the
containers with a test-tube brush Leave to soak for 2–3 hours.
Rinsing
Remove the articles one by one. Rinse each one thoroughly under the tap, then soak them all
in a bowl of ordinary water for 30 minutes.
Rinse each article in a stream of clean water. (Do not forget that traces of detergent left on
glassware can lead to false laboratory results.)
Draining
Place containers (beakers, flasks, measuring cylinders) on the pegs of a draining rack. Place
test-tubes upside-down in a wire basket.
Drying
Place the glassware in wire baskets and dry in a hot-air oven at 60 °C. Alternatively, place the
baskets in a sunny spot in the laboratory and cover them with a fine cloth.
Plugging
The clean dry glassware should be put away in a cupboard to protect it from dust. It is
recommended that glass containers be plugged with non-absorbent cotton wool or their
mouths covered with small caps made from newspaper or, preferably, thin sheets of paraffin
wax or clinging plastic, if available.
Pipettes
Immediate rinsing
Once a pipette has been used, rinse it immediately in a stream of cold water to remove blood,
urine, serum, reagents, etc.
Soaking in water
After rinsing, place the pipettes in a large, plastic measuring cylinder (or bowl) full of water.
If the pipettes have been used to measure infected material, leave them in a cylinder full of
disinfectant solution (e.g. a quaternary ammonium compound or
1% bleach solution; see pages 84 and 85) for 4 hours.
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Soaking in detergent and rinsing
Follow the instructions given above for soaking and rinsing of laboratory glassware.
Blocked pipettes
Put blocked pipettes in a cylinder filled with dichromate cleaning solution. Slide them
carefully into the solution and leave for 24 hours.
The next day, pour the dichromate solution into another cylinder (it can be used four times).
Hold the cylinder containing the pipettes under the tap and rinse thoroughly.
Remove the pipettes one at a time. Check that the obstruction has been washed away. Rinse
again.
Leave to soak in ordinary water for 30 minutes, then change the water and soak for a further
30 minutes.
Warning: Dichromate cleaning solution is highly corrosive and should be used with extreme
care. If it is accidentally splashed on the skin or clothing or into the eye(s), wash at once with
large quantities of water.
Drying
Dry heat-resistant glass pipettes in a hot-air oven at 60 °C and ordinary pipettes in an
incubator at 37 °C. Alternatively, leave pipettes to air-dry.
Using the vacuum pump
This is a small instrument made of metal, plastic or glass that is attached to the water tap.
Turn the water on hard to drive a strong jet through the pump. This causes air to be sucked
into the side arm of the pump and the rubber tubing attached to it.
Fit this rubber tubing over the tip of the pipette.
3. Dip the other end of the pipette into the rinsing liquid (water or detergent solution), which
is sucked through the pipette and discharged by the pump into the sink (Fig ).
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3.6 Disposal of Biohazard Waste
A. General rules
Prior to any treatment, all biohazard wastes, including those to be incinerated, should be
enclosed in a puncture-resistant, biohazard bag that is color-coded or labeled with the
biological hazard symbol.
Untreated biohazard waste is not to be disposed of in the municipal waste (system) stream.
All biohazard waste must be treated by chemical disinfection or autoclaving before in any
way being disposed of in the municipal waste stream.
After disinfection, but before disposal in the municipal waste stream, all treated biohazard
wastes (apart from liquids) should be enclosed in an unmarked outer bag that is not red or
labeled with the biohazard symbol. Any biohazard waste that has been treated as described
below and packaged such that it is clearly evident that the waste has been effectively treated,
is not subject to be treated as biohazard waste and may be collected, transported for
incineration or disposed of as municipal waste.
The person handling the emptying of waste bins, waste bottles or sharps containers must be
careful not to touch anything without protective clothing and must use gloves to avoid
contracting infections from the waste.
The waste bins, sharps containers, etc., must be clearly indicated/marked: Biohazard wastes.
B. Laboratory waste
Discarded swabs, cotton, sample containers and other biohazard but non-sharp objects used
for sample collection should be placed in a covered trash container lined with an autoclavable
biohazard yellow bag. Visibly soiled or splashed tourniquets and Vacutainer holders should
be discarded and new equipment used.
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Disposable plastics such as tips, culture plates, micro plates, petri dishes, test tubes, etc., used
for testing of samples should be disposed of in a covered waste bin lined with an autoclavable
biohazard yellow bag, autoclaved and transported for incineration.
Pipettes should be soaked in special pipette baskets in 10 % household bleach overnight,
before being thrown in a waste bin lined with an autoclavable biohazard yellow bag,
autoclaved and incinerated.
Reusable glassware such as cylinders, flasks and beakers should be disinfected with 10 %
household bleach over night before washed and autoclaved.
C. Sharps
All sharp implements used in the laboratory need to be handled carefully. They can easily
break the skin and increase the risk of infection with infectious agents.
Broken glassware must also be handled as sharps. The laboratory should have a special box
for broken glassware
If needles are used, they should never be recapped before disposal. Recapping needles may
lead to somebody accidentally pricking him or herself.
Dispose of all sharps directly into a secure perforated sharps disposal container lid. The lid
only allows the sharps into the container but not out. The sharps container should be
puncture-resistant, leak proof on the sides and bottom, and color-coded or labeled with a
biohazard symbol.
Note: When selecting sharps containers, look for special safety features such as lids that lock
tight for safe disposal and a container that can be sterilized by steam, gas, or chemicals. If
sharps containers are not specifically constructed to be autoclaved, the resulting mass of
melted plastic is extremely hazardous due to the needles that often protrude.
When full, seal top, autoclave, then arrange for incineration.
Decontamination is achieved by autoclaving the sharps container. Incineration destroys the
sharps completely hence eradicating the chances of accidental injury to people in the
community.
D. Residual body fluids
Ensure that all tubes/containers containing residual body fluids are properly sealed to avoid
spillage. These are then stored temporarily in waste bins lined with disposal bags.
Transfer the disposal bags, together with contents, into autoclaving bags. Then place a piece
of autoclavable tape on the disposal bag to seal it and autoclave as required.
After the autoclaving cycle is completed, incinerate the whole package to completely destroy
all contents.
E. Chemical wastes
The following procedure is adopted for most chemicals that are used in low concentrations.
Collect all liquid chemical waste in properly labeled bottles with a little concentrated
disinfectant (i.e., JIK).
Keep monitoring the rising level of waste in the trap bottles. Never fill the trap bottles to the
very top to avoid spillage.
Empty the contents of the trap bottle down the drain, preferably a special sink in the
laboratory, and wash down with more disinfectant, liquid soap and a large volume of water.
Minimum disinfection time of any liquid Biohazard Waste is 30 minutes.
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Note: Some disinfectants react with organic compounds, acids, or bases. Consider this when
selecting your disinfectant before commencing your work.
SAFETY PRECAUTIONS
Anybody handling Biohazard Wastes should always put on laboratory protective clothing and
gloves and keep to the laboratory safety practices to avoid viral and other infectious disease
transmissions.
Any spills or splashes of infectious material should be immediately cleaned up with absorbent
material using an approved disinfectant such as dilute 10% bleach or chlorhexidine solution.
Decontaminating bench tops and other surfaces
Bench tops should be wiped with a disinfectant (a phenolic disinfectant, 1% sodium
hypochlorite [bleach], or 70% isopropyl alcohol) routinely after working with infectious
agents or clinical specimens or after spills, splashes, or other contamination by infectious
materials. Solutions of disinfectants should be maintained at the work station.
Decontamination of spills
The following procedure is recommended for decontaminating spills.
• Isolate the area to prevent anyone from entering.
• Wear gloves and protective clothing (e.g., a gown or lab coat, shoes, and a mask [if the spill
may contain a respiratory agent or if the agent is unknown]).
• Absorb or cover the spill with disposable towels.
• Saturate the towels with an appropriately diluted intermediate or high-level disinfectant
(e.g., a phenolic formulation or household bleach).
• Place disinfectant-soaked towels over the area and leave them in place for at least 15
minutes before removing and discarding them.
• Wipe area using clean disinfectant-soaked towels and allow area to air dry.
• Place all disposable materials (including towels) used to decontaminate the spill into a
biohazard container.
• Handle the material in the same manner as other infectious waste.
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4. Hematology
4.1 Collection & Handling of blood
General precautions:




Technical errors:


Always wear disposable plastic gloves, especially if there is any hand cut.
Always be careful when handling syringes, needles & lancets.
Always use disposable syringes, needles & lancets.
Always take care of specimen identification & request forms.
Faults in sample Collection (See below).
Faults in sample Transport & Storage (identification error, how long delay & at
what temp.).
Technical errors from sample collection:

Insufficient or excess anticoagulant.

Inappropriate anticoagulant.

Inadequate mixing with anticoagulant.

Patient &/or specimen identification error.

Delay in transit to lab.
Venous blood collection:
The phlebotomist should enter a training program. Venous blood,
mixed with an anticoagulant, is preferred for most hematological examinations.
Best site is the antecubital vein, & in obese subject may use the dorsum of the hand.
Needle guage: for adults 21 (0.8mm), for children 23 (0.6mm).
Make sure of proximal & distal fixation of the vein.
Skin preferably cleaned with 70% alcohol.
Steps to facilitate obtaining a venous blood sample are:

Warming the patient’s arm.

Using a tourniquet (should be loosened once entered the vein to avoid
the skin over the site of the vein.

Asking the patient to open & clench his fist several times.
Advantages of using the tourniquet:
- Blood filling of the vein.
- Proximal fixation of the vein.
Tapping
Disadvantages of using the tourniquet:
- Hemoconcentration.
- Red cell hemolysis.
Always notice the blood level marker in tubes containing an anticoagulant.
After puncture: elevate the arm, apply pressure & adhesive dressing.
There is no difference in using glass or plastic tubes except in coagulation study (where plastic or
siliconized glass tubes should be used).
Avoid hemolysis of blood by:

Minimal use of the tourniquet.

Withdraw blood slowly.

Do not use very fine needled.

Deliver the blood gently into the tube.
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
Avoid frothing during mixing the tube.
Capillary (peripheral) blood:
It is often necessary to obtain blood by skin puncture in babies and infants and in adults with poor veins.
Capillary blood may be obtained from a freely flowing stab wound made with a sterile lancet on:
- The plantar surface of a warmed and cleansed heel (babies less than 3 months of age). The correct site
for puncture of the heel is shown in Fig. 1.4.
- The plantar aspect of the big toe (infants).
- A finger, thumb or ear lobe (older children and adults).
The lateral or posterior aspect of the heel should not be used in a baby, as the underlying bone is much
closer to the skin surface than it is on the plantar aspect. Osteomyelitis of the calcaneal bone has resulted
from puncture of the bone.
In older patients a finger (excluding the fifth finger) or the thumb is preferred. The palmar surface of the
distal phalanx is the preferred site on a digit, since the underlying bone is closer to the skin surface on other
aspects. The middle or ring finger of the non-dominant hand is preferred; these digits are less painful than
the index finger. Previous puncture sites should be avoided, to reduce the risk of infection.
In adults, skin punctures should ideally be more than 1.5 mm deep in order that the lancet passes through
the dermal– subcutaneous junction where the concentration of blood vessels is greatest, permitting a free
flow of blood. Lancets used for heel puncture in full-term babies must not exceed 2.4 mm in length, since
this is the depth below the skin of the calcaneal bone. Much shorter lancets are available and should be
selected for use in premature babies. Safety lancets, with a blade that retracts permanently after first use,
have been developed in order to reduce the risk of accidental injury to phlebotomy staff.
Capillary samples should be obtained from warm tissues so that a free flow of blood is more readily
obtained. If the area is cool then it should be warmed with a wet cloth. The skin should then be cleansed
with 70% alcohol and dried with sterile gauze (since traces of alcohol may lead to haemolysis of the
specimen). The first drop of blood may be diluted with tissue fluid and should be wiped away with sterile
gauze square. Flow of blood may be promoted by gentle pressure, but a massaging or pumping action
should not be employed, since this may lead to tissue fluid being mixed with blood.
Differences between capillary and venous blood:
Venous blood and “capillary” blood are not quite the same. Blood from a skin puncture is a mixture of
blood from arterioles, veins, and capillaries, and it contains some interstitial and intracellular fluid.
However, there are negligible differences when a free flow of blood has been obtained. The differences
may be exaggerated by cold with resulting slow capillary blood flow.
The packed cell volume (PCV), RBCs count, and Hemoglobin concentration (Hb) of capillary blood are
slightly greater than in venous blood. Also the total leukocyte and neutrophil counts are higher.
Conversely, the platelet count appears to be higher in venous than in capillary blood. This may be the result
of adhesion of platelets to the site of the skin puncture.
Serum:
The difference between plasma and serum is that the latter lacks fibrinogen and some of the coagulation
factors.
Blood collected to obtain serum should be delivered into sterile tubes with caps or evacuated collection
tubes and allowed to clot undisturbed for about 1 hour at room temperature (18-25°C). Then the clot should
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be loosened gently from the container wall by means of a wooden stick or a thin plastic or glass rod. Rough
handling will cause hemolysis. The tubes are centrifuged for 10 min at about 1200 rpm. The supernatant
serum then is pipetted into another tube and centrifuged again for 10 min at about 1200 rpm. The
supernatant serum is transferred to tubes for tests or for storage.
For most tests, serum should be kept at 4°C until used, but if testing is delayed, serum can be stored at 20°C for up to 3 months and at -40°C or less for long-term storage. Frozen specimens should be thawed
on the bench or in a water bath at room temperature, and then inverted several times to ensure homogeneity
before use for a test. Do not refreeze thawed specimens.
Figure 4:Anterior surface of the left arm showing veins most suitable for venepuncture.
Figure 5: Venepuncture technique using needle and syringe.
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Figure 6: Venepuncture technique using an evacuated container; the distal end of the needle has
been screwed into the holder and the proximal needle has then been unsheathed and inserted into a suitable
vein.
Figure 7:The areas of the foot of a baby or infant which are suitable for obtaining
capillary blood.
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4.2 Part One: Automated Blood Count Techniques
A variety of automated instruments for performing blood counts are in widespread use. semiautomated instruments require some steps (e.g., dilution of a blood sample) to be carried out
by the operator. Fully automated instruments require only that an appropriate blood sample is
presented to the instrument. Semiautomatic instruments often measure a small number of
components (e.g., WBC and Hb). Fully automated multichannel instruments usually measure
from 8 to 20 components, including some variables that have no equivalent in manual
techniques. Automated instruments usually have a high level of precision, which, for cell
counting and cell-sizing techniques, is greatly superior to that achievable with manual
techniques. If instruments are carefully calibrated and their correct operation is ensured by
quality control procedures, they produce test results that are generally accurate. When blood
has abnormal characteristics, the results for one or more parameters may be aberrant;
instruments are designed so that such inconsistent results are “flagged” for subsequent
review. The abnormal characteristics that lead to inaccurate counts vary between instruments,
so it is important for instrument operators to be familiar with the types of factitious results to
which their instruments are prone.
Blood cell counters may have automated procedures for sample recognition (e.g., by barcoding), for ensuring that adequate sample mixing occurs, for taking up the test sample
automatically, and for detection of clots or inadequately sized samples. Ideally, blood
sampling is carried out by piercing the cap of a closed tube so that samples that carry an
infection hazard can be handled with maximum safety.
Laboratories performing large numbers of blood counts each day require fully automated
blood counters capable of the rapid production of accurate and precise blood counts,
including platelet counts and differential counts, either three-part or five- to seven-part. The
sample throughput required varies with the workload and the timing of arrival of blood
specimens in the laboratory, but for most large laboratories a throughput of 100 or more
samples per hour is required. Sample size and the availability of a “predilute” mode are
particularly relevant if the laboratory receives many paediatric specimens.
Choice of an instrument for an individual laboratory, as well as for point-of-care sites outside
the laboratory, should take account of capital expenditure and running costs, including
maintenance and reagents; size of instrument; requirements of services such as water,
compressed air, drainage, and an electricity supply with stable voltage; environmental
disturbance by generation of heat, vibration, and noise; any influence on performance by the
ambient temperature and humidity; storage requirements for the often bulky reagents; ease of
operation; and the likely level of support that can be expected from the manufacturer.
Most automated instruments, however, count for a specified period of time rather than on an
exact volume of blood; they therefore require calibration by means of the direct counts
derived from instruments counting cells in a defined volume of diluted blood. For some
variables, instruments are calibrated by the manufacturer, but others require calibration in the
laboratory. Performance characteristics of an instrument vary over time, so periodic
recalibration is needed; both when quality control procedures indicate the necessity and when
certain components are replaced.
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4.2.1 Hemoglobin Concentration
Most automated counters measure haemoglobin by a modification of the manual HiCN
method with cyanide reagent or with a nonhazardous chemical such as sodium lauryl
sulphate, which avoids possible environmental hazards from disposal of large volumes of
cyanide-containing waste. Modifications include alterations in the concentration of reagents
and in the temperature and pH of the reaction. A nonionic detergent is included to ensure
rapid cell lysis and to reduce turbidity caused by cell membranes and plasma lipids.
Measurements of absorbance are made at a set time interval after mixing of blood and the
active reagents but before the reaction are completed.
4.2.2 Red Blood CELL Count
Red cells and other blood cells can be counted in systems based on either aperture impedance
or light-scattering technology. Because large numbers of cells can be counted rapidly, there is
a high level of precision. Consequently, electronic counts have rendered the RBC and the red
cell indices derived from it (the MCV and the MCH) of much greater clinical relevance than
was possible when only a slow and imprecise manual RBC was available.
4.2.3 Counting Systems
Impedance Counting
Impedance counting, first described by Wallace Coulter in 1956 depends on the fact that red
cells are poor conductors of electricity, whereas certain diluents are good conductors; this
difference forms the basis of the counting systems used in Beckman–Coulter, Sysmex,
Abbott, Roche, and a number of other instruments.
For a cell count, blood is highly diluted in a buffered electrolyte solution. The flow rate of this
diluted sample is controlled by a mercury siphon (as in the original Coulter system) or by
displacement of a tightly fitting piston. This result is a measured volume of the sample
passing through an aperture tube of specific dimensions (e.g., 100 mm in diameter and 70 mm
in length). By means of a constant source of electricity, a direct current is maintained between
two electrodes, one in the sample beaker or the chamber surrounding the aperture tube and
another inside the aperture tube. As a blood cell is carried through the aperture, it displaces
some of the conducting fluid and increases the electrical resistance. This produces a
corresponding change in potential between the electrodes, which lasts as long as the red cell
takes to pass though the aperture; the height of the pulses produced indicates the volume of
the cells passing through. The pulses can be displayed on an oscillograph screen. The pulses
are led to a threshold circuit provided with an amplitude discriminator for selecting the
minimal pulse height, which will be counted. The height of the pulses is used to determine the
volume of the red cells.
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Figure 8: Effect of threshold discrimination (horizontal axis) in separating cell
signals from background noise.
Light Scattering
Red cells and other blood cells may be counted by means of electro-optical detectors. A
diluted cell suspension flows through an aperture so that the cells pass, in single file, in front
of a light source; light is scattered by the cells passing through the light beam. The scattered
light is detected by a photomultiplier or photodiode, which converts it into electrical impulses
that are accumulated and counted. The amount of light scattered is proportional to the surface
area and therefore the volume of the cell so that the height of the electrical pulses can be used
to estimate the cell volume. The high-intensity coherent laser beams used in current
instruments have superior optical qualities to the noncoherent tungsten light of earlier
instruments. Sheathed flow allows cells to flow in an axial stream with a diameter not much
greater than that of a red cell; light can be precisely focused on this stream of cells. Electrooptical detectors are used for red cell sizing and counting in Bayer-Technicon systems and for
white cell differential counting in a number of other instruments.
4.2.4 Reliability of Electronic Counters
Electronic counts are precise, but care needs to be taken so that they are also accurate. The
recorded count on the same sample may vary from instrument to instrument and even
between different models of the same instrument. Inaccuracy may be introduced by :
1-coincidence (i.e., by two cells passing through an orifice simultaneously and
being counted as one cell).
2-pulse being generated during the electronic dead time of the circuit.
3-Recirculation of cells that have already been counted.
4-red cell agglutination (which causes a clump of cells to be counted as one
Cell).
28 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
5-by the counting of bubbles, lipid droplets, microorganisms, or extraneous
Particles as cells.
6-Faulty maintenance may lead to variation in the volume aspirated or the flow rate.
7-Single-channel instruments may have their thresholds set incorrectly, and multichannel
instruments may be incorrectly calibrated.
Coincidence correction; in some instruments, this is done automatically by electronic editing.
Errors of coincidence can be detected by carrying out a series of measurements at various
dilutions of the same specimen, plotting the data on graph paper, and then extrapolating the
graph to the baseline for the true value. Alternatively, the need for coincidence correction can
be avoided by having the dimensions and flow characteristics of the aperture through which
the cells pass such that cells can only pass in single file; this may be achieved by sheath flow
or hydrodynamic focusing in which diluted blood is injected into a sheath of fluid as it flows
into the sensing zone. This induces the cells to pass through the centre of the sensing zone in
single file and free of distortion. Coincidence can be more effectively reduced with sheathed
flow and precisely focused light in an electro-optical detector than in an impedance counter so
that less dilution of the blood sample is needed.
Electrical impulses generated by recirculation of cells can be eliminated by electronic editing.
recirculation of cells in the region of the aperture can be prevented by “sweep flow” in which
a directed stream of diluent sweeps cells and debris away from the aperture, thus preventing
cells from being recounted and debris from being counted as cells.
Inaccurate counts consequent on red cell agglutination are usually the result of cold
agglutinins. They are recognized as erroneous because of an associated marked factitious
elevation of the MCV. A correct count can be achieved by prewarming the blood sample and,
if necessary, also prewarming the diluent.
A correct RBC and, particularly, a correct measurement of the MCV are dependent on the use
of an appropriate diluent. For impedance counters, pH, temperature, and rate of ionization
have to be standardized and remain constant because changes alter the electrical field and
may lead to artefactual alterations in the size, shape, and stability of the blood cells in the
diluent. Diluents must be free of particles and give a background count of less than 50
particles in the measured volume. The correct diluent for each individual instrument must be
used; other diluents, even those made by the same manufacturer, may not be interchangeable.
Any laboratories using diluents other than those recommended by the manufacturer of the
instrument must satisfy themselves that no error is being introduced.
For red cell counting in simple single-channel counters a suitable diluent requires a pH of
7.0–7.5 and osmolality of 340 ± 10 mmol. Physiological saline (9 g/l NaCl) or phosphatebuffered saline, which have the advantages of simplicity and ready availability, can be used as
a red cell diluent, provided that the counts are performed immediately after dilution to avoid
errors owing to sphering. Commercial solutions of saline (for intravenous use) are usually
particle-free. Other solutions may require filtration through a 0.22- or 0.45-mm micropore
filter to remove dust.
Setting Discrimination Thresholds
An accurate RBC requires that thresholds be set so that all red cells, but a minimum of other
cells, are included in the count. Some counters have a lower threshold but no upper threshold
29 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
so that white cells are included in the “RBC.” Because the WBC is usually very low in
relation to the RBC, this is not usually of practical importance; however, an appreciable error
can be introduced if the WBC is greatly elevated, particularly if the patient is also anaemic.
The setting of the lower threshold is of considerable importance because it is necessary to
ensure that microcytic red cells are included in the count without also counting large platelets.
Current multichannel instruments, both impedance counters and counters using lightscattering technology, have thresholds that are either precalibrated by the manufacturer or are
automatically adjusted, depending on the characteristics of individual blood samples. Singlechannel impedance instruments capable of performing a direct RBC require setting of
thresholds so as to separate pulses generated by red cells from background noise and from
pulses generated by platelets. This is done by adjusting the aperture current and the pulse
amplification. A simple method is to dilute a fresh blood sample and carry out successive
counts on the suspension, while the lower threshold control is moved incrementally from its
maximum to its minimum position. At the maximum position, the count should be zero or
close to zero, and the counts will increase as the amplitude is reduced. The counts at each
setting are plotted on arithmetic graph paper.
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Figure 9: Setting Discrimination Thresholds
Method to establish working conditions of cell counters. The correct setting of the threshold
(at arrow) is intended to exclude noise pulses without loss of the signal pulses produced by
the blood cells.
The correct threshold setting is at the left of the horizontal part of the graph before the line
begins to slope. It is important to check that the setting selected is valid for microcytic cells.
The threshold can be defined more precisely for an individual sample by means of a pulse
height analyser linked to the counting system. The lower threshold is correctly set if beyond
this point there are less than 0.5% of the counts at the peak (mode) of the pulse size
distribution curve.
4.2.6 Packed Cell Volume and Mean Cell Volume
Modern automated blood cell counters estimate PCV by technology that has little connection
with packing red cells by centrifugation. It is sometimes convenient to use different terms to
distinguish the manual and automated tests, and for this reason the International Council for
Standardization in Haematology has suggested that the term “haematocrit” (Hct) rather than
PCV should be used for the automated measurement. However, it should be noted that, in the
past, the terms “packed cell volume” and “haematocrit” have been used interchangeably for
the manual procedure.
Sources of error include uneven cell distribution in the sample; too many or too few cells
in the sample; subjective judgments as to whether a given cell falls within the defined
counting area; contamination of the hemocytometer; variation in how a sample is
loaded; and differences in user technique.
With automated instruments, the derivation of the RBC, PCV, and MCV are closely
interrelated. The passage of a cell through the aperture of an impedance counter or through
the beam of light of a light-scattering instrument leads to the generation of an electrical pulse
the height of which is proportional to cell volume. The number of pulses generated allows the
RBC to be determined, as discussed earlier. Pulse height analysis allows either the MCV or
the PCV to be determined. If the average pulse height is computed, this is indicative of the
MCV, and the PCV can be derived by multiplying the estimated MCV by the RBC. Similarly,
if the pulse heights are summated, this figure is indicative of the PCV, and the MCV can, in
turn, be derived by dividing the PCV by the RBC.
Automated instruments require calibration before the PCV or MCV can be determined.
Calibration of the PCV can be based on manual PCV determinations. Alternatively, the MCV
can be calibrated by means of the pulse heights generated by latex beads, stabilize cells, or
some other calibrant containing particles of known size; however, unfixed human red cells
that are biconcave and flexible will not necessarily show the same characteristics in a cell
counter as latex particles or some other artificial calibrant. Aperture-impedance systems
measure an apparent volume that is greater than the true volume, being influenced by a
“shape factor; this factor is less than 1.1 for young, flexible red cells; is between 1.1 and 1.2
for fixed biconcave cells; and is about 1.5 for spheres, whether they be fixed cells or latex
spheres.
The MCV, and therefore the PCV, as determined by an automated counter, will vary with
certain cell characteristics other than volume. As indicated earlier, such characteristics include
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shape, which in turn is partly determined by flexibility. With impedance counters, the normal
disc-shaped red cell becomes elongated into a cigar shape as it passes through the aperture;
this is caused by deformation in response to shear force, which occurs in cells of normal
flexibility. Cells with a reduced haemoglobin concentration undergo more elongation than
normal cells; this leads to a reduced “shape factor,” a reduced pulse height in relation to the
true size of the cell, and underestimation of the MCV. Conversely, cells with abnormally rigid
membranes and cells such as spherocytes with a high haemoglobin concentration will
undergo less deformation than normal and the MCV will be overestimated. Earlier lightscattering instruments also underestimated the volume of red cells with a reduced
haemoglobin concentration because light scattering was affected by the haemoglobin
concentration. These artefacts are seen even with normal red cells of varying haemoglobin
concentration but are more apparent with red cells from patients with defects in haemoglobin
synthesis such as those from patients with iron deficiency. Light-scattering instruments have
been developed to avoid artefacts of this type. Cells are isovolumetrically sphered so that their
light-scattering characteristics are uniform and should follow the laws of physics.
The automated MCV and PCV are prone to certain errors that do not occur or are less of a
problem with manual methods. These include those resulting from microclots or partial
clotting of the specimen, extreme microcytosis, and the presence of cryoglobulins or cold
agglutinins; the last is a relatively common cause of factitious elevation of the MCV because
clumps of cells are sized as if they were single cells. Because the RBC is underestimated, the
PCV is less affected, although it is also inaccurate. It is rare for warm agglutinins to cause a
similar problem. Sickling may cause a factitious increase in MCV and PCV, whereas
alterations in plasma osmolarity occurring, for example, in severe hyperglycemias also cause
factitious elevation of the MCV and PCV.
4.2.7 Variation in Red Cell Volumes:
RED CELL DISTRIBUTION WIDTH
Automated instruments produce volume distribution histograms that allow the presence of
more than one population of cells to be appreciated. Instruments may also assess the
percentage of cells falling above and below given MCV thresholds and “flag” the presence of
an increased number of microcytes or macrocytes. Such measurements may indicate the
presence of a small but significant increase in the percentage of either microcytes or
macrocytes before there has been any change in the MCV.
The RDW is derived from pulse height analysis and can be expressed either as the standard
deviation (in fl) or as the coefficient of variation (CV) (%) of the measurements of the red cell
volume. Current Beckman-Coulter and Bayer-Technicon instruments express the RDW as the
SD, and Sysmex instruments express it as either the SD or the CV. The normal reference
range is in the order of 12.8 ± 1.2% as CV and 42.5 ± 3.5 fl as SD. However, widely different
ranges have been reported; therefore it is important for laboratories to determine their own
reference ranges. The RDW expressed as the CV has been found of some value in
distinguishing between iron deficiency (RDW usually increased) and thalassaemia trait
(RDW usually normal) and between megaloblastic anaemia (RDW often increased) and other
causes of macrocytosis (RDW more often normal).
32 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
4.2.8 Total White Blood Cell Count
The total WBC is determined in whole blood in which red cells have been lysed. The lytic
agent is required to destroy the red cells and reduce the red cell stroma to a residue that causes
no detectable response in the counting system without affecting leucocytes in such a manner
that the ability of the system to count them is altered. Various manufacturers recommend
specific reagents, and for multichannel instruments that also perform an automated
differential count use of the recommended reagent is essential.
Fully automated multichannel instruments perform WBCs by impedance or light-scattering
technology or both. Residual particles in a diluted blood sample are counted after red cell
lysis or, in the case of some light-scattering instruments, after the red cells have been rendered
transparent. Thresholds are set to exclude normal platelets from the count, although giant
platelets are included. Some or all of any nucleated red cells present are usually included, so
that when nucleated red cells are present the count approximates more to the TNCC than to
the WBC.
Factitiously low automated WBCs occasionally occur as a consequence of leukocyte
agglutination, prolonged sample storage, or abnormally fragile cells (e.g., in leukaemia).
Factitiously high counts are more common and usually result from failure of lysis of red cells.
With certain instruments this may occur with the cells of neonates or be consequent on
uraemia or on the presence of abnormal haemoglobin such as haemoglobin S or haemoglobin
C; high counts may also be the result of microclots, platelet clumping, or the presence of a
cryoglobulin.
4.2.9 Automated Differential Count
Most automated differential counters that are now available use flow cytometry incorporated
into a full blood counter rather than being stand-alone differential counters. Increasingly,
automated blood cell counters have a differential counting capacity, providing either a threepart or a five- to seven-part differential count. Counts are performed on diluted whole blood
in which red cells are either lysed or are rendered transparent. A three-part differential count
assigns cells to categories usually designated:
(a) “Granulocytes” or “large cells”;
(b) “Lymphocytes” or “small cells”; and
(c) “Monocytes,” “mononuclear cells,” or “middle cells.”
In theory, the granulocyte category includes eosinophils and basophils, but in practice it is
common for an appreciable proportion of cells of these types to be excluded from the
granulocyte category and to be counted instead in the monocyte category.
Five- to seven-part differential counts classify cells as neutrophils, eosinophils, basophils,
lymphocytes, and monocytes and in an extended differential count may also include large
immature cells (composed of blasts and immature granulocytes) and atypical lymphocytes
(including small blasts).
Automated instruments performing three-part or five-to seven-part differential counts are
able to “flag” or reject counts from the majority of samples with nucleated red cells,
myelocytes, promyelocytes, blasts, and atypical lymphocytes. To a lesser extent, instruments
incorporating a three-part differential count, although not capable of enumerating eosinophils
or basophils, are able to flag a significant proportion of samples that have an increased
number of one of these cell types.
33 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Automated differential counters using flow cytometry classify far more cells than is possible
with a manual differential count. Automated counts are consequently much more precise than
manual counts; however, with certain cell categories—specifically monocytes and
basophils—the degree of precision is sometimes less than would be expected for the number
of cells counted, indicating that such cells are not always classified in a consistent manner.
The accuracy of automated counters is less impressive than their precision. With all types of
counters, unusual cell characteristics or ageing of a blood specimen can lead to
misclassification of cells. Although the majority of samples containing abnormal cells are
“flagged,” this is not invariably so; the presence of nucleated red cells, immature
granulocytes, atypical lymphocytes, and blasts (even occasionally quite large numbers of
blasts) may not give rise to a “flag.” However, human observers performing a 100-cell
manual differential count also miss significant abnormalities. In general, automated counts
have compared favourably with routine manual counts, especially if the instruments are
assigned only two functions—performing differential counts on normal samples and
“flagging” abnormal samples. If morphological abnormalities are flagged, microscopic
examination of a stained blood film should always be undertaken.
In the presence of a significant number of NRBC, the total count is neither a true “WBC”
nor a true “TNCC” and the absolute WBC counts calculated from the total will necessarily be
somewhat erroneous.
Instruments currently in use that count NRBC and correct the WBC for NRBC interference
include the Abbott CellDyn 4000, the Sysmex XE2100, and the Beckman-Coulter LH750.
Differential counters based on pattern recognition in stained blood films were initially
preferred by many haematologists, but they were relatively slow, and because they could
count only a small number of cells in a reasonable time, the precision of the automated count
was no better than that of a manual count. However, with improved computing technology
and with the use of artificial neural networks, such instruments (e.g., DiffMaster, CellVision
AB, SE-223 70, and Lund, Sweden) are now capable of providing a useful differential count
on blood samples containing abnormal cells. Up to 30 films an hour can be processed and
reviewed, and abnormal cells can be reclassified if required.
4.2.10 New White Cell Parameters
Many instruments are able to “flag” the presence of atypical or “variant” lymphocytes by
features such as alteration in size and in impedance or light-scattering characteristics.
Automated white cell counters can also analyse cell characteristics by novel technologies and
identify cell types by features that differ greatly from those used when a blood film is
examined visually. It is possible, for example, to identify eosinophils by the ability of their
granules to polarize light or to detect a left shift or the presence of blasts by the reduced light
scattering of the nuclei of more immature granulocytes. There is also the potential to produce
information that is not directly analogous with that available from a manual differential count.
Instruments that incorporate a cytochemical reaction give information on enzyme activity
expressed as the mean peroxidase activity index (MPXI); standard range is -10 to +10. An
increased MPXI has been observed in infections, in some myelodysplasias and leukaemias, in
the acquired immune deficiency syndrome (AIDS), and in megaloblastic anaemia, whereas a
reduced MPXI occurs in inherited and acquired neutrophil peroxidase deficiency. It has been
suggested that the parameters used for defining leucocyte types might also allow detection of
the presence of malaria pigment as a screening test in areas where malaria is prevalent.
34 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
4.2.11 Platelet Count
Platelets can be counted in whole blood using the same techniques of electrical or electrooptical detection as are used for counting red cells. An upper threshold is needed to separate
platelets from red cells, and a lower threshold is needed to separate platelets from debris and
electronic noise. Recirculation of red cells near the aperture should be prevented, as pulses
produced may simulate those generated by platelets. Three techniques for setting thresholds
have been used:
(a) Platelets can be counted between two fixed thresholds (e.g., between 2 and 20 fl;
(b) Pulses between fixed thresholds can be counted with subsequent fitting of a curve and
extrapolation so that platelets falling outside the fixed thresholds are included in the computed
count.
(c) Thresholds can vary automatically, depending on the characteristics of individual blood
samples, to make allowance for microcytic or fragmented red cells or for giant platelets.
A new method for platelet counting by flow cytometry has been developed Platelets in a
blood sample are labelled fluorescently with a specific monoclonal antibody or combination
of antibodies, and by measuring the RBC :platelet ratio the platelet count can be calculated.
Suitable antibodies to platelet antigens are CD41, CD42, and CD61. This method using CD41
and CD61 has been adopted by the International Council for Standardization in Haematology
as the reference method some instruments now provide an automated immunological platelet
count for diagnostic use. Although these instruments can count platelets down to levels of 10
× 109/l or less, it should be noted that precision at these levels is often poor with CVs of 22–
66% being observed and with mean counts differing appreciably between instruments.
Factitiously low automated platelet counts may be the result of giant platelets being identified
as red cells, EDTA-induced platelet clumping, and satellitism. Misleadingly high platelet
counts may be due to markedly microcytic or fragmented red cells, to cell fragments in
leukaemia, or to bacteria or fungi.
4.2.12 Mean Platelet Volume
The same techniques that are used to size red cells can be applied to platelets. The calculated
mean platelet volume (MPV) is very dependent on the technique of measurement and on
length and conditions of storage prior to testing the blood. When MPV is measured by
impedance technology, it has been found to vary inversely with the platelet count in normal
subjects. If this curve is extrapolated, it has been found that data fit the extrapolated curve
when thrombocytopenia is caused by peripheral platelet destruction; however, the MPV is
lower than predicted when thrombocytopenia is caused by megaloblastic anaemia or bone
marrow failure. The MPV is generally greater than predicted in myeloproliferative disorders,
but differentiating essential thrombocythaemia from reactive thrombocytosis on this basis has
not been very successful.
Other platelet parameters that can be computed by automated counters include the platelet
distribution width (PDW), which is a measure of platelet anisocytosis, and the “plateletcrit,”
which is the product of the MPV and platelet count and, by analogy with the haematocrit,
may be seen as indicative of the volume of circulating platelets in a unit volume of blood. The
PDW has been found to be of some use in distinguishing essential thrombocythaemia (PDW
increased) from reactive thrombocytosis (PDW normal). The plateletcrit does not appear to
provide any information of clinical value.
35 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
4.2.13 Reticulocytes Count
Automated reticulocyte counts have been developed by using the fact that various dyes and
fluorochromes combine with the RNA of reticulocytes. Following binding of the dye,
fluorescent cells can be enumerated using a flow cytometer. Most fully automated blood
counters now incorporate a reticulocyte counting capacity so that use of a stand-alone
reticulocyte counter is no longer necessary and use of a general purpose flow cytometer is no
longer appropriate. An international standard for this method has been published by ICSH.
The dyes used in the different systems include auramine O (Sysmex), thiazole orange (ABX),
CD4K 530 (Abbott), as well as nonfluorescent dyes such as oxazine 750 (Bayer-Technicon)
and the traditional New methylene blue (Beckman-Coulter, Abbott).
After staining, it is necessary to separate the reticulocytes from unstained red cells, and,
because the dyes also combine with DNA of nucleated cells, these cells must also be
excluded.
The threshold for this exclusion is determined by the intensity of fluorescence and particle
sizing. Although the separation of reticulocytes from mature red cells is not always clearcut,
automated reticulocyte counts correlate well with manual reticulocyte counts, although
absolute counts may differ because automated counts are dependent on the conditions of
incubation and the method of calibrating the instrument. Precision is much superior to that of
the manual count because many more cells are counted and the subjective element inherent in
recognizing late reticulocytes is eliminated. Potential sources of inaccuracy are the inclusion
of some leucocytes and platelets and, less often, Howell–Jolly bodies or malarial parasites in
the “reticulocyte” count.
Automated reticulocyte counts are fairly stable in blood that has been stored for 1–2 days at
room temperature or up to 3–5 days at 4°C.
4.2.14 Immature Reticulocyte Fraction
Fully automated instruments provide a measure of the various degrees of reticulocyte
maturation because the most immature reticulocytes, produced when erythropoietin levels are
high, have more RNA and fluoresce more strongly than the mature reticulocytes normally
present in the peripheral blood. Parameters indicating reticulocyte immaturity have potential
clinical relevance. For example, an increase in mean fluorescence intensity indicative of the
presence of immature reticulocytes has been noted as an early sign of engraftment following
bone marrow transplantation.
The characteristics of reticulocyte output in different types of anemia’s can be especially
appreciated from an output bivariate graph relating fluorescent intensity to reticulocyte count.
As described earlier,
low total count with a relatively high immature reticulocyte fraction (IRF) is indicative of a
repopulating marrow,
whereas a reticulocytopenia with low IRF is typical of severe aplastic anaemia or renal failure
A high total count with high IRF occurs in acute haemolysis and blood loss,
whereas a low to normal total count with a high IRF occurs in dyserythropiesis and in early
response to haematinics. The appearance of reticulocytes with high fluorescence also heralds
response when severe aplastic anaemia is being treated with immunosuppressive therapy
4.2.15 Calibration of Automated Blood Cell Counters
The following methods are recommended for calibrating an automated blood cell counter
36 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
1.
By using fresh normal blood specimens to which values have been assigned for Hb,
PCV, RBC, WBC, and platelet count by standardized reference methods
2.
By use of a stable calibrant (either preserved blood or a substitute) to which values
appropriate for the instrument in question have been assigned by comparison with
fresh normal blood
3.
By use of a commercial calibrant with assigned values suitable for the instrument in
question
For reasons of convenience and economy, control materials are commonly used as calibrants;
but this practice is not recommended. Such materials are not sufficiently stable to serve as
calibrants and their stated values are often approximations that are not assigned by reference
methods. They are designed to give test results within a stated range over a stated period
rather than a specific result.
The procedure for assigning values to fresh blood samples and indirectly to a stable calibrant
is as follows:
1.
4 ml blood specimens are obtained from three haematologically normal volunteers
and are anticoagulated with K2 EDTA.
2.
The Hb value is assigned by using the haemiglobincyanide method and the mean of
two measurements.
3.
The PCV is assigned by the microhaematocrit method, taking the mean of
measurements in four microhaematocrit tubes.
4.
The RBC is assigned by performing counts on a single-channel aperture-impedance
counter capable of performing a direct cell count; the mean of two dilutions, each
counted twice, is used.
5.
The MCV is assigned by calculation from the RBC and PCV.
6.
The WBC is assigned by performing counts on a single-channel aperture-impedance
instrument capable of performing direct cell counts; the mean of two dilutions, each
counted twice, is used.
4.2.16 Flagging of Automated Blood Counts
“Flagging” refers to a signal that the specimen being analyzed may have a significant
abnormality because one or more of the blood count variables are outside specified limits
(usually 2SD) or there is a qualitative abnormality that requires a quality control check and/or
additional investigation. This usually includes a blood film review. Although it is
theoretically desirable for every blood count to include examination of a stained film, this is
being challenged by increasing workloads requiring time- and cost-effective rationalization,
as well as by the use of automated analyzers that report differential leucocyte counts on every
specimen. Consequently, fewer blood films are being examined microscopically. Thus, a
decision of when a blood film should be made, stained, and examined should take account of
flagging and the need to ensure analytic reliability. This includes a check of any significant
changes from a recent previous count (delta-check), as well as any specific clinical
circumstances. The following is a guide to this selection.
Blood count request: Is it a first time count or repeat count?
37 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
First time count: Is it a routine screening test or special category?
If Routine: Analyzer report for blood count alone
Film required if any flags are signalled
If Special category: Film required:
1.
Diagnosed blood disease patients
2.
Patients receiving radiotherapy and/or chemotherapy
3.
Renal disease
4.
Neonates
5.
Intensive care unit
6.
If special tests have also been requested for: infectious mononucleosis, haemolytic
anaemia, enzymopathy, abnormal haemoglobins
7.
If the clinical details on the request form indicate lymphadenopathy, splenomegaly,
jaundice or suggest the possibility of leukaemia or lymphoma
8.
Specific requests by clinician
Repeat count: Film required:
1.
Delta check positive when compared with previous record
2.
Any flag occurs in present count
3.
On each occasion for patients with known blood diseases, for neonates, and when
specifically requested by clinicians
4.3 Part Two: Manual Techniques
4.3.1 Haemoglobin Estimation
Introduction
The hemoglobin concentration (Hb) may be estimated by measurement of its colour, by its
power of combining with oxygen or carbon monoxide, or by its iron content. The procedures
to be described are all colour or light-intensity matching techniques, which also measure, to a
varying extent, any methaemoglobin (Hi) or sulphaemoglobin (SHb) that may be present.
Clinical significance
This test is done to detect if the patient is suffering from anaemia or rather high Hb
Concentration as in polycythaemia.
Principle:
To measure hemoglobin concentration (Hb), a known volume of carefully mixed whole blood
is added to a diluent which lyses red cells to produce a hemoglobin solution; lysis occurs
because of the hypotonicity of the diluent. The Hb is then determined from the light absorbance
(optical density) of the solution of hemoglobin or its derivative at a selected wavelength.
Specimen
Venous or free-flowing capillary blood
ethylenediaminetetra-acetic acid (EDTA).
that
has
been
anticoagulated
Glassware and equipment
EDTA tubes, dispenser or micropipette, test tubes, spectrophotometer or colorimeter.
38 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
with:
HAEMIGLOBINCYANIDE (CYANMETHAEMOGLOBIN) PROCEDURE
The haemiglobincyanide (Cyanmethemoglobin) procedure is the internationally recommended
procedure for determining the haemoglobin concentration of blood. The basis of the procedure
is dilution of blood in a solution containing potassium cyanide and potassium ferricyanide.
Haemoglobin, Hi, and HbCO, but not SHb, are converted to HiCN. The absorbance of the
solution is then measured in a spectrometer at a wavelength of 540 nm or a photoelectric
colorimeter with a yellow–green filter.
Reagents:
Diluent
The original (Drabkin's) reagent had a pH of 8.6. The following modified solution, Drapkintype reagent, as recommended by the International Committee for Standardization in
Haematology, has a pH of 7.0–7.4. It is less likely to cause turbidity from precipitation of
plasma proteins and requires a shorter conversion time (3–5 min) than the original Drabkin's
solution, but it has the disadvantage that the detergent causes some frothing:
Potassium ferricyanide (0.607 mmol/l)
200 mg
Potassium cyanide (0.768 mmol/l)
50 mg
Potassium dihydrogen phosphate (1.029 mmol/l) 140 mg
Nonionic detergent
1 ml
Distilled or deionized water
To 1 litre
The pH should be 7.0–7.4 and must be checked with a pH meter at least once a month. The
diluent should be clear and pale yellow in colour. When measured against water as a blank in a
spectrometer at a wavelength of 540 nm, absorbance must be zero. If stored at room temperature
in a brown borosilicate glass bottle, the solution keeps for several months. If the ambient
temperature is higher than 30°C, the solution should be stored in the refrigerator but brought to
room temperature before use. It must not be allowed to freeze. The reagent must be discarded
if it becomes turbid, if the pH is found to be outside the 7.0–7.4 range, or if it has an absorbance
other than zero at 540 nm against water blank.
Procedure
Make a 1 in 201 dilution of blood by adding 20 μl of blood to 4 ml of diluent, or according to
the manufactures instructions. Place a rubber stopper on the tube containing the solution and
invert it several times. Let the test sample stand at room temperature for at least 5 min (to ensure
the complete conversion of haemoglobin to haemiglobinocyanide), and then pour it into a
cuvette and read the absorbance in a spectrometer at 540 nm or in a photoelectric colorimeter
with a suitable filter, against a water blank. The absorbance of the test sample must be measured
within 6 hours of its initial dilution. The absorbance of a commercially available HiCN standard
(brought to room temperature if previously stored in a refrigerator) should also be compared to
a water blank in the same spectrometer or photoelectric colorimeter as the patient sample. The
standard should be kept in the dark, and, to ensure that contamination is avoided, any unused
solution should be discarded at the end of the day on which the ampoule is opened.
Calculation of Haemoglobin Concentration
Preparation of Standard Graph and Standard Table
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When many blood samples are to be tested, it is convenient to read the results from a standard
graph or table relating absorbance readings to haemoglobin in g/l for the individual instrument.
This graph should be prepared each time a new photometer is put into use or when a bulb or
other components are replaced. It can be prepared as follows:
Prepare five dilutions of the HiCN reference standard (or equivalent preparation) (brought to
room temperature) with the cyanide–ferricyanide reagent according to the Table below because
the graph will be used to determine the haemoglobin measurements, it is essential that the
dilutions are performed accurately.
Dilutions of haemiglobincyanide (HiCN) reference solution for preparation of standard graph
Haemoglobin [*]
HiCN volume
Tube (%)
(ml)
Reagent volume (ml)
1
100 (full strength)
4.0 (neat)
None
2
75
3.0
1.0
3
50
2.0
2.0
4
25
1.0
3.0
5
0
None
4.0 (neat)
*
As percent of haemoglobin in reference solution.
The haemoglobin concentration of the reference preparation in each tube should be plotted
against the absorbance measurement. For example, if the label on the reference preparation
states that it contains 800 mg/l, (i.e. 0.8 g/l) and the procedure for haemoglobin measurement
uses a dilution of 1:201, the respective haemoglobin concentrations of tubes 1–5 would be 160
g/l, 120 g/l, 80 g/l, 40 g/l, and zero.
Using linear graph paper, plot the absorbance values on the vertical axis and the haemoglobin
values on the horizontal axis. The points should fit a straight line that passes through the origin.
Providing that the standard has been correctly diluted, this provides a check that the calibration
of the photometer is linear. From the graph, it is possible to construct a table of readings and
corresponding haemoglobin values. This is more convenient than reading values from a graph
when large numbers of measurements are made. It is important that the performance of the
instrument does not vary and that its calibration remains constant in relation to haemoglobin
measurements. To ensure this, the reference preparation should be measured at frequent
intervals, preferably with each batch of blood samples.
Supplementary information
Inaccuracy in estimation of Hb due to:
The slow conversion of carboxyhemoglobin to methemoglobin leads to overestimation of the
Hb if the test is read at 3 minutes, since carboxyhemoglobin absorbs more light at 540 nm than
does cyanmethemoglobin. This difficulty can be overcome by prolonging the reaction time to
30 min before reading. The difference between the 5 and 30 min readings can be used as a
semiquantitative procedure for estimating the percentage of HbCO in the blood.
Spectrophotometers and photometers are both sensitive to the effect of turbidity, which may
be caused by a high WBC, high concentrations of lipids or plasma proteins, or non-lysed red
cells. Increased turbidity causes a factitiously elevated estimate of Hb.
40 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
When the WBC is high, turbidity effects are circumvented by centrifugation or filtration of
the solution prior to reading the absorbance.
When turbidity is due to a high level of plasma protein, it can be cleared by the addition of
either potassium carbonate or a drop of 25% ammonia solution.
When turbidity is due to hyperlipidemia, a blank can be prepared from the diluent and the
patient’s plasma or the lipid can be removed by diethyl ether extraction and centrifugation.
Calculation of Haemoglobin Concentration
Reference range
Haemoglobin
Men
150 ± 20 g/l
Women 135 ± 15 g/l
Units
g / l, mg/dl
4.3.2 Packed Cell Volume or Haematocrit
Introduction
The haematocrit ×1000 is about three times the haemoglobin expressed in g/l. In conjunction
with estimations of haemoglobin and red blood cell count (RBC), it can be used in the
calculation of red cell indices. However, its use in under-resourced laboratories may be limited
by the need for a specialized centrifuge and a reliable supply of capillary tubes.
Clinical significance
The packed cell volume (PCV) can be used primarily as a simple screening test for anaemia, as
a reference procedure for calibrating automated blood count systems, and as a rough guide to
the accuracy of haemoglobin measurements.
Principle
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The centrifuge used for the capillary tubes provides a relative centrifugal force (g) of about
12000 g, and 5 min centrifugation results in precipitation of RBC in the distal end.
Specimen
Anticoagulated blood sample, because the PCV gradually increases with storage, the test
should be performed within 6 hours of collecting the blood sample, but a delay of up to 24
hours is acceptable if the blood is kept at 4°C.
Failure to mix the blood sample adequately will produce an inaccurate result. The degree of
oxygenation of the blood also affects the result because the PCV of venous blood is ∼2% higher
than that of fully aerated blood (which has lost CO2 and taken up O2). To ensure adequate
oxygenation and sample mixing, the free air space above the sample should be >20% of the
container volume.
Glassware and equipment
Anticoagulant
K2-EDTA tube is recommended, because K3-EDTA causes shrinking of the red cells, reducing
the PCV by about 2%. Anticoagulant concentration in excess of 2.2 mg/ml may also cause a
falsely low PCV as a result of cell shrinkage.
Capillary Tubes
Variation of the bore of the tubes may cause serious errors if they are not within the narrow
limits of defined specifications that should be met by manufacturers: length 75 ± 0.5 mm;
internal diameter 1.07–1.25 mm, wall thickness 0.18–0.23 mm; and bore taper not exceeding
2% of the internal diameter over the entire length of the tube.
Centrifuge
Centrifuges should be checked at intervals (at least annually) by a tachometer for speed and by
a stopwatch for timer accuracy. Efficiency of packing should also be tested by centrifuging
samples of normal and polycythaemic blood for varying times from 5 to 10 min to determine
the minimum time for complete packing of the red cells.
Procedure
MICROHAEMATOCRIT PROCEDURE
The microhaematocrit procedure is carried out on blood contained in capillary tubes 75 mm
in length and having an internal diameter of about 1 mm. The tubes may be plain for use with
anticoagulated blood samples or coated inside with 1 iu of heparin for the direct collection of
capillary blood. The centrifuge used for the capillary tubes provides a centrifugal force of c
12000 g, and 5 min centrifugation results in a constant PCV. When the PCV is greater than 0.5,
it may be necessary to centrifuge for a further 5 min.
Allow blood from a well-mixed specimen, or from a free flow of blood by skin puncture, to
enter the tube by capillarity, leaving at least 15 mm unfilled. Then seal the tube by a plastic seal
(e.g. Cristaseal, Hawksley, and Lancing, Sussex). Sealing the tube by heating is not
42 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
recommended because the seals tend to be tapered and there is the likelihood of lysis. After
centrifugation for 5 min, measure the proportion of cells to the whole column (i.e., the PCV)
using a reading device.
Accuracy of Microhaematocrit
The microhaematocrit procedure has an adequate level of accuracy and precision for clinical
utility. Attention must be paid to a number of factors that may produce an inaccurate result.
Reading
The test should be read as soon as possible after centrifugation because the red cells begin to
swell and the interface becomes progressively more indistinct. To avoid errors in reading with
the special reading device, a magnifying glass should be used. White cells and platelets (the
buffy coat) must be excluded as far as possible from the reading of the packed red cells. If a
special reading device is not available, the ratio of red cell column to whole column can be
calculated from measurements obtained by placing the tube against arithmetic graph paper or
against a ruler.
Supplementary information
Plasma Trapping
The amount of plasma trapped between red cells, especially in the lower end of the red cell
column, and red cell dehydration during centrifugation generally counterbalance each other,
and the error caused by trapped plasma is usually not more than 0.01 PCV units. Thus, in routine
practice, it is unnecessary to correct for trapped plasma, but if the PCV is required for
calibrating a blood cell analyser or for calculating blood volume, the observed PCV should be
reduced by a 2% correction factor after it has been centrifuged for 5 min or for 10 min with
polycythaemic blood. It is, however, preferable to use the surrogate reference procedure.
Plasma trapping is increased in macrocytic anaemias, spherocytosis, thalassaemia,
hypochromic anaemias, and sickle cell anaemia; it may be as high as 20% in sickle cell anaemia
if all the cells are sickled.
Factors affecting the test:
43 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Reference range
Packed cell volume (PCV) or Haematocrit (Hct)
Men
0.45 ± 0.05 (l/l)
Women 0.41 ± 0.05 (l/l)
Units
% or L / L
4.3.3 Total White Blood Cell Count
Introduction
Visual counting of blood cells is an acceptable alternative to electronic counting for white cell,
while, it is not recommended for routine red cell counts because the number of cells that can be
counted within a reasonable time in the routine laboratory will be too few to ensure a
sufficiently precise result.
Principle:
The principles of manual cell counts, the use of the haemocytometer counting chamber for
manually counting white cells and platelets in a known surface area.
Clinical significance
Some of the common Causes of leucocytosis:
Bacterial infections
Inflammation and tissue necrosis
44 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Metabolic disorders (e.g. uraemia, eclampsia, acidosis, gout)
Neoplasms of all types (e.g. carcinoma, lymphoma, melanoma)
Acute haemorrhage or haemolysis
Drugs (e.g. corticosteroid therapy (inhibits margination): lithium, tetracycline)
Chronic myeloid leukaemia, myeloproliferative disease, polycythaemia vera, myelofibrosis,
essential thrombocythaemia
Treatment with myeloid growth factors (e.g. G-CSF, GM-CSF)
Infectious mononucleosis, rubella, pertussis,
Infectious hepatitis, cytomegalovirus, HIV, herpes simplex or zoster
tuberculosis, toxoplasmosis, brucellosis, syphilis
Chronic lymphoid leukaemias
Acute lymphoblastic leukaemia
Non-Hodgkin's lymphoma (some)
Thyrotoxicosis.
Some of the common Causes of leucopenia:
Selective neutropenia
Drug-induced
Systemic lupus erythematosus
Felty's syndrome
Hypersensitivity and anaphylaxis
Viral (e.g. hepatitis, influenza, HIV)
Fulminant bacterial infection (e.g. typhoid, miliary Tuberculosis)
Specimen
Venous or free-flowing capillary blood that has been anticoagulated with ethylenediamine
tetra-acetic acid (EDTA). Thorough mixing of the blood specimen before sampling is essential
for accurate test results. Ideally, tests should be performed within 6 hours of obtaining the blood
specimen because some test results are altered by longer periods of storage. However, results
that are sufficiently reliable for clinical purposes can usually be obtained on blood stored for
up to 24 hours at 4°C.
Glassware and equipment
Counting Chambers
The visibility of the rulings in the counting chamber is as important as the accuracy of
calibration, so that chambers with a “metallised” surface and Neubauer or Improved Neubauer
rulings are recommended. These have nine 1 mm × 1 mm ruled areas, which, when covered
correctly with the special thick cover glass, each contain a volume of 0.1 μl of diluted blood.
Coverslips designed for mounting of microscopy preparations must not be used with counting
chambers. The sample is introduced between the chamber and the coverglass using a pipette or
capillary tube, and the preparation is viewed using a ×40 objective and ×6 or ×10 eyepieces.
With Neubauer and Improved Neubauer chambers, count the cells in 4 or 8 horizontal
rectangles of 1 mm × 0.05 mm (80 or 160 small squares) or in 5 or 10 groups of 16 small
squares, including the cells that touch the bottom and left-hand margins of the small squares.
45 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 10: Counting chamber
 Microscope
 Micropipette
 Test tube
Reagents:
Diluent
To make the counting of white cells easier, diluted whole blood is mixed with a fluid to lyse
the red cells and stain the white cell nuclei deep violet-black. The diluent is 2% (20 ml/l) acetic
acid coloured pale violet with gentian violet.
Procedure
Make a 1 in 20 dilution of blood by adding 0.1 ml of well-mixed blood (lack of adequate mixing
is a major source of error) to 1.9 ml of diluent in a 75 × 10 mm plastic (or glass) tube. After
sealing the tube with a lid or tightly fitting bung, mix the diluted blood in a mechanical mixer
or by hand for at least 2 min by tilting the tube to an angle of about 120 degrees combined with
rotation, thus allowing the air bubble to mix the suspension. Fill a clean dry counting chamber,
with its coverglass already in position, without delay. This is simply accomplished with the aid
of a plastic Pasteur pipette or a length of stout capillary glass tubing that has been allowed to
take up the suspension by capillarity. Take care that the counting chamber is filled in one action
and that no fluid flows into the surrounding moat.
Leave the chamber undisturbed on a bench for at least 2 min for the cells to settle, but not much
longer, because drying at the edges of the preparation initiates currents that cause movement of
the cells after they have settled. The bench must be free of vibrations, and the chamber must
not be exposed to draughts or to direct sunlight or other sources of heat. It is important that the
coverglass should be of a special thick glass and perfectly flat, so that when laid on the counting
chamber, diffraction rings are seen. The coverglass should be of such a size that when placed
46 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
correctly on the counting chamber the central ruled areas lie in the centre of the rectangle to be
filled with the cell suspension.
Factors affecting the test:
If any of the following filling defects occur, the preparation must be discarded and the filling
procedure must be repeated using another clean dry chamber:
Overflow into moat
Chamber area incompletely filled
Air bubbles anywhere in chamber area
Any debris in chamber area
To obtain a coefficient of variation of 5%, it is necessary to count about 400 cells in practice,
it is reasonable to count 100 white cells. To minimise distribution errors, count the cells in the
entire ruled area (i.e., 9 × 0.1 μl areas in an Improved Neubauer counting chamber).
Counting in only one or two fields results in a wide variance that is reduced as more cells are
counted. However, high precision is achieved only when thousands of cells are counted, which
is only possible with automated cell counters.
Calculation
WBC/I
Thus, if N cells are counted in 0.1 μl, then the WBC/l is as follows:
(e.g., if 115 cells are counted, the WBC is 115 × 200 × 106/l = 23 × 109/l)
Range of White Blood Cell Count in Health
White blood cell count
4.0–10.0 × 109/l
Units
× 109/l
4.3.4 Platelet Count
Introduction
Manual counts are used routinely in under-resourced laboratories, and they are still needed even
in well-equipped laboratories for blood samples with a significant proportion of giant platelets.
However, for all other samples, automated full blood counters produce platelet counts with a
precision that is much superior to that of manual platelet counts.
47 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Principle:
Manual platelet counts are performed by visual examination of diluted, lysed whole blood
using a Neubauer or Improved Neubauer counting chamber as for total white cell counts.
Clinical significance
This test is used for detection of thrombocytosis or thrombocytopenia which occurs in many
diseases.
Specimen
Platelet counts are best performed on ethylenediaminetetra-acetic acid (EDTA)-anticoagulated
blood that has been obtained by clean venipuncture. They can also be carried out on blood
obtained by skin prick, but the results are less satisfactory than those on venous blood. Skinprick platelet counts are significantly lower than counts on venous blood and less constant; a
variable number of platelets are probably lost at the site of the skin puncture.
Glassware and equipment
Counting Chambers
The visibility of the rulings in the counting chamber is as important as the accuracy of
calibration, so that chambers with a “metallised” surface and Neubauer or Improved Neubauer
rulings are recommended. These have nine 1 mm × 1 mm ruled areas, which, when covered
correctly with the special thick cover glass, each contain a volume of 0.1 μl of diluted blood.
Coverslips designed for mounting of microscopy preparations must not be used with counting
chambers. The sample is introduced between the chamber and the coverglass using a pipette or
capillary tube, and the preparation is viewed using a ×40 objective and ×6 or ×10 eyepieces.
With Neubauer and Improved Neubauer chambers, count the cells in 4 or 8 horizontal
rectangles of 1 mm × 0.05 mm (80 or 160 small squares) or in 5 or 10 groups of 16 small
squares, including the cells that touch the bottom and left-hand margins of the small squares.
48 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure11: Counting Chamber
 Microscope
 Micropipette
 Test tube
Reagents:
1% ammonium oxalate, which lyses red cells, as a diluent produces a higher and more accurate
count than use of formal citrate, which leaves red cells intact.
Procedure
The diluent consists of 1% aqueous ammonium oxalate in which the red cells are lysed. This
procedure is recommended in preference to that using formal-citrate as diluent, which leaves
the red cells intact and is more likely to give incorrect results, when the platelet count is low.
Before diluting the blood sample, examine it carefully for the presence of blood clots. If these
are present, a fresh specimen should be requested because clots will cause the platelet count to
be artificially low. Make a 1 in 20 dilution of well-mixed blood in the diluent by adding 0.1 ml
of blood to 1.9 ml of ammonium oxalate diluent (10 g/l). Not more than 500 ml of diluent
should be made at a time, using carefully clean glassware and fresh glass-distilled or deionised
water. If possible, the solution should be filtered through a micropore filter (0.22 μm) and kept
at 4°C. For use, a small part of the stock is refiltered and dispensed in 1.9 ml volumes in 75 ×
12 mm tubes.
Mix the suspension on a mechanical mixer for 10–15 min. Fill a Neubauer counting chamber
with the suspension, using a stout glass capillary or Pasteur pipette. Place the counting chamber
in a moist Petri dish and leave untouched for at least 20 min to give time for the platelets to
settle.
Examine the preparation with the ×40 objective and ×6 or ×10 eyepieces. The platelets appear
under ordinary illumination as small (but not minute) highly refractile particles if viewed with
the condenser racked down; they are usually well-separated, and clumps are rare if the blood
sample has been skillfully collected. To avoid introducing into the chamber dirt particles, which
might be mistaken for platelets, all equipment must be scrupulously clean. Platelets are more
easily seen with the phase-contrast microscope. A special, thin-bottomed (1 μm) counting
chamber is best for optimal phase-contrast effect. The number of platelets in one or more areas
of 1 mm2 should be counted. The total number of platelets counted should always exceed 200
to ensure a coefficient of variation of 8–10%.
Calculation
Thus, if N is the number of platelets counted in an area of 1 mm2 (0.1 μl in volume), the
number of platelets per litre of blood is:
49 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Range of platelet Count
Platelet count 280 ± 130 × 109/l
Factors affecting the test:
The errors associated with manual cell counts are technical and inherent.
Technical errors can be minimized by avoiding the following:






Poor technique in obtaining the blood specimen
Insufficient mixing of the blood specimen
Inaccurate pipetting and the use of badly calibrated pipettes or counting
chambers
Inadequate mixing of the cell suspension
Faulty filling of the counting chamber
Careless counting of cells within the chamber
Units
× 109/l
4.3.5 Erythrocyte Sedimentation Rate
Introduction
Inflammatory response to tissue injury (the acute-phase response) includes alteration in serum
protein concentration, especially increases in fibrinogen, haptoglobin, caeruloplasmin,
immunoglobulins (Ig), and C-reactive protein (CRP), and decrease in albumin. The changes
occur in acute infection, during active phases of chronic inflammation, with malignancy, in
acute tissue damage (e.g., following acute myocardial infarction), or with physical injury.
Principle:
Essentially it is the measurement after 1 hour of the sedimentation of red cells in diluted blood
in an open-ended glass tube of 30 cm length mounted vertically on a stand.
Clinical significance
Measurement of the acute-phase response is a helpful indicator of the presence and extent of
inflammation or tissue damage and response to treatment. The usual tests are estimation of CRP
and measurement of the erythrocyte sedimentation rate (ESR);
Specimen
Either collect venous blood in ethylenediaminetetra-acetic acid (EDTA) and dilute a sample
accurately in the proportion of 1 volume of citrate to 4 volumes of blood,
or collect the blood directly into the citrate solution.
50 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Glassware and equipment
The recommended tube is a straight glass or rigid transparent plastic tube 30 cm in length and
not less than 2.55 mm in diameter. The bore must be uniform to within 5% throughout. A scale
graduated in mm extends over the lower 20 cm. The tube must be clean and dry.
Specially made racks with adjustable levelling screws are available for holding the
sedimentation tubes firmly in an exactly vertical position. The rack must be constructed so that
there will be no leakage of the blood from the tube.
Reagents:
Diluent prepare a solution of 109 mmol/l trisodium citrate (32 g/l Na3Ca6H5O7.2H2O). Filter
through a micropore filter (0.22 mm) into a sterile bottle. It can be stored for several months at
4°C but must be discarded if it becomes turbid through the growth of moulds.
Procedure
Conventional Westergren Procedure
The test should then be carried out on the diluted sample within 4 hours of collecting the blood,
although a delay of up to 6 hours is permissible provided that the blood is kept at 4°C. EDTA
blood can be used within 24 hours if the specimen is kept at 4°C, provided that 1 volume of
109 mmol/l (32 g/l) trisodium citrate is added to 4 volumes of blood immediately before the
test is performed.
Mix the blood sample thoroughly and then draw it up into the Westergren tube to the 200 mm
mark by means of a teat or a mechanical device; mouth suction should never be used. Place the
tube exactly vertical and leave undisturbed for exactly 60 min, free from vibrations and draughts
and not exposed to direct sunlight.
Calculation
Read to the nearest 1 mm the height of the clear plasma above the upper limit of the column of
sedimenting cells. The result is expressed as ESR = X mm in 1 h. A poor delineation of the
upper layer of red cells may sometimes occur, especially when there is a high reticulocyte count.
Range in Health
There is a progressive increase with age, but older than 70 years it is difficult to define a strictly
healthy population for determining normal values.
Erythrocyte sedimentation rate ranges in health
Age (years) 95% Upper limit (mm in 1h)
Men
17–50
10
51–60
12
61–70
14
>70
about 30
Women
17–50
12
51–60
19
61–70
20
51 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Age (years) 95% Upper limit (mm in 1h)
>70
about 35
Pregnancy
First half
48 (62 if anaemic)
Second half 70 (95 if anaemic)
In the newborn, the ESR is usually low. In childhood and adolescence, it is the same as for
normal men with no differences between boys and girls. It is increased in pregnancy, especially
so in the later stages, and dependent of anaemia.
Factors affecting the test:
Modified Procedures
Length of Tube
The overall length of the tube is not a critical dimension for the test provided that it fits firmly
in an appropriate holding device. The tube must, however, be long enough to ensure that
packing of the cells does not start before the test has been completed.
Temp: should be between 18-25 C
Hemolysis: affect the rate of sedimentation
Plastic Glass Tubes
A number of plastic materials (e.g., polypropylene and polycarbonate) are recommended as
substitutes for glass in Westergren tubes. Nevertheless, not all plastics have similar properties,
and it must be demonstrated that the ESR with the chosen tubes is reproducible and not affected
by the plastic.
Disposable Glass Tubes
Disposable glass tubes should be supplied clean and dry and ready for use. It is necessary to
show that neither the tube material nor the manufacturer's cleaning process affect the ESR.
Capillary Procedure
Short tubes of narrower bore than in the standard tube are available mainly for tests on infants.
These are, however, no longer in general use, and it is necessary to establish normal ranges or
a correction factor to convert results to an approximation of ESR by the Westergren procedure.
Time
Sedimentation is measured after aggregation has occurred and before the cells start to pack
usually at 18-24 min. From the rate during this time period the sedimentation that would have
occurred at 60 min is derived and converted to the conventional ESR equivalent by an
algorithm.
Sloping Tube
52 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Red cells sediment more quickly when streaming down the wall of a sloped tube. This
phenomenon has been incorporated into automated systems in which the end-point is read after
20 min with the tube held at an angle of 18 degrees from the vertical. This has been shown to
give results comparable to the conventional procedure.
Anticoagulant
EDTA blood can be used without citrate dilution, at least if packed cell volume (PCV) is below
0.36 (haemoglobin < 110 g/l); less precise results are obtained when the PCV is higher. The
readings from undiluted samples must then be adjusted as for the standardised procedure (see
below).
Because of the biohazard risk of blood contamination inherent in using open-ended tubes, it is
now recommended that, where possible, a closed system be used in routine practice. Manual
procedures are available that avoid transfer of the blood into the sedimentation tube. Automated
closed systems use either blood collected in special evacuated tubes containing citrate or EDTA
blood. A sample is taken up through a pierceable cap and then automatically diluted in the
system if this is required. Some systems use sloping tubes at an angle of 18 degrees to obtain
results rapidly, and one model of the Ves-matic (Diesse) also incorporates centrifugation.
Whenever a different procedure or tube is planned, a preliminary test should be carried out to
check precision and to compare results with those obtained by the standardised procedure
described in the following section.
Units
Mm / hour
4.3.6 Reticulocyte Count
Introduction
Reticulocytes are juvenile red cells; they contain remnants of the ribosomal ribonucleic acid
(RNA) that was present in larger amounts in the cytoplasm of the nucleated precursors from
which they were derived. Ribosomes have the property of reacting with certain basic dyes such
as azure B, brilliant cresyl blue, or New methylene blue (see below) to form a blue or purple
precipitate of granules or filaments.
Principle
On exposure of unfixed cells to certain dyes that contains only basic component of the stain,
such as brilliant cresyl blue or new methylene blue, the ribosomes are precipitated and stained
by the dye, to appear as a reticular network. With these stains red cells stain a pale greenishblue while the reticulum stains bluish-purple.
Clinical significance
The number of reticulocytes in the peripheral blood is a fairly accurate reflection of
erythropoietic activity, assuming that the reticulocytes are released normally from the bone
marrow and that they remain in circulation for the normal time period.
53 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Specimen
Venous blood that has been anticoagulated with ethylenediaminetetra-acetic acid (EDTA).
Thorough mixing of the blood specimen before sampling is essential for accurate test results.
Ideally, tests should be performed within 6 hours of obtaining the blood specimen because some
test results are altered by longer periods of storage. However, results that are sufficiently
reliable for clinical purposes can usually be obtained on blood stored for up to 24 hours at 4°C.
Glassware and equipment
EDTA tubes, dispenser or micropipette, test tubes, slides, Microscope.
Reagents
Staining Solution
Dissolve 1.0 g of New methylene blue or azure B in 100 ml of iso-osmotic phosphate buffer
pH 6.5.
Procedure
Deliver 2 or 3 drops of the dye solution into a 75- × 10-mm plastic tube by means of a plastic
Pasteur pipette. Add 2–4 volumes of the patient's EDTA-anticoagulated blood to the dye
solution and mix. Keep the mixture at 37°C for 15–20 min. Resuspend the red cells by gentle
mixing, and make films on glass slides in the usual way. When dry, examine the films without
fixing or counterstaining.
The exact volume of blood to be added to the dye solution for optimal staining depends on the
RBC. A larger proportion of anaemic blood, and a smaller proportion of polycythaemic blood,
should be added than of normal blood. In a successful preparation, the reticulofilamentous
material should be stained deep blue and the nonreticulated cells should be stained diffuse
shades of pale greenish blue. Films should not be counterstained. The reticulofilamentous
material is not better defined after counterstaining, and precipitated stain overlying cells may
cause confusion. Moreover, Heinz bodies will not be visible in fixed and counterstained
preparations. If the stained preparation is examined under phase contrast, both the mature red
cells and reticulocytes are well defined. By this technique, late reticulocytes characterized by
the presence of remnants of filaments or threads are readily distinguished from cells containing
inclusion bodies. Satisfactory counts may be made on blood that has been allowed to stand
(unstained) for as long as 24 hours, although the count will tend to decrease after 6–8 hours
unless the blood is kept at 4°C.
Counting Reticulocytes
An area of film should be chosen for the count where the cells are undistorted and where the
staining is good. A common fault is to make the film too thin; however, the cells should not
overlap. To count the cells, use the ×100 oil-immersion objective and, if possible, eyepieces
provided with an adjustable diaphragm. If eyepieces with an adjustable diaphragm are not
available, a paper or cardboard diaphragm, in the centre of which has been cut a small square
with sides about 4 mm in length, can be inserted into an eyepiece and used as a less convenient
substitute.
54 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
The counting procedure should be appropriate to the number of reticulocytes present. Very
large numbers of cells have to be surveyed if a reasonably accurate count is to be obtained when
only small numbers of reticulocytes are present. When the count is less than 10%, a convenient
procedure is to survey successive fields until at least 100 reticulocytes have been counted and
to count the total red cells in at least 10 fields to determine the average number of red cells per
field.
Calculation
Number of reticulocytes in n fields = x
Average number of red cells per field = y
Total number of red cells in n fields = n × y
Reticulocyte percentage = [x ÷ (n × y)] ×
100%
Absolute reticulocyte count = % × RBC
Thus, when the reticulocyte percentage is 3.3 and the RBC is 5 × 1012/l, the absolute reticulocyte
count per litre is as follows: [3.3/100] × 5 × 1012 = 165 × 109
It is essential that the reticulocyte preparation be well spread to ensure an even distribution of
cells in successive fields.
When the reticulocyte count exceeds 10%, only a relatively small number of cells will have to
be surveyed to obtain a standard error of 10%.
Factors affecting the test:
It is essential that the reticulocyte preparation be well spread and well stained. Other important
factors that affect the accuracy of the count are the visual acuity and patience of the observer
and the quality and resolving power of the microscope. The most accurate counts are carried
out by a conscientious observer who has no knowledge of the supposed reticulocyte level, thus
eliminating the effect of conscious or unconscious bias.
Range of Reticulocyte Count in Health
The range of reticulocyte in adults and children is 50–100 × 109/l (0.5–2.5%). In infants (full
term, cord blood) it is 2–5%.
Carry out a rough platelet count to determine the approximate concentration, and add an
appropriate amount of the suspension to preserved blood. Mix well for 20 min and, with
continuous mixing, dispense into sterile containers. Cap and seal. At 4°C, the preparation
should have a shelf life of 3-4 months. Before use, resuspend by thorough shaking by hand,
followed by mechanical mixing for approximately 15 min.
4.2.7 PREPARATION OF BLOOD FILMS ON SLIDES
Introduction
55 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Blood films should be made on clean glass slides. Films made on cover glasses have negligible
advantages and are unsuitable for modern laboratory practice. Films may be spread by hand or
by means of an automated slide spreader, the latter being either a stand-alone instrument or a
component of an automated blood cell counter.
Clinical significance
It is used for diagnosis of various blood diseases, such as…..
Principle
Although we can judge the proportions of red cells and white cells in a tube of sedimented
blood, we get far more information if the blood is carefully mixed and a thin layer is spread on
a glass slide to form a blood film. The blood cells are then preserved by exposure to the alcohol
methanol, a process known as fixation. The fixed film of blood is stained with a mixture of
several dyes so that the individual cells can be recognized when they are examined with a
microscope.
Specimen
Blood films can be prepared from fresh blood with no anticoagulant added or from
ethylenediaminetetra-acetic acid (EDTA)-anticoagulated blood. Heparinized blood should not
generally be used because its staining characteristics differ from those of EDTA-anticoagulated
blood. Good films can be made in the following manner, using clean slides, if necessary wiped
free from dust immediately before use. Slides should measure 75 × 25 mm and approximately
1 mm thick; ideally, they should be frosted at one end to facilitate labelling, but these are more
expensive.
Glassware and equipment
EDTA tubes, dispenser or micropipette, spreader, slides, Microscope.
Reagents
Leishman's Stain
Weigh out 0.2 g of the powdered dye, and transfer it to a conical flask of 200–250 ml capacity.
Add 100 ml of methanol and warm the mixture to 50°C for 15 min, occasionally shaking it.
Allow the flask to cool and filter. It is then ready for use, but it will improve on standing. For
daily use, store the stain in an airtight (prevent moisture entering the stain) amber (semiopaque) container. Keep in a cool place (not refrigerated) and never in direct sunlight.
The stock stain should be kept in a tightly stoppered light opaque (e.g. amber)
container in a cool dark place. Renew every 3 months or earlier if indicated. To obtain
optimum color reaction, some suggest that 3–5 days should be allowed before using
freshly made stain.
56 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Buffered Water
Make up 50 ml of 66 mmol/l Sörensen's phosphate buffer of the required pH to 1 litre with
water at a pH of 6.8. An alternative buffer may be prepared from buffer tablets, which are
available commercially. Solutions of the required pH are obtained by dissolving the tablets in
water and testing with a pH meter.
Manual Procedure
Spreading Blood Films
First, make a spreader from a glass slide that has a smooth end. Using a glass cutter, break off
one corner of the slide, leaving a width of about 18 mm as the spreader. A spreader can be used
repeatedly unless the edge becomes chipped, but it must be thoroughly washed and dried
between films.
Place a small drop of blood in the centre line of a slide about 1 cm from one end. Then, without
delay, place a spreader in front of the drop at an angle of about 30 degrees to the slide and move
it back to make contact with the drop. The drop should spread out quickly along the line of
contact. With a steady movement of the hand, spread the drop of blood along the slide. The
spreader must not be lifted off until the last trace of blood has been spread out; with a correctly
sized drop, the film should be about 3 cm in length. It is important that the film of blood finishes
at least 1 cm before the end of the slide.
The thickness of the film can be regulated by varying the pressure and speed of spreading and
by changing the angle at which the spreader is held. With anaemic blood, the correct thickness
is achieved by using a wider angle, and, conversely, with polycythaemic blood, the angle should
be narrower. The ideal thickness is such that on microscopy there is some overlap of red cells
throughout much of the film's length. The leucocytes should be easily recognizable throughout
most of the film. With poorly made films the leucocytes will be unevenly distributed, with
monocytes and other large leucocytes being pushed to the end and the sides of the spread. An
irregular streaky film will occur if the slide is greasy, and dust on the surface will cause patchy
spots.
The films should be allowed to dry in the air. In humid conditions the films may be exposed to
a current of warm air (e.g., from a hairdryer), but this should be in a microbiological safety
hood.
Labeling Blood Films
The film should be labelled immediately after spreading. Write either a laboratory reference
number or the name of the patient and the date in pencil on the frosted end of the slide or on
the film itself (writing on the thickest part, which is least suitable for microscopic examination).
A label written in pencil will not be removed by staining. A paper label should be affixed to the
slide later. If blood films are to be stored for future reference, apply the paper label in such a
manner that it is easily read when the slides are filed.
Fixing Blood Films
Dry the films in the air. Leave for 3 min in the fixative this must be done without delay and the
films should never be left unfixed for more than a few hours. If films are sent to the laboratory
by post, it is preferable that, when possible, they are thoroughly dried and fixed before dispatch.
57 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
STAINING BLOOD FILMS
Romanowsky stains are used universally for routine staining of blood films, and satisfactory
results can be obtained.
Air dry the film and then flood the slide with the stain. After 2 min, add double the volume of
water and stain the film for 5–7 min. Then wash it in a stream of buffered water until it has
acquired a pinkish tinge (up to 2 min). After the back of the slide has been wiped clean, set it
up right to dry.
When several batches of films are being stained in succession, the staining solution should be
renewed at intervals (e.g., after each 50 slides). Loss of staining power is usually the result of
precipitation of the eosin Y, and this will result in the nuclei staining blue instead of purple.
Carry out a rough platelet count to determine the approximate concentration, and add an
appropriate amount of the suspension to preserved blood . Mix well for 20 min and, with
continuous mixing, dispense into sterile containers. Cap and seal. At 4°C, the preparation
should have a shelf life of 3-4 months. Before use, resuspend by thorough shaking by hand,
followed by mechanical mixing for approximately 15 min.
4.3.11 Antiglobulin or Coombs' Test
The antiglobulin test is probably the most important test in serology and blood banking. The antiglobulin
test (Coombs' test) was introduced by Coombs, Mourant, and Race in 1945 as a method for detecting
“incomplete” Rh antibodies (i.e., IgG antibodies capable of sensitizing red cells but incapable of causing
agglutination of the same cells suspended in saline) as opposed to “complete” IgM antibodies, which do
agglutinate saline-suspended red cells. Coombs' test is divided into direct and indirect antiglobulin tests:

In the direct antiglobulin test (DAT), the patient's cells, after careful washing, are
tested for sensitization that has occurred in vivo.

In the indirect antiglobulin test (IAT), normal red cells are incubated with a serum
suspected of containing an antibody and subsequently tested, after washing, for in vitro–
bound antibody.
The DAT is used to demonstrate in vivo attachment of antibodies to red cells, as in autoimmune hemolytic
anemia, alloimmune HDN, and alloimmune hemolysis following an incompatible transfusion.
The IAT has wide application in blood transfusion serology, including antibody screening and
identification and cross-matching.
DAT:
A spin tube technique is recommended for the routine antiglobulin test.
The test should be carried out in glass tubes. Plastic tubes are not recommended because they may adsorb
IgG, which could neutralize anti-IgG of the antiglobulin reagent. Use a blood sample in EDTA
anticoagulant.
If a cold-reacting autoantibody is present, the patient's red cells should be washed three times in a large
volume of saline warmed to 37°C to wash off cold antibodies and obtain a smooth suspension of cells.
58 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
There is no risk of washing off adsorbed complement components. However, the washing process should
be accomplished as quickly as possible and the test should be set up immediately afterward because bound
warm antibody occasionally elutes off the cells when they are washed and false-negative results may be
obtained. “Saline” refers to 9 g/l NaCl buffered to pH 7.0.
DAT Method:Make 5% suspension of red cells that have been washed three times in saline. If failed to
wash the red cells properly then the antihuman globulin antisera may be neutralized by immunoglobulins
or complement in the surrounding serum or plasma and cause a false negative result.
Add 1 drop of the cell suspension to 2 drops of anti-human globulin reagent. Centrifuge for 1 minute at
150g.
Examine for agglutination after gently re-suspending the button of cells. A concave mirror and good light
help in macroscopic readings. If the result appears to be negative, confirm this microscopically.
59 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 12: Coombs Test
60 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
4.3.12 ABO & Rh Group
The test used to determine the blood ABO and Rh group
Materials
12345-
Slide
Anti-A Monoclonal sera
Anti-B Monoclonal sera
Anti-D Monoclonal sera
EDTA blood
Method
123456-
Add one drop of each colored anti-sera on the slid
To each anti-sera add one drop of EDTA blood
Mix the blood with anti-sera
Rotating the slide by hand many times read the result within 2-5 mintes
Record the result
If Rh negative, test it by tube method:
A. Add one volume of antiD sera to test tube.
B. Add one volumeof 2% red cells suspension.
C. mix suspension by traping the tube.
D. leave the suspension undisturbed for 15 min and read result.
Discrepancies
A- False Positive
1- Rouleaux formation
2- Auto-immune hemolytic anemia
B- False Negative
Occurred when the anti-sera is impotent or expired
5. Serology
5.1Rapid Plasma Reagin (RPR) TEST
Principle
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Syphilis is a chronic venereal infection caused by the spirochaete micro-organism Treponema
pallidum. As the organism cannot be cultured on artificial media, diagnosis of syphilis
depends on the detection of specific antibodies by serological tests in combination with
clinical findings.
The RPR test is “non-treponemal” in that the antibodies detected are not specific for T.
pallidum, although their presence in patient’s serum or plasma is strongly associated with
infection by the organism. This test measures antibody (IgG and IgM) produced in response
to lipoidal material released from damaged host cells as well as to lipoprotein-like material
released from the spirochaetes. These antibodies tend to disappear after successful treatment
of the infection.
The RPR test consists of modified VDRL antigen containing microparticulate carbon, which
aggregates in the presence of reagin type antibodies in serum or plasma, indicating a positive
result. The aggregation can be read macroscopically. Non-reactive samples typically appear
as a smooth non-aggregated pattern, which may form buttons in the centre of the test area .
Material
1. Carbon antigen reagent
2. Positive Control
3. Negative Control
4. Disposable Test Slides
5. Disposable Pipette Stirrers (pipstirs)
6. Dispenser
7. Needle
8. Specimen collection container
9. Timer
10. Automatic card rotator (100 rpm)
11. (0.85%) physiological saline (semi quantitative test only).
12. Test tubes for sample dilution (semi quantitative test only).
PROCEDURE
a.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Qualitative Test
Allow all reagents, controls and samples to reach room temperature before use.
Draw the sample into the pipstir taking care not to transfer any cellular elements.
Hold the pipstir over a test card circle and allow one drop (50µl) of specimen to fall
onto the test card. It is important to hold the dispenser in a vertical position whilst
dispensing the sample.
Spread the specimen evenly over the entire test circle using the mixing (broad) end of
the pipstir.
Shake the vial of carbon antigen reagent to ensure even mixing.
Attach the needle to the dispenser. Withdraw sufficient carbon antigen for the number
of tests being performed.
Keeping the dispenser in a vertical position allow one drop to fall onto the specimen.
Do not mix. Return unused antigen from the dispenser to the glass vial.
Rotate the tile for 8 minutes preferably on an automatic rotator at 100 rpm.
Read and interpret results macroscopically in good light.
62 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
b. Semi Quantitative Test
1. Prepare doubling dilutions of the sample from the undiluted specimen to 1:32 using
physiological saline. Mix well.
2. Using the pipstirs , place one drop (50µl) of each dilution onto a separate test card
circle.
3. Using the mixing (broad) end of the pipstir spread each dilution evenly over the test
circle, starting at the highest dilution (1:32), proceeding to the lowest (1:2).
4. Continue as from 5 in the Qualitative Test.
5. After 8 minutes rotation, read the test and note the last circle in the dilution series that
has a positive result.
6. If the highest dilution tested (1:32) is reactive, proceed with a further dilution series
by preparing doubling dilutions of the sample from 1:32 to 1:512 using physiological
saline. Mix well and continue as from 2 in the semi quantitative test.
INTERPRETATION
Quality Control
It is recommended that the kit positive and negative controls are run with each batch of test
specimens. For the assay to be valid the positive control provided should give a strong
positive pattern and the negative control provided should give a clearly negative result.
Results
Qualitative Test
Positive result
Reactive (positive) samples display characteristic agglutination ranging from slight (weakreactive) to intense (reactive). A strong positive reaction is seen as large aggregates in the
centre of the test circle. Weakly positive reactions are seen as small aggregates around the
edge of the test circle.
Negative result
Negative results show no aggregates. The carbon antigen either remains in a smooth
suspension or forms a distinct button.
63 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 13: Appearance of Positive and Negative RPR Results
Semi Quantitative Test
Results may be graded from strong to non-reactive and the titre expressed as the reciprocal of
the last dilution showing a positive reaction.
1. Strong Reactive (SR): Large clumps of carbon particles with a clear background.
2. Reactive (R): Large clumps of carbon particles, more dispersed than strong reactive.
3. Weak Reactive (WR): Small clumps of carbon particles with light grey background.
4. Trace Reactive (TR): Slight clumping of carbon particles, typically seen as a button of
aggregates in the centre of the test circle or dispersed around the edge of the test circle.
5. Non-Reactive (NR): A smooth grey pattern or a button of non-aggregated carbon particles
in the centre of the test circle.
Reactive samples should be recorded as antibody positive and must be subjected to further
tests to determine the presence or absence of specific anti-Treponemal antibody.
Limitations



As with all reagin tests the RPR test may give a small proportion of false positive
results. Diseases such as infectious mononucleosis, leprosy, lupus erythematous,
vaccinia and viral pneumonia can cause such reactions.
Reactive RPR test specimens should be tested with further serological tests (i.e.
TPHA and FTA-abs) since, as with any serological procedure, the diagnosis should
not be made on a single reactive result.
As with other serological tests, the RPR test cannot distinguish between syphilis and
other pathogenic Treponemal infections e.g. Yaws.
5.2 TPHA(Treponema pallidum Haemagglutination Assay ) TEST
Principle
Serological screening tests for syphilis using cardiolipin and lecithin as antigens are simple to
perform but biological false positive reactions occur frequently because the tests use nontreponemal antigens.
TPHA reageants are used to detect human serum antibody to T.pallidum by means of an
indirect haemagglutination (IHA) method. Preserved avian erythrocytes are coated with
antigenic components of pathogenic T.pallidum . These Test Cells agglutinate in the presence
of specific antibodies to T.pallidum, and show characteristic patterns in microtitration plates.
Any non-specific reactions occurring are detected using the Control Cells, which are avian
erythrocytes not coated with T.pallidum antigens. Non-specific reactions may also be
absorbed out using these Control Cells.
Antibodies to non-pathogenic treponemes are absorbed by an extract of Reiter’s treponemes,
included in the cell suspension. Test results are obtained in 45-60 minutes and the cell
agglutination patterns are both easily read and long lasting.
TPHA test has been shown to be a convenient and specific test for the diagnosis of
treponemal infection, having a specificity and a sensitivity comparable to that of the FTA64 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
ABS test (Fluorescent treponemal antibody absorption Test) . It requires minimum laboratory
equipment and is very simple to perform.
Material
1.
2.
3.
4.
Test cells; preserved avian erythrocytes sensitised with T.pallidum antigen.
Control cells;preserved avian erythrocyte.
Diluent.
Positive control serum; (prediluted 1:20)This will give an equivalent titre of
1/640:/2560 in the quantitative test.
5. Non-reactive control serum; (prediluted 1:20).
6. Accurate pipettes for delivering 10,25,75 and 190 microlitres.
7. U-Well microtitration plates.
Procedure
Qualitative method
Each sample requires 3 wells of a microtitration plate.
1.
2.
3.
4.
Add 190µl of diluent to Well 1.
Add 10µl serum to Well 1.
Using a micropipette, mix contents of Well 1 and transfer 25µl to Wells 2 and3.
Ensure that the Test and Control Cells are thoroughly resuspended. Add 75µl of
control cells to Well 2. Add 75µl of Test Cells to Well 3.
5. Tap the plate gently to mix the contents thoroughly.
6. Incubate 45-60 minutes at room temperature. ( Keep the plate away from heat, direct
sunlight and any source of vibration).
7. Read results. Results are stable for 24hrs if the plate is covered and the above
precautions are observed.
Quantitative test
Each sample requires 8 Wells of a microtitration plate. Labelled A through to H.
1. Add 25µl of diluent to Wells B to H inclusive.
2. Transfer 25µl of 1:20 serum dilution from screening test to Wells A and B.
3. Take 25µl of diluted serum from Well B and serially dilute from Wells B to H
inclusive in 25µl aliquots, discarding 25µl of diluted serum from Well H.
4. Ensure that the Test Cells are thoroughly resuspended. Add 75µl of Test cells to wells
A to H inclusive. This will give a dilution of serum of 1/ 80 in Well A through 1/
10240 Well H.
5. Shake the plate gently to mix the contents thoroughly.
6. Incubate for 45-60 minutes at room temperature.( Keep the plate away from heat,
direct sunlight and any source of vibration).
7. Read results. Results are stable for 24hrs. if the plate is covered and the above
precautions are observed.
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Table1: TPHA results
RESULTS
TEST CELLS
CONTROL CELLS
Strong Positive
Full cell pattern covering
the bottom of the well.
No agglutination tight
button
Weak Positive
Cell pattern covers
approx. 1/3 of well
bottom
No agglutination tight
button
Indeterminate
Cell pattern shows a
distinctly open centre
No agglutination tight
button
Negative
Cells settled to a
compact bottom,
typically with a small
clear centre.
No agglutination tight
button
Positive reaction
Non-specific *
Non-specific absorption
Positive reaction
1. Add 100µl of test serum to a small tube then add 400µl of Control Cells.
Mix well and stand for 1 hour.
2. Centrifuge for 15 minutes at 1000 rpm and test the supernatant by the
qualitative method.
Note: The sample is now at 1/5, this should be taken into account when preparing the
dilutions. If the result is repeatedly non-specific the sample should be tested by another
method eg . FTA-ABS.
INTERPRETATION OF RESULTS
Strong positive reactions may show some folding at the edge of the cell mat.
When the Test well is positive , the Control well should be observed.
The Control cells should settle to a compact button. They should not be used as a comparison
for Non-Reactive serum patterns since the Control Cells will give a more compact pattern
than the Test Cells.
Agglutination in the Control well indicates the presence of non-specific agglutinins in the
sample, the test should be reported as INVALID. A serum that gives this result may be
absorbed using the Control Cells as detailed under Non-specific absorption. A doubtful
reaction with Test Cells should be reported as INDETERMINATE.
This result may indicate a low level of antibody in early primary syphilis or yaws.
This sample should be first retested in the qualitative test then a further sample should be
tested at a later date to determine whether or not there is a rising titre. It is also advisable to
perform another confirmation test (FTA-ABS) to complete the profile of the test serum.
5.3 Widal Test ((agglutination test))
Introduction
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The widal agglutination test for the diagnosis of the enteric fever, The patients' serum is tested
directly for antibody by entire a slide of tube method, These test are either qualitative and semi
quantitative.
The rapid slide test is used primarily as a screening procedure especially useful when large
numbers of sera must be examined. The tube test should be used to confirm positive result
obtained by the slide test.
The degree to which agglutination occurs depends upon the concentration of the antigen, the
amount of antibody present, the composition of the salt solution and the temperature.
Sera from normal patients may show positive agglutination with widal antigen due the
previously immunization, past infection, or the present of antibodies to related antigen. titer
detected as a result of active infection or recent immunization.
It is therefore, necessary to evaluate two or more serum samples, taken at 3 to 5 days intervals
after the onast of disease. a progressive increase in titers is the prime evidence of recent
infection or immunization.
Martials
1. antigens: Antigen are nonviable bacteria cells(salmonella)in 0.5% phenolized saline
and stained
2. Serological pipettes, 0.2 ml with 0.1 ml gradation. 0.1 ml capacity
3. Class agglutination slide with bow
4. Wooden applicator sticks
5. Physiological saline (0.85% NaCl)
6. Rotatory shaker (if available)
Procedure:
- Specimen collection: Collect 5-10 ml of blood in the sterile plain tube without
anticoagulant. Allow the blood to clot for 20-30 min, serum maybe kept for up to
24 hours at 2.8 c. If sample to be kept for longer periods , it should be frozen, do
not refreeze one it thawed serum
The antigen should be allowed to warm to room temperature prior to use in test.
A- Slide method
1- deliver 0.08,0.04,0.02,0.01 and 0.005 ml of test serum on to separate ring, using 0.2 ml
pipette
2- Shake the antigen vial in order to thoroughly mix the contents.
3- Add one drop of antigen to each circle, mix antigen and serum, using a new tooth pick or
applicator for each circle.
4- Rock the slide for 1-3 min (lock the kit procedure) or using rotatory shaker if available.
5- The degree of agglutination observe in each ring is estimated as follow.
6- Dilution giving a 2+ agglutination is the end point or titer of that serum.
Note: storage kit: Widal kit, store at 2-8°C, antigen are ready for use as supplied
Measure titer:
The titer is the reciprocal of the highest final dilution procedure a 2 +agglutination
67 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Table 2:Record agglutination
Degree of agglutination
100%
back ground clear to slightly hazy
4+
75%
back ground slightly cloudy
3+
50%
back ground moderately cloudy
2+
25%
back ground cloudy
1+
None
negative
Table 3: Determination of antibody titer
The titer is reciprocal of the highest dilution producing (2+) agglutination
Serum ml
correlation dilution
0.08
1:20
0.04
1:40
0.02
1:80
0.01
1:160
0.005
1:320
Table 4: Example of slide test are shown in the following table
Slide test results
Slide test result
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Serum ml
readying
0.08
0.04
0.02
0.01
0.005
1:20
1:40
1:80
1:160
1:320
Serum
titers or
endpoint
dilution
Agglutination reaction
Serum No 1
++++
++++
++++
++
-
1:160
Serum No 2
++++
+++
++
-
-
1:80
Serum No 3
+++
++
++
_
_
1:80
Note:
1-If auto-agglutination of antigen is suspected (Show by low power microscope) the vial
antigen is unstable and should be discarded.
2-patints, occasionally fail to develop any serum agglutinin
The chart below give approximate indication of the significant of serum titers.
Table 5: indication of significant serum titers
Serum agglutinins
Disease
Typhoid fever
Para typhoid
fever
salmonella
infection
Febric antigen
widal antigen
Salmonella O group D
Salmonella O group A
Salmonella O group B
appear
maximum
7-10 Days
3-5 week
titer and significance
1:80 (in early
stage)=suspicious
1:160 and rising= strong
suspicious
Those characterized by
prolonged fever and
typhoid like symptoms
present antibodies of titers
similar to above
Note:
1-The minimal titers defined as positive of the O typhoid antigen and H antigen must be
determined for individual geographic areas.
2-Cross-reaction occur with both non Styphi group dsalmonella and salmonella from other
groups
69 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
3-A single elevated titer for (O) equal to or greater than (1:320) or H equal to or greater then
(1:640) is considered positive.
5.4 Rose Bengal Test for Brucellosis
Introduction
Human brucellosis –undulant or Malta fever:
Brucellosis is systemic infection in which any organ or system of body can be involved.
Human become infected by three primary routes
1-ingestion of infected unpasteurized animal milk products
2-inhaltion of infected aerosolized particles
3-Direct contact with infected animal parts through rupture of skin and mucous membrane
including the conjunctiva.
Brucellosis is one of the most commonly reported bacterial infection acquired in laboratory ,
within laboratory aerosolizing is primary mechanism of the transmission, so it is required
biosafety level 3 precaution for isolation in Lab .
Most species of pathogenic for human are:
1- Br abroteus from cows
2- Br melitens from goats and sheep
3- Br suis from pigs (USA)
4- Br canis from dog
Definitive diagnosis of brucellosis require isolation of the organism in culture of blood , bone
marrow or other tissues.
the cultures , should be incubated in 5% to 10 % CO2 in humidified atmosphere incubated for
3 weeks before discarded as negative.
The mean way of diagnosis of brucellosis is by serological test.
Become of the difficulty of isolating the organism (serological test) the serum agglutination test
(SAT) is widely used and detected antibodies for Br abroteus and Br melitensis.
Rose Bengal test (agglutination test) for brucellosis it is remain an inexpensive sensitive and
specified test for acute case , but it is not useful in following up of patients or in diagnosis
relapses .
Principle
The rose Bengal an agglutination test reaction used for the diagnosis of brucellosis.
The patients’ serum is tested directly for antibody by either a slide or tube method.
The test are qualitative and semi qualitative.
A number of antigens are available commercially for use is the rapid slide test.
The test is performed by mixing serum and antigen in ruled squired on a glass slide.
This test is widely used and detected antibodies to Brucellosis aborteas, Br. Melitensis.
In case of a cute infection a titer of 1:160 or greater in rapid slide method are consider
diagnosis if this result fits the clinical and epidemiologic findings.
Materials:
1. Antigen Brucellosis aborteas, nonviable. Bacterial cells in 0.05% phenolized saline
and stained.
2. Serological pipettes 0.2 ml with 0.1 ml graduation. 0.1 ml capacity
3. Class agglutination slide with row
4. Wooden applicator sticks
70 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
5. Physiological saline(0.85% NaCl)
6. Rotator shaker if available
Procedure
Specimen collection
Collect 5-10 ml of blood in the sterile plain tube without anticoagulant. Allow blood to
clot for 20-30 mint, serum may be kept for up to 24 hours at 2-8 C° e . If samples to be
kept for longer period, it should be frozen don’t refreeze once themed serum
The antigen should be allow to warm to room temperature prior to use in test
A- Slide method.
1. delivers 0.08,0.04,0.02,0.01 and 0.005 ml of test serum on to separate ring using 0.2 ml
pipette.
2. Shake the antigen vial in order to thoroughly mix the contents.
3. Add one drop of antigen to each circle mixer antigen and serum , using a new tool pick or
applicator for each circle.
4. Rock the slide for 1-3 min (lock the kit procedure) or using rotatory shaker if available.
5. The degree of agglutination observe in each ring is estimated as follow.
Record agglutination
Degree of agglutination
100%
back ground clear to slightly hazy
75%
back ground slightly cloudy
50%
back ground moderately cloudy
25%
back ground cloudy
None
4+
3+
2+
1+
negative
Note: Storage kits Rose Bengal kit store at 2-8C° antigen are ready for use as supplied.
Table 6: Determination of antibody titer
The titer is reciprocal of the highest dilution producing (2+) agglutination
The serum
a 2+
is the end
of the serum.
Serum Ml
correlation dilution
0.08
1:20
0.04
1:40
0.02
1:80
0.01
1:160
0.005
1:320
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dilation giving
agglutination
point or titer
Measure titer is reciprocal of the highest final dilution producing (2+) agglutination
Example of rose bagel test result are shown in the flowing table
Table 7: Examples of Rose Bengal results
Slide test result
Serum (ml)
readying
0.08
0.04
0.02
0.01
0.005
1:20
1:40
1:80
1:160
1:320
Serum
titers or
endpoint
dilution
Agglutination reaction
Serum No 1
++++
++++
++++
++
-
1:160
Serum No 2
++++
+++
++
-
-
1:80
Serum No 3
+++
++
++
_
_
1:80
Limitation of the procedure
1- The serum agglutination test SAT measure both antibodies IgG and IgM in serum of patient
2- After cure the antibodies for brucellosis, usually persistent for more than one year
3- Use 2-mercaptoethanol (detect IgG only) to identify chronic or relapse Brucellosis cases.
4- Any titer high than 1:40 with 2-mercaptoethanol lgG indicative active infection (i.e.
relapses or chronic infection).
5- A single titer of greater than 160 or more is considered significant.
6- Prozone phenomena may give false negative result which is possible because of the high
antibody concentration so serum dilution is recommended to avoid this.
7- False positive reaction may result from cross-reaction with antibodies of cholera, yersinia
or francisella.
8- False negative and false positive reaction can be avoided by routinely dilution the serum
beyond at 1:320
5.5 C-Reactive Protein (CRP) Test
PRINCIPLE
C-Reactive Protein (CRP) in patient’s sera has been found in association with acute
infections, necrotic conditions and a variety of inflammatory disorders. There is a strong
correlation between serum levels of CRP and the onset of the inflammatory process.
Monitoring the levels of CRP in patient’s sera indicates the effectiveness of treatment and the
assessment of patient recovery.
72 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
CRP latex particles are coated with antibodies to human CRP. When the latex suspension is
mixed with serum containing elevated CRP levels on a slide, clear agglutination is seen
within 2 minutes.
MATERIAL
1- Suspension of polystyrene latex ( approximately 1%) particles coated with Anti-CRP
antibodies.
2- Positive Control. Serum containing CRP antibodies.
3- Negative Control. Serum free of CRP antibodies.
4- Stirrers
5- Plastic slide
6- Micro-pipettes capable of dispensing 50μl.
7- Isotonic saline (0.9% NaCI)
REAGENT PREPARATION
o
o
All reagents should be brought to room temperature (20 C to 25 C) and mixed gently to
resuspend latex prior to use. Do not induce foaming.
The test slide should be thoroughly cleaned before use as traces of detergent or prior
specimen may affect the result.
Recommended cleaning procedure:
1. Used cards must be immediately immersed in a disinfectant solution. Follow disinfectant
manufactures guidelines.
2. The reaction circles must be physically rubbed with non-abrasive material to ensure
removal of possible adhering particles.
3. Thoroughly rinse in purified water.
4. Allow reaction card to dry.
5. Spray cards with a 70% alcohol solution.
6. Allow the alcohol to evaporate prior to re-use.
PROCEDURE
Qualitative Method
1. Allow kit reagents and patient serum to come to room temperature.
2. Transfer one drop (50μl) of patient’s serum to the test circle on the slide.
3. Shake the latex reagent, then using the dropper provided, add one drop of suspension to the
test circle.
4. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the
mixture.
5. Gently and evenly, rock and rotate the test slide for 2 minutes whilst examining the test
slide for agglutination.
Semi Quantitative Method
1. Using isotonic saline prepare serial dilutions of the patients serum (1/2, 1/4, 1/8, 1/16, 1/32,
1/64 and so on)
73 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
2. Transfer one drop (50μl) of each serum dilution to the test circle on the slide.
3. Shake the latex reagent, then using the dropper provided, add one drop of suspension to the
test circle.
4. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the
mixture.
5. Gently and evenly, rock and rotate the test slide for 2 minutes whilst examining the test
slide for agglutination.
RESULTS AND INTERPRETATION
Examine the test slide under a strong light source after 2 minutes. Kit controls or known level
value samples should be tested with each test run. The kit negative control should give a
negative result after 2 minutes. The kit positive control should give a positive result at a titre
of 1/4 +/- one double dilution after 2 minutes. If levels of controls or users known samples do
not give expected results, test results must be considered invalid.
Qualitative Method
A positive result is indicated by the obvious agglutination pattern of the latex, in a clear
solution. A negative result is indicated by no change in the latex suspension on the test slide.
Latex CRP kit has a detection limit of 6mg/litre of CRP in the patient’s serum. Positive results
will be obtained at a CRP serum concentration above 6mg/litre and negative results will be
obtained at 6mg/Litre and below.
Semi-Quantitative Method
The serum CRP concentration can then be calculated approximately by multiplying the
dilution factor (i.e 2, 4, 8 or 16) by the detection limit, i.e. 6, to give the number of mg/Litre
concentration e.g. if the agglutination titre appears at 1/8 the approximate serum CRP
concentration is 8 x 6 = 48 mg/Litre.
Titres of 500 mg/Litre have been detected with CRP with no prozone ( hook ) effect.
5.6 ASO latex slide agglutination test
Principle
Streptococcal infections are very common in all populations, and a high percentage of people
will have antibodies against streptococci. The b-haemolytic group A streptococci produce two
haemolysins: oxygen-labile streptolysin O and oxygen-stable haemolysin S. Only reduced
(non-oxidized) streptolysin O is immunogenic and is used for the test. The antistreptolysin O
test is based on the fact that patients with Streptococcus pyogenes (group A streptococcal)
infections develop antibodies that inhibit the haemolytic activity of streptolysin O. The
antibodies are usually long-lasting and a single increased titreis not an indication of a current
infection. Only a fourfold or greater rise in titre on successive serum samples taken 10–14
days apart should be considered indicative of recent infection. This test is mainly used in the
diagnosis of acute rheumatic fever, acute glomerulonephritis and other post-streptococcal
diseases.
There are two types of commercial antistreptolysin O test kits:
The ASO latex slide agglutination test is used to screen sera to identify those with raised ASO
titres (200 IU or higher).
74 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
The ASO tube test is a haemolysis inhibition test that is used to determine ASO antibody titre
in serum samples that are positive in the ASO latex slide agglutination test. Atitre of less than
50 IU does not confirm the diagnosis of acute rheumatic fever.
Materials and reagents
1234-
Disposable cards, with 6 wells each Disposable dropper
Positive control serum
Sensitized latex reagent (with streptolysin O)
Applicator sticks
Procedure
1- Dilute the serum 1: 20. (Or according to kit literature).
2- Place 1 drop of the serum solution in a well on the disposable card.
3- Use a new dropper to add 1 drop of sensitized latex reagent.
4- Use an applicator stick to mix the two drops and spread them over the entire well.
5- Examine for agglutination within 2 minutes.
 A positive reaction appears as a fine agglutination within 2 minutes.
 A negative reaction shows no agglutination.
5.6 Rheumatoid Factor (RF)
PRINCIPLE
Rheumatoid Factor (RF) is found in sera of patients with Rheumatoid Arthritis and is believed
to be IgM antibodies directed against the patient’s own immunoglobulin G.
RF is a rapid latex agglutination test kit for the detection of (RF) in human serum. RF latex
particles are coated with specially purified human gamma globulin, When the latex
suspension is mixed with serum containing elevated RF levels on a slide, clear agglutination
is seen within 2 minutes. RF has a detection limit of 8 IU/ml of RF in the patient serum .
Material
1- Suspension of Polystyrene latex particles ( approximatly 1.25% ) coated with suitably
modified Fc fraction of IgG in stabilizing buffer.
2- Positive Control. Serum containing Rheumatoid Factor antibodies. Working Strength.
3- Negative Control. Serum free of Rheumatoid Factor antibodies.
4- Stirrers
5- Plastic slide
6- Micropipettes capable of dispensing 50 μl.
7- Isotonic saline (0.9% NaCI)
SPECIMEN COLLECTION AND PREPARATION
Obtain a sample of venous blood from the patient and allow a clot to form and retract.
Centrifuge clotted blood sample and collect clear serum. Fresh serum samples are required.
Do not use haemolysed, contaminated or lipaemic serum for testing as this will adversly
affect the results.
75 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
o
o
Serum may be stored at 2 C to 8 C for up to 48 hours prior to testing. If longer storage is
o
required, store at –20 C for up to 6 weeks. Thawed samples must be mixed prior to testing.
Do not repeatedly freeze-thaw the specimens as this will cause false results.
REAGENT PREPARATION
o
o
All reagents should be brought to room temperature (20 C to 25 C) and mixed gently prior to
use. Do not induce foaming.
The test slide should be thoroughly cleaned before use as traces of detergent or prior
specimen may affect the result.
Recommended Cleaning procedure:
1- Used cards must be immediately immersed in a disinfectant solution. Follow
disinfectant manufactures guidelines.
2- The reaction circles must be physically rubbed with non-abrasive material to ensure
removal of possible adhering particles.
3- Thoroughly rinse in purified water.
4- Allow reaction card to dry.
5- Spray cards with a 70% alcohol solution.
6- Allow the alcohol to evaporate prior to re-use.
ASSAY PROCEDURE
Qualitative Method
1. Allow kit reagents and patient serum to come to room temperature.
2. Transfer 50μl of patient’s serum to the test circle on the slide.
3. Shake the latex reagent, then using the dropper provided, add one drop of suspension to the
test circle.
4. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the
mixture.
5. Gently and evenly, rock and rotate the test slide for 2 minutes whilst examining the test
slide for agglutination.
Semi Quantitative Method
1. Using isotonic saline prepare serial dilutions of the patients serum (1/2, 1/4, 1/8, 1/16, 1/32,
1/64 and so on)
2. Transfer 50μl of each serum dilution to the test circle on the slide.
3. Shake the latex reagent, then using the dropper provided, add one drop of suspension to the
test circle.
4. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the
mixture.
5. Gently and evenly, rock and rotate the test slide for 2 minutes whilst examining the test
slide for agglutination.
76 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
RESULTS AND INTERPRETATION
Examine the test slide under a strong light source after 2 minutes. Kit controls or known level
value samples should be tested with each test run. The kit negative control should give a
negative result after 2 minutes. The kit positive control should give a positive result at a titre
of 1/4 +/- one double dilution after 2 minutes. If levels of controls or users known samples do
not give expected results, test results must be considered invalid.
QUALITATIVE METHOD
A positive result is indicated by the obvious agglutination pattern of the latex, in a clear
solution. A negative result is indicated by no change in the latex suspension on the test slide.
Positive results will be obtained at a RF serum concentration of 8 IU/ml or more and negative
results will be obtained at a RF concentration below 8 IU/ml.
SEMI – QUANTITATIVE METHOD
The serum RF concentration can then be calculated approximately by multiplying the dilution
factor (i.e 2, 4, 8 or 16) by the detection limit, i.e. 8, to give the number of IU/ml
concentration e.g. if the agglutination titre appears at 1/8 the approximate serum RF
concentration is 8 x 8 = 64 IU/ml.
Titres of 1074 IU/ml have been detected with Avitex RF with no prozone ( Hook ) effect.
5.7 Toxoplasmosis Test
Toxoplasma IgG ELISA
Introduction
Toxoplasma gondii is an intracellular protozoan parasite with a worldwide distribution.
Although cats are the definitive host, human infection results from ingestion of contaminated
soil, careless handling of cat litter, ingestion of raw or undercooked meat or transmission
from mother to foetus through the placenta. When a seronegative woman becomes infected
with T. gondii during pregnancy, the organism is often transmitted to the foetus. Infection
during the first trimester may lead to spontaneous abortion, stillbirth, or overt disease in the
neonate.
The Toxoplasma IgG kit is a rapid ELISA designed for the qualitative or quantitative
detection of IgG antibodies to Toxoplasma gondii in human serum.
Principle of the test
Diluted serum or plasma specimens (1:100) are incubated for 20
minutes to allow specific antibodies to T. gondii to bind to the antigencoated wells. After
washing away unbound antibodies and other serum constituents, T. gondii specific IgG is
detected using rabbit anti-human IgG conjugated to horseradish peroxidase. After 20 minutes
incubation, unbound conjugate is removed by washing, and TMB enzyme substrate is added
for 10 minutes. A blue colour develops if antibodies to T. gondii are present. Addition of stop
77 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
solution gives a yellow colour and the optical densities of controls, standard(s) and samples
are measured using a microplate reader.
Materials
1. Microplate 96 wells in 12 X 8 break-apart strips, pre-coated with T.gondii purified antigens
enriched for P30 antigen
2. Reagent 1: Sample Diluent 100mM Tris-buffered saline, pH 7.2 with antimicrobial agent,
10ml, (blue), concentrate (x10)
3. Reagent 2: Wash Buffer 100mM Tris-buffered saline with detergent, pH 7.2, 100 ml,
concentrate (x10)
4. Reagent 3: Conjugate (peroxidase conjugated rabbit anti-human IgG), 12 ml, (red), ready to
use
5. Reagent 4: TMB Substrate, 12 ml, ready to use
6. Reagent 5: Stop solution, 12 ml, ready to use
7. Standards1: (for quantitative assays), 15 IU/ml, 50 IU/ml; 150 IU/ml (blue), ready to use
8. Standard1: (for qualitative assays) 8 IU/ml, (yellow), ready to use
9. Positive control1: 100 IU/ml (red), 1ml, ready to use
10. Negative control: 1 ml (green), 1ml, ready to use
11.Distilled or de-ionised water
Equipment
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10mm X 60mm tubes for dilution,
pipettes 10μl, 100μl, 1000μl;
repeating dispenser 100μl,
microplate reader with 450nm filter,
microplate washing device.
Samples
Only freshly drawn and properly refrigerated sera or plasma should be used in this assay.
Avoid haemolysed, lipemic or bacterial contaminated sera. Sera should be stored at 2-8ºC for
no longer than 5 days. If delay in testing is anticipated, store test sera at –20ºC. Avoid
multiple freeze-thaw cycles.
Method
Ensure that all materials are at room temperature before beginning the procedure. We
recommend that the standards and the controls are always run in duplicate. Samples may be
run singly or in duplicate.
78 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
1. Assemble the number of strips required for the assay.
2. The sample diluent X10 concentrate contains 0.09% sodium azide as preservative. Prepare
sufficient working strength diluent for the assay run. However, if the working strength diluent
is to be stored for more than 1 week, add sodium azide (0.9g/L). Store unused sample diluent
concentrate and dilute sample diluent at 2 – 8oC. Dilute the Sample Diluent (Reagent 1) 1:9
in distilled water to make sufficient buffer for the assay run e.g. add 10ml sample diluent
concentrate to 90 ml water.
3. Dilute patient samples 1:100 (e.g. 5μl serum plus 0.5 ml diluent). It is important to dispense
all samples and controls into the wells without delay. Therefore ensure that all samples are
ready to dispense.
4. For qualitative determinations, dispense 100 μl of the negative control, the 8 IU/ml
standard, the positive control and the diluted patient sample into the wells. For quantitative
determinations, use sample diluent as 0 IU/ml and additionally dispense the 15 IU/ml, 50
IU/ml and 150 IU/ml standards.
5. Place the strips into the incubation bag provided and incubate for 20 minutes at room
temperature. During all incubations, avoid direct sunlight and close proximity to any heat
sources.
6. Dilute the Wash Buffer (Reagent 2) 1: 9 in distilled water to make sufficient buffer for the
assay run e.g. add 50ml wash buffer concentrate to 450ml water. The diluted wash buffer is
stable for two months at 2 - 8°C.
7. After 20 minutes, decant or aspirate the well contents and wash the wells 3 times using an
automatic plate washer or the manual wash procedure (see below). Careful washing is the key
to good results. Blot the wells on absorbent paper before proceeding. Do not allow the wells
to dry
Out .
Manual Wash Procedure:
Empty the wells by inversion. Using a multi-channel pipette or wash bottle, fill the wells with
ash buffer. Empty by inversion and blot the wells on absorbent paper. Repeat this wash
process two more times.
8. Dispense 100μl of Conjugate (Reagent 3) into each well.
This reagent is color coded red. Keep all pipettes and other equipment used for Conjugate
completely separate from the TMB Substrate reagent! Incubate the wells for 20 minutes in the
incubation bag at room temperature.
9. After 20 minutes, discard the well contents and carefully wash the wells four times with
wash buffer. Ensure that the wells are completely washed. Blot the microplate on absorbent
paper to remove final drops of wash fluid. Do not allow the wells to dry out.
10. Using a repeating dispenser, rapidly dispense 100μl of TMB Substrate (Reagent 4) into
each well. Incubate the plate for 10 minutes.
79 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
11. Add 100μl of Stop Solution (Reagent 5) to each well. To allow equal reaction times, the
stop solution should be added to the wells in the same order as the TMB Substrate.
12. Read the optical density in a microplate reader within 10 minutes.
Interpretation
Qualitative determinations
Negative samples: OD < 8 IU/ml OD
Positive samples: OD >/= 8 IU/ml OD
Quantitative determinations
Plot the optical densities of the standards against their respective concentrations. Draw a line
to join the points. Read the concentrations of unknowns from this graph. Concentrations
below 8 IU/ml are considered negative; concentrations above 8 IU/ml are considered positive
for anti-toxoplasma IgG. A negative result indicates no current or previous infection with
T.gondii. Such individuals are presumed to be susceptible to primary infection. However see
Limitations below. A positive result indicates a current or previous infection with T. gondii.
5.8 Infectious Mononucleosis test
Principle
Is a qualitative determination of the nonspecific heterophile antibodies that result from
primary infection by Epstein – Barr Virus and causing the clinical presentation of Infectious
mononucleosis.
The IM- Latex is a slide agglutination test also can get semi-quantitative detection of
Heterophile antibodies . Latex particles coated with antigenic extract of beef erythrocyte
membranes and are agglutinated when mixed with samples containing IM heterophile
antibodies.
Materials and reagents Preparedness:

Reagents
Table 8: Reagents of Infectious Mononucleosis Test
Latex
Latex particles coated with antigenic extract of beef
erythrocytes membranes , phosphate buffer, pH 7.2. Sodium
azide , 0,95 g/L.
Control +ve
Human serum with anti – IM antibodies titer ≥ 1/4 . Sodium
azide , 0.95g/L
Control -ve
Animal serum . Sodium azide , 0.95g/L
Disposable slides
Storage: when stored tightly closed at 2-8 oC do not freeze. (according to kit literature)
80 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e


Samples : Fresh serum . Stable 7 days at 2-8 oC or 3 months at – 20 oC. Samples
with presence of fibrin should be centrifuged. Do not use highly hemolized or lipemic
samples.
Equipment :
1- Mechanical rotator with adjustable speed at 80-100 r.p.m.
2- Centrifuge.
3- Automatic micropipette
Methods:
 Qualitative Method :
1- Allow reagents and samples to reach room temperature.
2- Place 50 ul of the sample and one drop of each Positive and Negative controls into
separate circles on the slide test.
3- Swirl the IM – Latex reagent gently before using and add one drop ( 50ul) next to the
samples to be tested.
4- Mix the drops with stirrer , spreading them over the entire surface of the circle . Use
different stirrers for each sample.
5- Place the slide on a mechanical rotator at 80-100 r.p.m. for 2 minutes .False positive
results could appear if the test is read later than two minutes.
 Semi quantitative method:
1- Make serial two ford dilutions of the sample in 9 g/L saline solution.
2- Proceed for each dilution as in the qualitative method.
Results and Interpretations in reports:
Examine macroscopically the presence or absence of visible agglutination immediately after
removing the slide from the rotator. The presence of agglutination indicates a titer ≥ 1/28 of
anti IM Heterophil antibodies by the Davidson method.
The titer in the semi- quantitative method is defined as the highest dilution showing a positive
result.
The following are the limitations of the test:
1- False positive results may be obtained in some geographical areas where the horse serum is
used as a prophylactic measure (vaccination). Also this could be seen in patients with
leukemia, Burkitts Lymphoma, Pancreatic carcinoma, viral hepatitis, CMV infections and
others.
2- False negative results also encountered and in such Patients either repeat testing after
interval of several days to get a high heterophil antibody titer or look for specific antibodies
against viral structural antigens like anti VCA IgM .
Quality Control:
Positive and Negative controls are recommended to monitor the performance of the
procedure, as well as the comparative pattern for a better result interpretation.
5.9 Rota Virus Latex Test
Principle of the method and clinical significance:
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Rapid latex agglutination assay for the detection of rotavirus in faecal samples this virus has
been shown to be principle causative agent of gastro-enteritis. Patients may harbor up to 108
virus particles per gram of faeces.
Materials and reagents Preparedness:
 Material insert in kit: Test reagent latex, control negative ,Positive bovine rota-virus
control inactivated ( ready to use ),5 x concentrated extraction buffer pH
7.2,Agglutination slides ,pipette
 Specimen collection and storage: Prepare a 10% suspension of the faecal sample by
adding 0.1 g /0.1 ml of sample to 1 ml of extraction buffer in screw capped vial. Mix
well and stand in room temperature for 2 minutes. Specimen may be stored 24 hours
at the time of the test .If storage is more than 24 hour the specimen should be stored
at -20 oC in freezer .
 Safety warnings: Do not pipette by mouth, use disinfectant 70% alcohol, waste must
be treated as biohazardous and incinerated.
Method:
123456-
Add 10 ml of extraction buffer to 40ml de-ionize water.
Add 2 gms of faecal sample to 2 ml of extraction buffer.
Mix well on vortex mixer.
Stand at room temperature for 10 minutes.
Centrifuge test sample for 10 minutes about 800 g.
Place one drop of the supernatant from the faecal sample onto the circle of test slide
and use the 2 circle for control reagent.
7- Add one drop of control latex reagent next to the drop of faecal sample on circle 1.
8- Add one drop of test latex reagent next to drop of faecal sample on circle 2.
9- Spread the control latex reagent and test latex reagent in circle 1, 2 by disposable
pipette.
10- Gently tilt the test slid backward and forward for 2 minutes.
Results and Interpretations in reports:
1- Positive result is indicated by the visible agglutination of the latex particles which
occur within few seconds.
2- Negative result is indicated by milky appearance without any visible aggregation of
the latex particles.
5.10 Pregnancy Test
5.10.1 Rapid test for detection of human Chorionic
Gonadotrophin in Urine
Human chorionic gonadotrophin (hCG), a glycoprotein hormone secreted by viable placental
tissue during pregnancy, is excreted in urine approximately 20 days after the last menstrual
period. The levels of hCG rise rapidly reaching peak levels after 60-80 days. The appearance
of hCG in urine soon after conception and its rapid rise in concentration makes it an ideal
marker for the early detection and confirmation of pregnancy. Test kit utilizes the principle of
Immunochromatography, a unique two-site immunoassay on a membrane. As the test sample
flows through the membrane assembly within the test device in place of the dipstick, the
82 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
coloured monoclonal anti-hCG-colloidal gold conjugate complexes with the hCG in the
sample. This complex moves further on the membrane to the test region where it is
immobilised by the monoclonal anti-hCG coated on the membrane, leading to the formation
of a pink coloured line which confirms a positive test result. Absence of this coloured line in
the test region indicates a negative test result. The unreacted conjugate, unbound complex, if
any, and the colloidal gold conjugated rabbit IgG moves further along the membrane and are
subsequently immobilised by the goat anti-rabbit IgG coated on the control region forming a
pink line. This control line serves to validate the test results.
SPECIMEN COLLECTION AND PREPARATION
Urine samples collected at any time may be used, however, it is recommended that to
maximize hCG concentration, the first voided morning specimen should be used. The urine
should be collected in a clean dry container (plastic or glass) which must be free from
detergent. Urine specimens should be as fresh as possible and it is preferable to test within 24
hours of collection. The sample may be stored for longer periods (72 hours) prior to use, if
stored, store at 2oC to 8oC.
Filtration or centrifugation is generally not necessary for urine used in this test, however, if a
sample is very turbid, centrifugation or filtration may be necessary. (The use of supernatant
from turbid samples allowed to sediment naturally prior to use may negate the need for
sample preparation – this does not affect the hCG concentration.)
REAGENT PREPARATION
Devices and samples should be brought to room temperature (20 to 25) °C and mixed gently
prior to use.
In case the pouch has been stored at 4°C to 8°C, allow at least 30 minutes for the device to
come to room temperature. Check the color of the desiccant. It should be blue. If it has turned
colorless or faint blue, discard the device and use another device.
ASSAY PROCEDURE
1. Open the pouch and remove the device. Once opened, the device must be used
immediately.
2. Dispense two drops of urine specimen into the sample well „S‟ using the dropper
provided.
3. Read the results at the end of fifteen minutes for serum or five minutes for urine.
RESULTS AND INTERPRETATION
Negative: Only one colored line appears on the control region „C‟ only
Positive: A distinct colored line appears on the control region „C‟ and on the test region “T”.
83 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 14: Results of PT
The test should be considered invalid if no line appears. Repeat the test with a new device.
Depending on the concentration of hCG in the specimen, positive results may appear as early
as 30 seconds. Negative results must be confirmed only at the end of fifteen minutes for
serum or five minutes for urine.
5.10.2 Direct HCG Latex Pregnancy Kit
Detectable levels of Chorionic Gonadotropin (hCG) in urine start at 5 mIU/ml during the first
week of gestation and rises to 100,000 mIU/ml at 2 to 3 months. The hCG level doubles
approximately every 2.2 days during the first trimester1. Values decline from 10% to 15% of
peak concentrations during 2nd and 3rd trimesters2.
Direct hCG Latex kit contains one reagent of latex particles coated with monoclonal
antibodies to hCG. The reagent is mixed with the urine samples.
MATERIALS
1. Latex reagent.
2. Positive and negative controls.
3. Reaction slide and stirring sticks.
SPECIMEN COLLECTION AND PREPARATION
Generally, the first morning urine contains the highest concentration of the hCG hormone and
therefore, it is more recommended for testing. However, urine collected at other periods can
also be used. The urine in this case should have been kept in 2-8°C and used within 72 hours
from collection time.
PROCEDURES
1. Bring reagents to room temperature.
2. Place 50μl of pa�ent urine, one drop positive and one drop negative controls into
different circles of the slide.
3. Add one drop of latex reagent directly to each sample.
4. Mix using the supplied sticks and spread the mixture over the entire circle.
5. Gently rock the slide. Agglutination may be observed after two minutes. Direct light
source may help to observe the results.
READING THE RESULT
Presence of agglutination within two minutes indicates positive reaction.
Lack of agglutination within two minutes indicates negative reaction.
84 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
6. Bacteriology
6.1 General urine examination
Introduction:
The examination of urine provides a wide variety of useful medical information regarding the
disease involving the kidney and lower urinary tract . both functional (physiologic ) and
structural (anatomic disorders of the kidney and lower urinary tract my be elucidated . and
diagnostic .monitoring, we believe laboratory urine tests will remain an essential part of
clinical medicine
The basic routine analysis consist of 4 parts : specimen evaluation , gross (physical)
examination , chemical screening , and sediment examination
Clinical significance
Examination of urine is a fundamental investigation in patients in whom kidney disorders or
infections of the urinary tract are suspected. There are also many patients who exhibit no
clinical symptoms, but in whom previously unrecognized urinary tract infections can be
diagnosed by urine examination
Collection of urine specimens
Containers for the collection of urine should be wide-mouthed, clean and dry. And we can
also be used the adhesive bag for infants .If the urine specimen has to be transported for any
length of time it should contain an appropriate preservative to prevent bacterial overgrowth or
hatching of viable ova The specimen can be preserved for microscopic examination of the
deposit by adding 8–10 drops of formaldehyde, 10% solution per 300 ml of urine. Urine
preserved in this way is not suitable for other tests.
Urine to be examined under the microscope must be freshly passed into a clean dry vessel. A
midstream urine specimen is the most useful. Urine stored in a refrigerator may contain an
excess of precipitated salts and will not be suitable for microscopy
principle
the elements of routine urinalysis include macroscopical examination the evolution of
physical characteristics ( color, odor, and specific gravity ) pH and chemical examination (
protein , glucose. Keton bodies, bilirubin, urobilinogen. Bile pigment and bile salt) and
microscopic inspection of centrifuge sediment for R.B.C pus, cast. crystal. etc..
Material( glassware & Equipment):
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centrifuge
Microscope
Urine cup
conical centrifuge tube
slides
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6- cover slide
7- Watch glasses
8- Dropper
9- Forceps
10- Indicator paper of limited pH range: for the 5.0–7.0 range and for the 6.0–8.0 range.
11- Test tube
12- burner
13- Urinometer
14- Pasteur pipette
REAGENTS
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20 % 5-sulfosalicylic acid
Ehrlich reagent
Acetic acid
Lougal iodine
Dipstick strip (reagent strip): should be considered as a first choice because of its
practical, easy and cost effective.
6.1.1 Macroscopical Examination of Urine
Appearance
Urine is normally clear straw-yellow in color. More concentrated urine may appear
dark yellow, the presence of blood cells or excess salts may make the urine appear
cloudy. Pigments from bile substances may make the urine appear deep yellow or
brown. Urine can occasionally appear colorless. Report the appearance as clear or
cloudy, colorless, pale yellow, deep yellow or brown.
Color
Urine varies in appearance, depending principally upon a body's level of hydration, as well as
other factors. Normal urine is a transparent solution ranging from colorless to amber but is
usually a pale yellow. In the urine of a healthy individual the color comes primarily from the
presence of urobilin. Urobilin in turn is a final waste product resulting from the breakdown of
heme from hemoglobin during the destruction of aging blood cells.
Colorless urine indicates over-hydration, generally preferable to dehydration (though it can
remove essential salts from the body). Colorless urine in drug tests can suggest an attempt to
avoid detection of illicit drugs in the bloodstream through over-hydration.





Colorless very dilute urine caused by polyuria, diabetes insipidus
Dark yellow urine is often indicative of dehydration.
Yellowing/light orange may be caused by removal of excess B vitamins from the
bloodstream.
Certain medications such as rifampin and phenazopyridine can cause orange urine.
Reddish urine is termed hematuria, a symptom of a wide variety of medical
conditions may be caused by porphyria (not to be confused with the harmless
temporary pink or reddish tint caused by beeturia).
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
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Dark orange to brown urine can be a symptom of jaundice, rhabdomyolysis, or
Gilbert's syndrome.
Black or dark-colored urine is referred to as melanuria and may be caused by a
melanoma.
Pinkish urine can result from the consumption of beets.
Greenish urine can result from the consumption of asparagus.
Milky caused by lipiduria
Blue urine stains can be caused by blue diaper syndrome
Odor
Normally with aromatic odor.
Abnormally:
Offensive odor due to pus increase.
Acetone odor due to ketones increase ( ketonuria).
Reaction (PH)
Principle
Colored indicator paper is dipped in the urine (or placed in a watch glass and a few drops of
urine are added to it). The color changes according to the PH. The paper is then compared
with a standard control chart giving the corresponding pH value.
Method
The urine specimen must be tested within 1 hour of collection
1- Place a strip of universal indicator paper in a watch glass, let a few drops of fresh
urine fall from the dropper on to the paper alternatively, and dip the test paper
directly into the urine in the receptacle.
2- Pick the strip of paper up with forceps. Compare the color obtained with those shown
on the standard chart .Read off the pH unit given for the color that matches the test
paper most closely.
3- According to the result obtained, select a strip of indicator paper for the
corresponding limited range. For example, if the pH is 6, use indicator paper for the
range 5.0–7.0. If the pH is 7 or more, use indicator paper for the range 6.0–8.0. The
pH of urine is normally about 6.0 (range 5.0–7.0). Acid pH values (4.5–5.5) are
observed in some forms of diabetes, muscular fatigue and acidosis. Alkaline pH
values (7.8– 8.0) are common in patients with infections of the urinary tract and in
people on a vegetarian diet. Determination of the pH of urine is useful for the
identification of crystalline
Detection of Glucose in urine
Introduction:
Glucose may appear in urine at different blood glucose level , varying in individuals blood
level, Glomerular, blood flow, tubular reabsorption rate and urine flow influence its
appearance. Glucosuria usually occur when blood level is more than 180 to 200 mg/dl
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Principle
Glucose is the most commonly found sugar substance in urine, particularly in diabetic
patients and patients suffering from chronic renal failure.
Method
Glucose in urine can be detected using a urine dipstick, the dipsticks are placed into the urine
and immediately removed. They are then compared with a comparison chart after an
appropriate time that is also specified on the chart
The color changes observed on the dipstick will give a semi-quantitative estimation of the
amount of substance present. This can be reported as negative, Trace , +(160-195) , ++( >200)
, +++ (250-300) . According to the read of glucose strip or as an approximate value of the
concentration of the substance tested for. Dipsticks must be stored according to the
manufacturer’s instructions
Detection and estimation of protein
Principle:
Protein is not normally found in urine, this is because kidneys ensure that large molecules
such as protein remain in the blood. However, the kidneys sift out the smaller impurities and
expel them through the urine. In case some protein does get through, the body usually reabsorbs it and uses it as an energy source. However, if there is too much protein in the blood,
then some amount of protein will be present in the urine. This can happen even if the kidneys
are functioning properly Elevated protein levels are observed in the urine of patients with:





urinary Schistosomiasis
chronic renal disease
pyelonephritis
diabetes mellitus systemic disorders (lupus erythematosus) multiple myeloma
However, orthostatic proteinuria, a form of functional proteinuria usually seen in
young men, which occurs on standing up and disappears on lying down, has no
pathological significance
METHOD
Urine for Albumin test done by one of the following: acetic acid test
1. Prepare 1 ml of urine in a test tube then heat gently till ebullition 2.If the solution
becomes turbid that indicates the presence of albumin or amorphous urate.
So you must put acetic acid for insurance if the turbidity precipitated so it is
amorphous urate, if still turbid so it is albumin in urine.
 Sulfosalicylic acid test
1-To approximately 3ml of supernatant urine in test tube and one equal amount of
3%SSA invert in the mix
2- Let to stand exactly 10 minute
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3-Invert again twice
 Rapid test Protein can also be detected using a urine dipstick
1- Observe the degree of precipitation and grade the results according to the following:
Table 9: Degree of Precipitation
Negative
Trace
+
++
+++
++++
No turbidity
perceptible
Distinct turbidity but no discrete
granulation
Turbidity with granulation but no
flocculation
Turbidity with granulation and flocculation
Clumps of precipitated protein or solid
precipitate
Specific gravity
clinical significance
urinalysis is a parameter commonly used in the evaluation of kidney function and can aid in
the diagnosis of various renal diseases ( eg diabetes insipidus).
principle
Specific gravity (which is directly proportional to urine osmolality which measures solute
concentration) measures urine density, or the ability of the kidney to concentrate or dilute the
urine over that of plasma. Dipsticks are available that also measure specific gravity in
approximations. Most laboratories measure specific gravity with a refractometer Specific
gravity between 1.002 and 1.035 on a random sample should be considered normal if kidney
function is normal. Since the specific gravity of the glomerular filtrate in Bowman's space
ranges from 1.007 to 1.010, any measurement below this range indicates hydration and any
measurement above it indicates relative dehydration
Method

The Urinometer vessel is filled three fourth full with urine ( minimum volume of
urine 15 ml)
 The urinometer is inserted with spinning motion
 Make sure that is floating freely ( which reading the urinometer be sure that it is not
touching the sides or the bottom of cylinder avoid surface bubble read a bottom of the
meniscus
 we can identifying the specific gravity by using dipstick
Bilirubin
Introduction
Bilirubin, a product of hemoglobin catabolism, is characterized by its distinctive yellow
pigment. The presence of bilirubin in urine is always abnormal. In most healthy individuals
the amount of conjugated bilirubin excreted is not detected by the strips. In cases when
bilirubin is elevated and is conjugated, it will be detected by the test strip. It is important to
note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to
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albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid
through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide.
Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the
intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately
half of the urobilinogen is excreted in the feces; most of the other half is recirculated through
the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine
Normally bilirubin is not found in a routine urine test . this test is done to detect abnormally
high urine concentration of direct (conjugated ) bilirubin and help to identify the cause of
jaundice .
Clinical significance
The presence or absence of bilirubin in urine must be correlated with serum bilirubin test, the
specimen appearance is dark with yellow foam
Detection Bilirubin in urine
1. Prepare 1 ml of urine in a test tube then put 2 -3 drops Iodine on the wall of test tube
gently.
2. A violet ring appeared on the surface of urine
3. Shake the tube, then solution becomes violet or brownish
4- if violet ring appeared on the surface of urine mean positive result
Urobilinogen test
Urobilinogen test is directly associated with the function of liver and any disorder or infection
related to this organ can be traced in the urobilinogen test normal specimen contain small
amount of urobilinogen (0.1 to 1 Ehrlich units per dl of urine) this test done to aid diagnosis
of extra hepatic obstruction such as blocking of the common bile duct and to aid differential
diagnosis of hepatic and pre hepatic.
Method
Urobilinogen Analysis
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Add 2.5ml of afresh specimen of urine into a tube
Add o.25ml of Ehrlich reagent
Allow to stand for 5 minute
A tea color of the urine indicate urobilinogen is present in increased amounts . A
fine pink or brown color indicates that urobilinogen present in normal amount. We
can also be use the dipstick to identifying the urobilinogen in urine
The test for urobilnogen is based on the Ehrlich Aldehyde Reaction. Pdimethylaminobenzaldehyde in an acid medium with a color enhancer reacts with
urobilinogen to form a pink-red color. The urine chemical reagent strip reactivity increases
with increasing temperature. The optimum temperature for testing is 22° - 26°C. A freshly
voided sample is always best for ensuring optimal results
False-Positive and False-Negative Urobilinogen Results
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False-positive results: A false positive urobilinogen reaction may occur with the chemical
reagent strip method when substances known to react with Ehrlich's reagent such as
sulfonamides and p-aminosalicylic acid are present in the urine. Drugs that contain Azo dyes,
such as Azo Gantrisin, have a gold color that masks the reaction, causing a false positive
reaction. Atypical color reactions may be obtained in the presence of high concentrations of
p-aminobenzoic acid. The chemical reagent strip urobilinogen test cannot detect
porphobilinogen in a urine specimen. Porphobilinogen is a molecule formed during the
synthesis of the heme portion of hemoglobin. False-negative resultsDue to the instability of
urobilinogen, a false negative result may occur using the chemical reagent strip method if the
urine specimen has remained at room temperature for an extended period of time exposed to
light. A false negative result may also occur if formalin is present.
Detection of ketones bodies
Introduction
Normal urine does not contain ketone bodies. Acetone and other ketones bodies may appear
in urine:
 In severe or untreated diabetes( diabetic ketoacidosis )
 In certain other conditions (dehydration, vomiting, starvation and following strenuous
exercise).
Principle
When sodium nitroprusside is added to urine containing ketone bodies, a purple color is
produced.
Materials and reagents
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Test-tubes
Test-tube rack
Measuring cylinder, 10ml
Dropping pipette
Sodium nitroprusside crystals
Acetic acid
Ammonia.
Method
1- Just before carrying out the test, place a sufficient amount of sodium
nitroprusside crystals into a test-tube to cover the bottom
2- Add 5ml of distilled water. Shake well until the crystals are almost dissolved.
(Not all the crystals are expected to dissolve as the solution is saturated.)
3- Measure 10ml of urine into another test-tube.
4- Add four drops of acetic acid to the urine, followed by 10 drops of freshly
prepared Sodium nitroprusside solution and mix well
5- Holding the tip of the pipette against the side of the tube, let 20 drops (1 ml) of
ammonia solution flow on to the surface of the liquid Wait for 5 minutes
before reading a positive result may be obvious before this time
RESULT
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If the result is positive purple ring appears on top of the urine. If the result is negative, no
color change occurs
Reporting the results of the test for detection of ketone bodies in urine
a: Positive reaction;
b: negative reaction.
substances in urine Color change Result
None = Negative Pink ring = +
Red ring= ++
Purple ring =+++
Ketone bodies in urine can also be detected using a urine dipstick
6.1.2 Microscopical Examination of Urine
Detection of abnormal elements
Urine contains cells and crystals in suspension that can be collected by centrifugation or by
allowing the urine to stand and the suspended particles to form a sediment. The resulting
urinary deposit can be examined under the microscope
In certain diseases of the urinary tract, the urinary deposits are considerably altered.
Method
Preparation of the deposit
1- Mix the urine specimen gently and pour 10 ml into a centrifuge tube.
2- Centrifuge the specimen at medium speed (1500RPM) for 5 minutes.
3- Pour off the supernatant by quickly inverting the tube without shaking. (The
supernatant may be used for biochemical testing.)
4- Tap of the tube to re-suspend the deposit.
5- Transfer one drop of the deposit on to a slide using a Pasteur pipette and cover with a
cover slip
6- Label the slide with the patient’s name or identification number.
Microscopic examination
Using the X10 objective and with the condenser lowered, scan the cover slip all over to look
for ova of Schistosoma haematobium when indicated.
Using the X40 objective and with the condenser lowered or aperture reduced, scan the
coverslip area again and report any findings as a quantitative value for each high-power field
The following may be found in urine
1- Erythrocytes
2- Leukocytes
3- epithelial cells
4- casts
5- fungi
6- bacteria
7- crystals
8- parasite eggs and larvae( Schistosoma haematobium )
9- Trichomonas vaginalis
10- spermatozoa.
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 Erythrocytes
Erythrocytes in urine may be:
(a) Intact: small yellowish discs, darker at the edges (8um);
(b) Crenated: spiky edges, reduced diameter (5–6um);
(c) Swollen: thin circles, increased diameter (9–10um).
The shape of the cells often changes during storage of urine and does not have any diagnostic
importance.
In normal individual s occasional red blood cell (0-2HPF) may be seen in microscopic
examination of the sediment
Note: Erythrocytes may be found in the urine of women if the specimen has been taken
during the menstrual period.
 Leukocytes
Found in urine may be:
(a) Intact: clear granular discs, 10–15mm (the nuclei may be visible);
(b) Degenerated: distorted shape, shrunken, less granular;
(c) Pus: clumps of numerous degenerated cells.
The presence of many leukocytes, especially
in clumps, indicates a urinary tract infection.
How to express the quantity leukocytes found in urine deposits
Place one drop of urine deposit on a slide and cover with a cover slip.
Using the x40 objective, examine the deposit and count the number of leukocytes per
microscope field.
Report the results according to cells / high power field count.
EPETHELIAL CELL
Different types of epithelial cells can be observed in urine. Some of these are readily
identifiable; however it is difficult to distinguish small transitional epithelial cells
from WBC and renal tubular epithelial cells from transitional epithelial cells. Thus, all
non-squamous cells in urine are considered to be of transitional origin. When we are
unsure about the origin of the cells in urine, we can stain a urine sediment with
Wright's stain (or Diff-quik) and perform a cytological examination on the urine
sediment.
Epithelial cells are subjectively semi-quantified in urine (usually under low power
using the 10x objective) as: none seen, few, moderate, many
Transitional epithelial cells
The urinary tract from the pelvis down the ureters to the bladder and the proximal urethra is
lined by transitional epithelial cells. These cells vary in size and shape depending on the
location from which they originate, e.g. those from the renal pelvis are more caudate whereas
those from the bladder are more round to polygonal and vary in size. These cells naturally
slough into the urine in quite low numbers, so none to a few transitional epithelial cells are
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seen in the urine from healthy animals. Note that this depends on the method of urine
collection, since these cells will be sloughed (traumatically) when the bladder is catheterized.
Transitional epithelial cells must be distinguished from WBC, because they both have the
same granular appearance. In general, transitional epithelial cells (arrow in above image) are
larger and have more irregular borders than WBC (which are uniformly more round,
arrowhead in above image).
Squamous epithelial cells
These can be keratinized or non-keratinized.
Non-keratinized squamous epithelial cells originate from the distal urethra, prepuce and/or
vagina. They are larger than transitional cells and have small central nuclei. They can be
round or have one or more flat border. Keratinized squamous epithelial cells are from the skin
or vulva and are large cells with angular borders. They may or may not have nuclei (see upper
image to the right). Nuclei are more visible in cells when the urine is stained with Sedi-stain
(see central panel on right). If in doubt about the origin of the cells, a Wright's stain (routine
hematologic stain) can be performed on a urine sediment and demonstrates the central nuclei
and angular borders of squamous epithelial cells (lower panel on right).Squamous cells are
frequently seen as contaminants in voided urine samples and can also contaminate samples
collected by catheterization
Renal tubular epithelial cells
These are rarely seen in the urine and, as mentioned above, are very difficult to distinguish
from transitional epithelial cells. If large numbers of smaller epithelial cells of uniform
appearance (size and shape) are observed in the urine, a renal origin for these cells is
suspected. Transitional epithelial cells tend to be more variable in size and shape (to some
extent). Sloughing of large numbers of renal tubular epithelial cells would indicate renal
tubular injury
RBCs
WBCs
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Squamous epithelial cells
Transitional epithelial cells
Neoplasia
Fig15: common urine cells
 CAST
Hyaline casts: These can be quite difficult to see in wet preparations of urine sediments with
light microscopy, even with the condenser of the microscope racked down. They are much
easier to visualize using phase contrast; however phase is usually not available on most
microscopes. They become more visible with regular light microscopy if fat sticks to the
protein matrix (Tamm-Horsfall mucoprotein) that makes up the hyaline cast (hyaline with fat)
or particulate material from degenerating cells is present within the cast matrix (hyaline to
finely granular cast).
Granular casts: are rather short casts filled with large granules, pale yellow in color, with
rounded ends. The granules come from degenerated epithelial cells from the tubules of the
kidney.


Fine granular casts have smaller granules that do not fill the cast
Do not confuse with hyaline casts, partly covered by amorphous phosphate crystals
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Blood casts are filled with more or less degenerated erythrocytes, brownish in color.
They are found in acute kidney disease.
Pus casts are completely filled with leukocytes
Hyaline casts, which may contain a few leukocytes suffering from kidney infection.
Epithelial casts are filled with pale yellow epithelial cells (To make the cells more
distinct, add a drop of 10% acetic acid to the deposit.)
Epithelial casts have no diagnostic significance.
Fatty casts are very shiny yellowish casts; the edges are indented and distinct and the
ends are rounded They are soluble in ether but not in acetic acid Fatty casts are
found in patients with severe kidney disease.
Pseudo casts Do not mistake for casts:
1- clumps of phosphate crystals, short and clear-cut
2- aggregations of translucent mucus, the ends tapering into threads
Fig16: different casts seen in urine
Crystals
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Crystals have regular geometric shapes unlike amorphous debris, which is made
up of clumps of small granules with no definite shape Except in very rare diseases, crystals in
urine have no diagnostic significance.
1- Calcium oxalate (acid urine)
 Size: 10–20µ or about 50µ
 Shape: envelope-shaped or peanut-shaped
 Color: colorless, very shiny.
2- Uric acid (acid urine)
 Size: 30–150µ.
 Shape: varies (square, diamond-shaped, cubical or rose-shaped).
 Color: yellow or brownish-red.
3- Triple phosphates (neutral or alkaline urine)
 Size: 30–150µ.
 Shape: rectangular or like a fern leaf or star
 Color: colorless, shiny
4- Urate crystals
 Urates (alkaline urine) (Size: about 20µ.
 Shape: like a cactus or a bundle of needles
 Colour: yellow, shiny.
 Urates are often found together with phosphates.
 Calcium phosphate (neutral or alkaline urine) Size: 30–40µ.
 Shape: like a star. Colour: colourless.
5- Calcium carbonate (neutral or alkaline urine)
 Size: very small.
 Shape: similar to millet or corn grains, grouped in pairs.
 Colour: colourless.
 If acetic acid, 10% solution is added, the crystals dissolve, giving off
 bubbles of gas.
6- Calcium sulfate (acid urine)
 Size: 50–100µ.
 Shape: long prisms or flat blades, separate or in bundles.
 Calcium sulfate crystals can be distinguished from calcium phosphate crystals by
measuring the pH of the urine.
7- Amorphous phosphates (alkaline urine)
 Amorphous phosphates appear as small, whitish granules, often scattered.
8- Amorphous urates (acid urine)
Amorphous urates appear as very small, yellowish granules, which are grouped in
compact clusters. They are not soluble in acetic acid, 10% solution, but dissolve if the
urine is gently heated
(Urine kept in the refrigerator often shows a heavy precipitate of urates.)
Other crystalline deposits
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The following are rarely found in the urine. When present, however, they are found
9


Cystine (acid urine)
Size: 30–60µ.
Shape: hexagonal plates.
Color: colorless, very shiny.
Cystine crystals are found only in fresh urine as they are soluble in ammonia. & they are
soluble in acetic acid, 10% solution (one drop per drop of deposit)
They are found in patients with cystin uria, a very rare hereditary disease.
10- Acetyl sulfonamides (neutral or acid urine)
 Shape: varied, but often similar to sheaves of needles.
 Acetyl sulfonamide crystals are found in the urine following treatment with
sulfonamide drugs. The presence of these crystals should be reported as they can
cause kidney damage.
Ammonium biurate
Amorphous urate
Bilirubin
Calcium carbonate
Calcium oxalate
dihydrate
Magnesium ammonium
phosphate
Uric acid
Calcium oxalate
monohydrate
Calcium phosphate
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Cystine
Tyrosine
Flat plates resembling
cholesterol
Fig17: common urine crystals
Fungi



Size: 5–12µ.
Shape: round or oval bodies of various sizes found together. Do not confuse with
erythrocytes. Budding may be seen. Fungi are not soluble in acetic acid.
Fungi are occasionally present in urine containing glucose. Check that the urine
specimen is fresh.
Bacteria
In healthy persons the urine contains practically no organisms. Bacteria may be found in
patients who have an infection of some part of the urinary tract.
Urethritis, cystitis or nephritis), or where bacteria from an infection elsewhere in the body are
excreted in the urine
The urine is centrifuged at high speed and the resulting deposit is examined under the
microscope.
Parasite eggs and larvae
The following may be found:

eggs of Schistosoma haematobium: found together with erythrocytes

Trichomonas vaginalis
Bacteria: Bacilli
Bacteria: Cocci
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Schistosoma haematobium( egg)
Trichomonas vaginalis (trophozoites)
Fig18: Infectious agents
6.2 General Stool Examination
Introduction
Many diseases are caused by infection with parasites. They are an important cause of diarrhea
(which is a major health problem in developing countries. If acute diarrhea is caused by
parasitic infection, this can be determined by examination of a stool specimen.
Clinical Significance
The Inspection of feces is important because it may lead to a diagnosis of parasitic infestation,
obstructive jaundice, diarrhea, malabsorption, rectosigmoidal ,obstruction , dynsentry ,
ulcerative colitis , or gastrointestinal tract bleeding.
Materials and reagents:







Microscope
Microscope slides
Coverslips
Wooden applicators or wire loops (0.45mm, nickel–chromium alloy wire)
Grease pencils
Sodium chloride, 0.85% solution
Lugol iodine, 0.5% solution
Examination of stool specimens for parasites
Collection of specimens
Inspect approximately 100 g of faeces in a clean, dry container without preservatives.
A screw-top wide mouth container is most suitable. Make sure that any adult worms
or segments passed are included.
For collection of stool specimens for bacteriological examination (e.g. for culture
of cholera and other bacteria that cause dysentery),.
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The container with specimen should be labeled, the urine and dirt should be excluded,
urine will destroy any amoebic trophozoites and dirt will interfere with diagnosis
 never leave the stool specimen exposed to the air in container without lead
 never keep the stool specimen artificially warm and don’t leave in the sun
Using Cary–Blair transport medium
Cary–Blair transport medium will preserve many kinds of enteric bacteria (cholera vibrios,
other vibrios, salmonella, shigella, etc.) for up to 4 weeks.
The uninoculated medium may be stored in a sealed bottle at room temperature for 8–12
weeks.
1. Dip a sterile cotton wool swab in the stool specimen
2. For infants or other patients who cannot produce a stool specimen, take a rectal swab.
Moisten the swab with sodium chloride solution and introduce the swab into the rectum. Turn
the swab several times with a circular movement
3. Place the swab in a bottle containing Cary–Blair medium (three-quarters full)
and send it to the bacteriology laboratory. If you cannot send the swab immediately,
store it at room temperature.
Important:


Never store the swab in the incubator.
Never store the swab in the refrigerator.
6.2.1 Macroscopical examination
Faecal samples are best described by their colour, consistency and presence or absence of
macroscopic blood, exudate and adult parasite.
1-Colour
The color can be described as:
 Black (bleeding from upper gastrointestinal tract is more likely to cause stool black)
 Brown
 pale yellow (fat)
 white (obstructive jaundice)
 green
 bloody (especially blood originating from lower gut may cause the stool to be red )
 clay (suggested diminution or absence of bile or presence of barium sulfate )
2-Consistency
The consistency can be described as:
 formed (normal shape)
 soft formed
 unformed or liquid (watery).
The presence of external blood or mucus, usually seen as streaks of red or white, should be
noted. Blood may be present in certain medical conditions (e.g. ulcerative colitis,
schistosomiasis.
3- Pus
101 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Patient with chronic ulcerative colitis and chronic bacillary dysentery frequently pass large
quantity of pus with the stool
 Large amount of pus never accompany amoebic colitis .
 No pus seen in watery stool ….. viral gastroenteritis.
4- Mucus
Even in slightest quantity is abnormal
Bloody mucus = inflammation of rectal canal
Associated with pus and blood is found in stool of patient with ulcerative colitis , bacillary
dysentery , intestinal tuberculosis
Mucus with pus and blood = ulcerative colitis , bacillary dysentery . ulcerative carcinoma of
colon . intestinal tuberculosis
5 - blood
Blood in stool should be never ignored
 Bleeding upper GIT may give black- tarry appearance to stool.
 Bleeding from lower GIT may give red color
6- Fecal fat

It is raised in malabsorption.
6.2.2 Microscopic Examination
Direct microscopic examination of faeces in saline or iodine suspension is useful for the
following reasons:
 to detect motile trophozoites
 to detect ova and cysts present in moderate numbers
 to detect erythrocytes, cellular debris or excess fat
Select unformed or liquid faeces when using direct microscopy for detection of trophozoites.
Formed stools rarely contain motile trophozoites. Also perform a direct examination of any
external blood or mucus.
Method
1. Prepare a 1:1 mixture of 0.5% Lugol iodine solution.
2. Take a dry microscope slide and label it with the name or number of the patient.
3. Put: one drop of sodium chloride solution warmed to 37°C in the middle of the left
half of the slide; and one drop of the iodine–acetic acid solution in the middle of the
right half of the slide
4. Using an applicator or wire loop, take a small portion (about 2–3mm diameter) of the
stool.
a) If the stools are formed, take the portion from the centre of the sample and
from the surface to look for parasite eggs.
b) If the stools contain mucus or are liquid, take the portion from the mucus on
the surface or from the surface of the liquid to look for amoebae.
5.
Mix the sample with the drop of sodium chloride solution on the slide.
102 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
6. Using the applicator or wire loop, take a second portion of stool from the specimen as
described above and mix it with the drop of iodine, acetic acid solution, discard the
applicator (or flame the wire loop) after use.
7. Place a coverslip over each drop (apply the coverslips as shown in to avoid the
formation of air bubbles).
8. Examine the preparations under the microscope. For the saline preparation use the
x10 and x40 objectives and a x10 eyepiece. As the eggs and cysts are colorless,
reduce the amount of light using the condenser aperture or lower the condenser to
increase the contrast. Examine the first preparation with the x10 objective, starting at
the top left hand corner as indicated in Focus on the edge of one coverslip using the
x10 objective and examine the whole area under each coverslip for the presence of
ova and larvae of parasit . Then switch to the x40 objective and again examine the
whole area of the coverslip over the saline for motile trophozoites and the area of the
coverslip over the iodine for cysts.
9. Lugol iodine–acetic acid solution causes the trophozoite forms to become nonmotile
The nucleus is clearly stained but it may be difficult to distinguish between
trophozoite and cystic forms.
10. Using a fine Pasteur pipette, allow a drop of 5% methylene blue solution to run under
the coverslip over the saline preparation This will stain the nuclei of any cells present
and distinguish the lobed nuclei of polymorphs from the large single nuclei of
mucosal cells.
Intestinal protozoa
Protozoa are microorganisms consisting of a single cell. Intestinal protozoa may be
found in stools in their motile form (trophozoites) or as cysts. Some intestinal protozoa
are pathogenic (others are harmless. All these protozoa are found throughout the world.
Identification of motile forms (trophozoites)
The trophozoites of protozoa are motile
 either because of slow movements of the cell (amoebae)
 or because they have rapidly moving flagella (long whip-like threads) or cilia
(numerous short hairs).
Trophozoites are chiefly found in:
 watery stools
 stools containing mucus
 soft formed stools.
The following features are useful for the identification of motile forms of intestinal protozoa







size
cytoplasm
pseudopodia
nuclei
Ectoplasm
Endoplasm
Vacuoles
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




Inclusion bodies containing erythrocytes, bacteria, yeast cells, debris, etc.
nuclear membrane (chromatin)
nuclear karyosome
flagella
Undulating membrane.
Identification of cysts
Cysts are the resistant forms of certain intestinal amoebae, flagellates and ciliates.
They are small, round and non-motile and may have one or several nuclei.
Measurement of cysts is useful for the correct identification of species.
Importance of cysts
The clinical importance of cysts varies from country to country. The cyst is the infective form
of the organism. Healthy persons may be asymptomatic carriers of cysts and are therefore, a
public health hazard.
The most important problem in the laboratory is the precise identification of cysts
Taenia saginata
Hymenolepis nana
Diphyllobothrium latum
Ascaris lumbricoides
Ancylostoma duodenale
Trichuris trichuria
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Strongyloides stercoralis
Clonorchis sinensis
Paragonimus westermani
Schistosome mansoni ova
Schistosome japonicum
Schistosoma haematobium
Figure19: Ova in GSE
Entamoeba histolytica cyst
Entamoeba histolytica trophozoites
Giardia lamblia cyst
Giardia lamblia trophozoites
Figure 20: Entamoeba histolytica & Giardia lamblia (HPF)
105 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
6.3 Urine Culture
Introduction
Urine is the specimen most frequently submitted for culture. It also presents major problems
in terms of proper specimen collection, transport, culture techniques, and interpretation of
results. As with any other specimen submitted to the laboratory, the more information
provided by the submitting physician the more able is the laboratory to provide the best
possible culture data.
The most common sites of urinary tract infection (UTI) are the urinary bladder (cystitis) and
the urethra. From these sites the infection may ascend into the ureters (ureteritis) and
subsequently involve the kidney (pyelonephritis). Females are more prone to infection of the
urinary tract and also present the greater problem in the proper collection of specimens.
In both males and females, UTI may be asymptomatic, acute, or chronic. Asymptomatic
infection can be diagnosed by culture. Acute UTI is more frequently seen in females of all
ages; these patients are usually treated on an outpatient basis and are rarely admitted to
hospital. Chronic UTI in both males and females of all ages is usually associated with an
underlying disease (e.g., pyelonephritis, prostatic disease, or congenital anomaly of the
genitourinary tract) and these patients are most often hospitalized. Asymptomatic, acute, and
chronic UTI are three distinct entities and the laboratory results often require different
interpretation.
Asymptomatic pyelonephritis in females may go undetected for some time, and is often only
diagnosed by carefully performed quantitative urine culture. Chronic prostatitis is common
and difficult to cure, and is often responsible for recurring UTI. In most UTI, irrespective of
type, enteric bacteria are the etiological agents, Escherichia coli being isolated far more
frequently than any other organism. In about 10% of patients with UTI, two organisms may
be present and both may contribute to the disease process. The presence of three or more
different organisms in a urine culture is strong presumptive evidence of improper collection
or handling of the urine specimen. However, multiple organisms are often seen in UTI in
patients with indwelling bladder catheters
Expected pathogens
- Enterococci
- Escherichia coli
- Mycobacterium tuberculosis
- Other Enterobacteriaceae
- Other staphylococci
- Pseudomonas and other non-fermenters
- Staphylococcus saprophyticus
Media and diagnostic reagents
- Isolation and quantitative media
Blood agar
CLED agar or chromogenic media
MacConkey agar, with crystal violet, )
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- Identification media and diagnostic reagents
Kligler iron agar (KIA)
Kovacs reagent for indole
motility–indole–urease (MIU) medium
oxidase reagent
lysine–decarboxylase broth (Möller)
ONPG test
Simmons citrate agar
For staphylococci and enterococci
catalase test (H2O2)
coagulase plasma
bile–aesculin agar (for enterococci)
novobiocin (5mg) disc for differentiating negative-coagulase staphylococci
Specimen collection
It cannot be overemphasized that considerable importance is attached to the method of
collection of urine specimens, transport to the laboratory, and the initial efforts by the
laboratory to screen and culture the urine. It is the responsibility of the laboratory to provide
the physician with sterile, wide-mouthed, glass or plastic jars, beakers, or other suitable
receptacles. Urine specimens may have been collected by a surgical procedure, e.g.,
suprapubic aspiration, cystoscopy, or catheterization. If not, the laboratory must insist on a
clean-catch midstream urine specimen, particularly in females and children. Since urine itself
is a good culture medium, all specimens should be processed by the laboratory within 2 hours
of collection, or be kept refrigerated at 4 °C until delivery to the laboratory and processed no
longer than 18 hours after collection.
Whenever possible, urine specimens for culture should be collected in the morning. It is
advisable to ask the patient the night before to refrain from urinating until the specimen is
collected.
Women
A woman who is ambulatory should:
1. Wash her hands thoroughly with soap and water and dry them with a clean towel.
2. Undress in a suitable room, spread the labia, and cleanse the vulva and labia thoroughly,
using sterile cotton gauze pads and warm soapy water wiping from front to rear.
3. Rinse thoroughly with warm water and dry with a sterile gauze pad. During the entire
process the patient should keep the labia separated and not touch the cleansed area with the
fingers.
4. Pass urine, discarding the first part of the stream. Collect the remaining urine in the sterile
container, closing the lid as soon as the urine has been collected.
5. Hand the clean midstream urine, in the closed container, to the nursing personnel for
prompt delivery to the laboratory.
For bedridden patients, the same procedure is followed, except that a nurse must assist the
patient or, if necessary, do the entire cleansing procedure before requesting the patient to pass
urine.
In both situations every effort must be made to collect a clean-catch urine specimen in a
sterile container and to ensure that it is delivered promptly to the laboratory together with
information on the patient, clinical diagnosis, and the requested procedures.
Men
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A man who is ambulatory should:
1. Wash his hands.
2. Pull back the foreskin (if not circumcised) and pass a small amount of urine.
3. Still holding back the foreskin, pass most of the remaining urine into a sterile container.
This is a midstream urine specimen.
4. Place the cover on the container and hand to nursing staff for prompt delivery to the
laboratory.
For bedridden patients:
1. If necessary, nursing personnel should pull back the foreskin, wash and dry the glans with
soapy water and gauze pads.
2. With foreskin pulled back, the patient should pass a small amount of urine into a urinal.
3. The patient should then pass most of the remaining urine into the sterile container. The
cover should be placed on the container and the specimen transported to the laboratory.
Infants and children
Collection of a clean-catch urine specimen from infants and children who are ill in bed or
uncooperative can be a problem. Give the child water or other liquid to drink. Clean the
external genitalia. The child can be seated on the lap of the mother, nurse, or ward attendant,
who should then encourage the child to urinate and collect as much urine as possible in a
sterile container. The container should then be covered and delivered to the laboratory for
prompt processing.
Culture and interpretation
All urine specimens brought to the microbiology laboratory should be examined at once, or
placed in a refrigerator at 4 °C until they can be examined. The examination procedure
includes the following steps:
1. Examination of a Gram-stained smear.
2. A screening test for significant bacteriuria.
3. A definitive culture for urine specimens found to be positive in the screening test (step 2),
and for all specimens obtained by cystoscopy, Suprapubic aspirate (SPA) , or catheterization.
4. Susceptibility tests on clinically significant bacterial isolates.
Preparation and examination of a Gram-stained smear is a necessary part of the laboratory
process. Using a sterile Pasteur pipette (one for each sample), place one drop of well mixed,
uncentrifuged urine on a slide. Allow the drop to dry without spreading, heat-fix and stain.
Examine under an oil-immersion lens for the presence or absence of bacteria,
polymorphonuclear leukocytes, and squamous epithelial cells.
One or more bacterial cells per oil-immersion field usually implies that there are 105 or more
bacteria per milliliter in the specimen. The presence of one or more leukocytes per oilimmersion field is a further indication of UTI. Non-infected urine samples will usually show
few or no bacteria or leukocytes in the entire preparation. In specimens from females, the
presence of many squamous epithelial cells, with or without a mixture of bacteria, is strong
presumptive evidence that the specimen is contaminated with vaginal flora and a repeat
specimen is necessary, regardless of the number of bacteria per oil-immersion field. If results
are required urgently, the report of the Gram-stain findings should be sent to the physician
with a note that the culture report is to follow.
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Screening method
The absence of leukocytes and bacteria in a Gram-stained smear of a clean-catch urine sample
prepared as described above is good evidence that the urine is not infected. A urine specimen
that is “negative” on careful examination of the Gram-stained smear does not need to be
cultured. An alternative simple and effective screening test is the test strip for leukocyte
esterase/nitrate reduction. The strip is dipped into the urine specimen as instructed in the
package literature. Any pink colour is a positive reaction indicating the presence of leukocyte
esterase and/or bacteria in excess of 105 per ml. Urine samples that are positive in the
screening test should be cultured as soon as possible to prevent possible overgrowth by
nonsignificant bacteria. If the strip does not develop a pink colour it is interpreted as a
negative screening test, is so reported, and no culture is indicated. The test strip may not be
sensitive enough to detect bacterial counts of less than 105 per ml of urine.
Quantitative culture and presumptive identification
Calibrated loop technique
There are several culture methods for the quantification of bacteria in urine. The easiest and
most commonly used are the calibrated loop technique. Of these method using CLED or
chromogenic media, are considered to be the most versatile and efficient for large numbers of
specimens.
Chromogenic media contain various substrates which permit presumptive identification of
several common species through a change in either colony pigmentation or colour of agar.
They perform satisfactorily compared to CLED and have the advantage that mixed cultures
are easier to detect. However, chromogenic media from different manufacturers can vary in
specificity, and are relatively expensive Calibrated loop/surface streak method
Mix the urine gently to avoid foaming.
Dip the end of a sterile calibrated loop (eg 1 μL, 2 μL or 10 μL) in the urine to just below the
surface and remove vertically, taking care not to carry over any on the shank.
Use this to inoculate CLED or chromogenic agar plate and spread according to the number of
specimens . A maximum of four samples per 9 cm plate is recommended for this method
with a 1 μL or 2 μL loop, or two samples if using a 10 μL loop. If a 1 μL loop is used, one
6
colony equals 1000 cfu/mL (ie 1 x 10 cfu/L).
SPA, other surgically obtained urine, and urine samples with expected significant bacteriuria
5
as low as 10 cfu/L (increased inoculum sizes are required) ,Inoculate 100 μL (0.1 mL) of
specimen aseptically to a full CLED or chromogenic agar plate.
Spread inoculum over entire surface of plate with a sterile loop or a spreader. Do not use a
sterile swab which will absorb much of the inoculum. To isolate individual colonies, spread
inoculum with a sterile loop.
No. of cfu/L = No. of cfu on plate x 104.
This semi quantitative method is only sensitive for screening down to106 cfu/L if a 5 μL or
10 μL loop is used (eg 5 or 10 colonies), or107 cfu/L if a 1 μL or 2 μL loop is used (eg 10 or
20 colonies). (See table below).
Guidance on assessing colony counts :
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Interpretation of culture
The basis studies for interpreting urine culture results showing that bacterial counts of ≥108
cfu/L (≥105 cfu/mL) are indicative of an infection and counts below this usually indicate
contamination.
In specific patient groups , counts between 108 cfu/L (105 cfu/mL) and 105 cfu/L (102 cfu/mL)
may be significant. A pure isolate with counts between 107 and 108 cfu/L (104-105 cfu/mL)
should be evaluated based on clinical information or confirmed by repeat culture. Overall the
confirmation of a UTI requires the demonstration of significant bacteriuria by quantitative
culture (defined according to patient group or specimen type). Routine culture methods may
not be sensitive enough to detect low bacteria levels (eg ≥107 cfu/L / ≥104 cfu/mL) and
increased sensitivity will be achieved by increasing the inoculum size .
The following is a list of imperial units and their equivalents.
≥105 cfu/mL equivalent to ≥108 cfu/L
<105 cfu/mL equivalent to <108 cfu/L
104 cfu/mL equivalent to107
cfu/L
<104 cfu/mL equivalent to <107 cfu/L
103 cfu/mL equivalent to 106 cfu/L
3
6
<10 cfu/mL equivalent to <10 cfu/L
102 cfu/mL equivalent to 105 cfu/L
Increased inoculum sizes are also required for persistently symptomatic patients without
bacteriuria if the patient has recurrent ‘‘sterile pyuria’’, or for specimens where lower counts
are to be expected, such as SPA or other surgically obtained urine.
Identification
Identification should be performed as rapidly as possible. Isolation of uropathogens should
be performed by a surface streak procedure on both blood and MacConkey agar using
calibrated loops for semi-quantitative method and incubated aerobically at 37 °C for 24 hours,
and those cultures which becomes negative at the end of 24 hrs incubations will be further
incubated for 48 hours. Bacterial identification should be made using biochemical tests,
namely indole, citrate, oxidase, H2S production, lysine decarboxylase, lactose fermentation,
110 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
urea hydrolysis, gas production, catalase, coagulase, manitol fermentation and novobiocin
susceptibility test (See Flowcharts below ).
Clinical specimen
Primary isolation plate
(blood, CLED, Staph/Strep medium or fastidious anaerobe agar)
Gram stain
Gram positive cocci in pairs and/or short chains
Positive
(Probable Staphylococcus)
A weak catalase or pseudocatalase reaction may be
produced by some strains of Aerococcus &
Enterococcus species
Catalase
Negative
non-haemolytic
a-haemolytic
(Consider Gemella)
b-haemolysis
(Consider Leuconostoc,
Gemella, Helcococcus)
Suspected Enterococcus
(1-2 mm may be a, β or non-haemolytic.
Consider clinical details)
Optochin
Sensitive
Lancefield Group
Resistant
Rapid Aesculin hydrolysis
(or PYR or group)
A,B,C,D,F,G
Positive
S. pneumoniae:
Some S. pneumoniae
may be resistant to
optochin: if there is a
clinical suspicion of
pneumococcal infection,
confirm by performing
bile solubility
(A,C,G, consider
S.anginosus
group)
Negative
“viridans”
Streptococci:
Occasional strains of
S. oralis may be
optochin sensitive:
S. pseudopneumoniae
optochin sensitive, bile
solubility
inconclusive
Enterococcus spp
(PYR & LAP positive)
PYR positive, LAP positive
consider Aerococcus, Lactococcus,
Facklamia, Dolosigranulum.
PYR positive, LAP negative
consider Globicatella, Aerococcus
viridans, Dolosicoccus
PYR
Consider
Aerococcus,
Pediococcus,
Lancefield Group B
S. anginosus group
(Aesc+, PYR-)
Negative
Positive
S. anginosus group
Group A
Further identification if clinically indicated
Commercial identification system or other biochemical identification or send to the
Reference Laboratory
Fig 21: Identification of Streptococci and Enterococci Flowchart
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Non groupable
(repeat, consider Listeria,
check previous
tests)
Clinical specimens
Primary isolation plate
Opaque, white, cream, yellow or orange colonies on blood agar
Gram stain
Gram positive cocci in clusters
If there is a different Gram stain
appearance refer to the appropriate
SMI
Negative
(S. anaerobius is catalase
negative)
Consider other
organisms
Modified oxidase
Resistant
Positive
Micrococcus
(Aerobic growth only)
Suspected
S. aureus
Catalase Positive
Novobiocin
Negative
Sensitive
DNase, clumping factor
(slide or commercial
latex kit) Protein A or
thermostable nuclease
Negative
S. sciuri Group
(Aesculin Positive)
(Meticillin resistant strains may be
mistaken for MRSA and will grow
well on many chromogenic
selective agars).
S. saprophyticus
Group
Coagulase-negative
Staphylococcus
Positive
Confirm with tube
coagulase if required
S. aureus
Further identification
if clinically indicated
Commercial identification
system
Confirm with tube
coagulase if required
DNA +ve: S. aureus, S. intermedius, S. schleiferi, S. hycius, S. sciuri, S. chromogenes
Protein A latex +ve: S.aureus (S. saprophyticus and S. sciuri may give false positive)
Coagulase +ve: S. aureus, S. intermedius, S. hyicus, S. schleiferi
Fig 22: Presumptive Identification of Staphylococcus Species Flowchart
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Clinical Specimens
Primary isolation plate
BA
CLED B or CLED A, MAC
DCA, XLD, CT-SMAC, TCBS, CIN agar
Carbohydrate
fermenting
Carbohydrate
non fermenting
Further identification if
clinically indicated
Oxidase
Performed from non selective
medium
Negative
Further identification
Serology for possible:
Salmonella / Shigella species (XLD / DCA)
E. coli O157 (CT-SMAC) all presumptive (locally confirmed)
E. coli O157 should be sent to the Reference Laboratory Y. enterocolitica (CIN)
(see ID 20, 21, 22, 24)
Commercial identification system
or
other biochemical identification
or
send to the Reference Laboratory
Positive
Possible Pseudomonas species
or
Pasteurella species
(see ID 17 & 13)
Fig 23: Identification of Enterobacteriaceae Flowchart
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Clinical specimens
Primary isolation plate
Pseudomonas selective agar
Blood agar
(or other primary isolation media)
Burkholderia cepacia
selective agar
Typical colonies of GNR at 16-48 hr (see Sections 3.2 and 3.3)
Burkholderia cepacia complex may require up to 5 days incubation
Pigment production
Blue-green colonies
No blue-green colonies
Pink colonies on B. cepacia
selective agar
Oxidase test
Oxidase test
Gram stain of
pure culture
Positive
P. aeruginosa
growth at
42°C
Negative
Positive
Negative
Gram
negative rod
Further identification if clinically indicated
Commercial identification kit or other biochemical
identification or send to the Reference Laboratory
If required, save the pure isolate on an agar slope
Fig 24: Identification of Glucose Non-Fermenting Gram negative
Rods Flowchart
Susceptibility tests
Susceptibility tests should only be performed on well isolated colonies of similar appearance
that are considered significant according to the guidelines presented above. Susceptibility
tests are generally more important on cultures obtained from patients who are hospitalized or
114 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Other
Discard
have a history of recurring UTI. Cultures from patients seen in the clinic with a primary UTI
may not require a susceptibility test.
6.4 Throat Swab
The upper respiratory tract can be the site of several types of infection:





pharyngitis, sometimes involving tonsillitis, and giving rise to a “sore throat”
nasopharyngitis
otitis media
Sinusitis
epiglottitis.
Of all those infections, pharyngitis is by far the most frequent; in addition, the untreated
infection may have serious sequelae. Only pharyngitis will be considered here.
Bacteriological diagnosis of pharyngitis is complicated by the fact that the oropharynx
contains a heavy, mixed, normal flora of aerobic and anaerobic bacteria. The normal flora
generally outnumbers the pathogens and the role of the bacteriologist is to distinguish
between the commensals and the pathogens. Where possible only the latter should be reported
to the physician.
Normal flora of the pharynx
The normal flora of the pharynx includes a large number of species that should be neither
fully identified nor reported when observed in throat cultures:
• viridans (a-haemolytic) streptococci and pneumococci
• Nonpathogenic Neisseria spp.
• Moraxella (formerly Branhamella) catarrhalis (this can also be a respiratory pathogen)
• Staphylococci (S. aureus, S. epidermidis)
• diphtheroids (with the exception of C. diphtheriae)
• Haemophilus spp.
• Yeasts (Candida spp.) in limited quantity
• Various strictly anaerobic Gram-positive cocci and Gram-negative rods, spirochaetes and
filamentous forms.
The throats of elderly, immunodeficient, or malnourished patients, particularly when they
have received antibiotics, may be colonized by Enterobacteriaceae (Escherichia coli,
Klebsiella spp., etc.) and by the nonfermentative Gram-negative groups (Acinetobacter spp.
and Pseudomonas spp.). Such patients may also have in their pharynx a proliferation of S.
aureus or of
Candida spp., or other yeast-like fungi. Although these microorganisms do not cause
pharyngitis, except in association with granulocytopenia, it is advisable to report such isolates
to the clinician, as they occasionally indicate the existence of (or may sometimes give rise to)
a lower respiratory tract infection
(e.g. pneumonia) or bacteraemia. However, an antibiogram should not be performed routinely
on these colonizing microorganisms.
Expected pathogens
 Candida albicans (oropharynx)
 Corynebacterium diphtheriae (throat and nose)
 Haemophilus influenzae (ear and sinus)
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





Moraxella catarrhalis (ear and sinus)
Neisseria meningitides
Pseudomonas
Staphyloccoccus aureus (ear and sinus)
Streptococcus pneumoniae (ear and sinus)
Streptococcus pyogenes (group A, throat)
Media and diagnostic reagents
Isolation media
Isolation media
Priority
grading
1
2
2
2
3
Blood agar (prepared from a glucose-free base)
Chocolate agar
Löffler coagulated serum or Dorset egg medium
Tellurite blood agar
Modified Thayer–Martin medium (for gonococci andmeningococci)
Diagnostic reagents
Diagnostic reagents
Bacitracin disc
Catalase and coagulase reagents
Optochin disc
Carbohydrate degradation media for Neisseria spp.
Oxidase reagent
V and XV factors (discs or strips)
Tributyrin
Priority
grading
1
1
1
2
2
2
3
Rapid diagnostic tests
Grouping kit for haemolytic streptococci
3
Bacterial agents of pharyngitis
Streptococcus pyogenes (Lancefield group A) is by far the most frequent cause of bacterial
pharyngitis and tonsillitis. This infection is particularly prevalent in young children (5–12
years).
Non-group-A, b-haemolytic streptococci (e.g. groups B, C and G) are uncommon causes of
bacterial pharyngitis and if detected should be reported.
Corynebacterium diphtheriae is the cause of diphtheria, a disease that is endemic in many
countries. It can reach epidemic proportions in countries where the vaccination programme
has been interrupted. Characteristically (with a few exceptions), C. diphtheriae causes a
typical form of infection, characterized by a greyish-white membrane at the site of infection
(pharynx, tonsils, nose, or larynx). Diphtheria is a serious disease and the diagnosis is made
on the basis of clinical findings. The physician would then generally make a specific request
to culture for diphtheria bacilli.
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(Vincent angina) is a rare condition characterized by a necrotic ulceration of the pharynx with
or without formation of a pseudomembrane. It is associated, at the site of infection, with a
heavy mixed flora of strict anaerobes dominated by Gram negative fusiform rods and
spirochaetes, generally referred to as Fusobacterium spp. and Treponema vincentii, and
possibly others. Although both species belong to the normal mouth flora, their presence in
large numbers in a Gram-stained smear of ulcerated lesions should be reported as a
“fusospirochaetal complex”. This microscopic diagnosis need not be confirmed by anaerobic
culture, which is difficult and time-consuming. However, the presence of this complex does
not exclude the need to search for other pathogens, particularly S. pyogenes.
Although small numbers of C. albicans or other Candida species may be part of the normal
oral flora, oral candidiasis results when the number of organisms increases considerably in
certain pathological conditions, e.g. in malnourished premature babies, in immunodeficient
adults (e.g. patients with HIV/AIDS), or in patients who have received broad-spectrum
antimicrobials or cancer therapy. The affected area—tongue, tonsils, throat or buccal mucosa
—may be extremely red, or covered with white patches or a confluent grey-white membrane
(thrush). The diagnosis of candidiasis is best made by finding numerous yeast cells, some of
them forming long mycelium-like filaments, in a Gram-stained smear of the exudate. Swabs
from the upper respiratory tract may be submitted to the laboratory, not for the diagnosis of a
clinical infection, but to detect a potential pathogen in a healthy subject, a pharyngeal or a
nasal “carrier”. This should only be done as part of well-defined epidemiological surveys.
The following pathogens can give rise to a carrier state in the upper respiratory tract:
• Staphylococcus aureus. Sampling of patients and staff for nasal carriers is sometimes
performed as part of an investigation of hospital outbreaks of Meticillin-resistant S. aureus
(MRSA).
• Neisseria meningitidis. Carriage of meningococci may be very prevalent (20% or more)
even at non-epidemic times. Identification of pharyngeal carriers of meningococci is rarely
needed, and need not be performed prior to the administration of prophylactic antibiotics to
family or other close contacts of patients with meningococcal disease.
• Streptococcus pyogenes. Carriage of this organism in low numbers may be prevalent,
especially among schoolchildren (20–30%).
• Corynebacterium diphtheriae. The carrier rate of the diphtheria bacillus is high in nonvaccinated populations. In such communities, it may be justified to identify and treat carriers
among the close contacts of a patient with proven diphtheria. Carriers are rare when an
immunization programme is correctly implemented.
Collection and dispatch of specimens
Ideally, specimens should be collected by a physician or other trained personnel.
The patient should sit facing a light source. While the tongue is kept down with a tongue
depressor, a sterile cotton-wool swab is rubbed vigorously over each tonsil, over the back
wall of the pharynx, and over any other inflamed area. Care should be taken not to touch the
tongue or buccal surfaces.
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It is preferable to take two swabs from the same areas. One can be used to prepare a smear,
while the other is placed into a glass or plastic container and sent to the laboratory.
Alternatively, both swabs may be placed in the container and dispatched to the laboratory. If
the specimen cannot be processed within 4 hours, the swab should be placed in a transport
medium (e.g. Amies or Stuart).
Direct microscopy
The fusospirochaetal complex of necrotizing ulcerative pharyngitis (Vincent angina) and
Candida are best recognized on a Gram-stained smear, which should be prepared if the
physician makes a special request. The Gramstained smear is not useful for the detection of
streptococci or Neisseria spp.
Moreover, the direct smear has poor sensitivity and specificity for the detection of the
diphtheria bacillus, unless the specimen has been collected with care and is examined by an
experienced microbiologist. In the absence of a physician’s request or of clinical information,
a Gram-stained smear should not be made for throat swabs.
Culture and identification
Culture for Streptococcus pyogenes
Immediately upon receipt in the laboratory, the swab should be rubbed over one-quarter of a
blood agar plate, and the rest of the plate streaked with a sterile wire loop. The blood agar
should be prepared from a basal agar medium without glucose (or with a low glucose
content), e.g. tryptic soy agar (TSA). Acidification of glucose by S. pyogenes inhibits the
production of haemolysin. Blood from any species, even human blood (fresh donor blood),
can be used at a concentration of 5%. The plates should be filled to a depth of 4–5 mm. Sheep
blood is preferred because it does indicate haemolysis of some commensal Haemophilus spp.
and it gives no haemolysis with the zymogenes variant of Enterococcus faecalis.
The recognition of b-haemolytic colonies can be improved, and their presumptive
identification hastened, by placing a co-trimoxazole disc (as used for the susceptibility test)
and a special low-concentration bacitracin disc over the initial streaked area. Because S.
pyogenes is resistant and many other bacteria are susceptible to co-trimoxazole, this disc
improves the visibility of b-haemolysis. Incubation in a candle-jar will detect most bhaemolytic streptococci.
A simple way to increase haemolysis is to stab the agar surface perpendicularly by inserting
the loop deep into the medium to encourage growth
of subsurface colonies. After 18 hours and again after 48 hours of incubation at 35–37 ∞C,
the blood plates should be examined for the presence of small (0.5–2 mm) colonies
surrounded by a relatively wide zone of clear haemolysis.
After Gram-staining to verify that they are Gram-positive cocci, the colonies should be
submitted to specific identification tests for S. pyogenes. For clinical purposes, presumptive
identification of S. pyogenes is based on its susceptibility
to a low concentration of bacitracin. For this purpose, a special differential disc is used
containing 0.02–0.05 IU of bacitracin. The ordinary discs used in the susceptibility test, with
a content of 10 units, are not suitable for identification. A b-haemolytic streptococcus
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showing any zone of inhibition around the disc should be reported as S. pyogenes. If the
haemolytic colonies
are sufficiently numerous, the presence or absence of an inhibition zone may be read directly
from the primary blood agar plate. If the colonies are less numerous, one or two should be
picked from the primary plate, streaked on one-fifth of another plate to obtain confluent
growth, and each inoculated area covered with a bacitracin disc. After overnight incubation,
the subcultures should be read for inhibition zones.
In some laboratories this presumptive identification is confirmed by serological
demonstration of the specific cell wall polysaccharides. This can be done either by the
classical precipitin method, or more rapidly by using a commercial
kit for the rapid slide coagglutination or latex agglutination tests. If desirable, bacitracinresistant b-haemolytic streptococci can be further identified using some simple physiological
tests (see Table 19). Minute colonies of b-haemolytic streptococci may be encountered,
which, when grown and serologically grouped, react with group A antiserum. These
streptococci are not
considered to be S. pyogenes and are not associated with the serious infections caused by
group A streptococci.
In reporting the presence of S. pyogenes in a throat culture, a semiquantitative answer should
be given (rare, +, + +, or + + +). Patients with streptococcal pharyngitis generally show
massive growth of S. pyogenes, with colonies over the entire surface of the plate. Plates of
carriers generally show fewer than 20 colonies per plate. Even rare colonies of b-haemolytic
streptococci should be confirmed and reported.
Table 10: Differentiation of b-haemolytic streptococci
Culture for Corynebacterium diphtheriae
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Although the diphtheria bacillus grows well on ordinary blood agar, growth is improved by
inoculating one or two special media:
• Löffler coagulated serum or Dorset egg medium. Although not selective, both of these
media give abundant growth of the diphtheria bacillus after overnight incubation. Moreover,
the cellular morphology of the bacilli is more “typical”: irregularly stained, short to long,
slightly curved rods, showing metachromatic granules, and arranged in a V form or in parallel
palisades. Metachromatic granules are more apparent after staining with methylene blue or
Albert stain than with the Gram stain.
• A selective tellurite blood agar. This medium facilitates isolation when the bacilli are few in
number, as is the case for healthy carriers. On this medium, colonies of the diphtheria bacillus
are greyish to black and are fully developed only after 48 hours. Suspicious colonies,
consisting of bacilli with a coryneform morphology on the Gram-stained smear, should
be subcultured to a blood agar plate to check for purity and for “typical” morphology. It
should also be remembered that colonies of the mitis biotype of C. diphtheriae, which is the
most prevalent, show a marked zone of b-haemolysis on blood agar.
A presumptive report on the presence of C. diphtheriae can often be given at this stage.
However, this should be confirmed or ruled out by some simple biochemical tests and by
demonstration of the toxigenicity. As the latter requires inoculation of guinea-pigs or an in
vitro toxigenic test (Elek) and has to be performed in a central laboratory, only rapid
biochemical identification will be covered here. C. diphtheriae is catalase- and nitratepositive. Urea is not hydrolysed. Acid without gas is produced from glucose and maltose,
generally
not from saccharose. The fermentation of glucose can be tested on Kligler medium. Urease
activity can be demonstrated on MIU and nitrate reduction in nitrate broth in the same way as
for Enterobacteriaceae. For the fermentation of maltose and saccharose, Andrade peptone
water can be used as a base
with a 1% final concentration of each carbohydrate. Results can usually be read after 24
hours, although it may be necessary to reincubate for one night.
It must be emphasized that the microbiology laboratory’s role is to confirm the clinical
diagnosis of diphtheria. Therapy should not be withheld pending receipt of laboratory reports.
Susceptibility testing
Routine susceptibility tests on throat or pharyngeal isolates are most often not required, and
may even be misleading. The major pathogens involved in bacterial pharyngitis are S.
pyogenes and C. diphtheriae benzylpenicillin and erythromycin are considered as the
antimicrobials of choice to treat both types of infection. In cases of diphtheria, treatment with
antitoxin is also indicated.
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6.5 Nasal Swab
Nasal colonization with Staphylococcus aureus increases the risk of staphylococcal infections
at other sites of the body such as postoperative wounds and dialysis access sites3. It is also
associated with recurrent skin infections and nosocomial infections in nurseries and hospital
wards. S. aureus is a major cause of morbidity and mortality in haemodialysis patients4 as
most patients carry the organism in their anterior nares.
Eradication of nasal carriage of S. aureus may be beneficial in certain clinical conditions such
as recurrent furunculosis. Systemic, in addition to topical, treatment is appropriate for nasally
colonised patients who have infection elsewhere. Topical antibacterial agents such as
mupirocin and chlorhexidine/neomycin are preferred to systemic formulations when a patient
is identified as a carrier.
Nose swabs may be used to investigate carriage of Lancefield group A streptococcus and
Meticillin Resistant Staphylococcus aureus (MRSA)
Specimen Collection, Transport and Storage
Safety considerations
 Use aseptic technique.
 Collect specimens in appropriate CE marked leak proof containers and transport
specimens in sealed plastic bags.
 Collect swabs into Amies transport medium with charcoal22 and transport in sealed
plastic bags.
 Compliance with postal and transport regulations is essential.
 Collect specimens before antimicrobial therapy where possible.
 Specimens should be transported and processed as soon as possible.
 If processing is delayed, refrigeration is preferable to storage at ambient
temperature20. Delays of over 48hr are undesirable.
Correct specimen type and method of collection
 Plain sterile cotton wool swab. Sample the anterior nares by gently rotating the swab
over the mucosal surface.
 Unless otherwise stated, swabs for bacterial and fungal culture should then be placed
in amies transport medium with charcoal.
 Numbers and frequency of specimen collection are dependent on clinical condition of
patient.
Specimen Processing
Inoculate each agar plate with a swab For the isolation of individual colonies, spread
inoculum with a sterile loop.
Table 11: Culture media, conditions and organisms for all specimens
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Staphylococci Identification
Staphylococci are the bacteria most frequently associated with the production of pus.
Staphylococci grow well aerobically on blood agar and form opaque white to cream colonies,
1–2mm in diameter, after overnight incubation. They are unique in growing on media with a
high salt content, such as MSA. They can be differentiated from streptococci by their
morphology and by the production of catalase. Catalase production by staphylococci is shown
by placing it in a drop of 3% hydrogen peroxide onto the colonies deposited on a clean glass
slide. The appearance of bubbles of oxygen is an indication of catalase production.
For clinical purposes, staphylococci can be divided into those that produce coagulase and
those that do not. The coagulase-producing staphylococci belong to the species S. aureus,
which is the species of greatest medical interest.
Of the several coagulase-negative species, only two will be considered here—S. epidermidis
and S. saprophyticus.
Although S. aureus is part of the commensal microbial flora of the nose (40% of healthy
adults are positive), skin, and intestinal tract, this species causes impetigo, boils, abscesses,
wound infection, infection of ulcers and burns, osteomyelitis, mastitis (breast abscess), pleural
empyema, pyomyositis, toxic shock syndrome, and other types of pyogenic infection. S.
epidermidis is also a common commensal of the skin, nose, and other mucous membranes and
possesses a very low pathogenicity. However, its presence in pus should not always be
dismissed as skin contamination. Despite its low infectivity, S. epidermidis can cause skin
infections at the site of an in-dwelling catheter, cannula, or other device. Infections with S.
epidermidis are particularly troublesome in cardiac and orthopaedic surgery involving the
insertion of prosthetic devices (artificial heart valves or artificial hips).
S. saprophyticus is recognized to be a common cause of urinary tract infections in young
women, being second only to E. coli in some populations.
The distinctive features of the three main species of Staphylococcus are given in Table ( 1).
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Table 12: Differentiation of medically important species of Staphylococcus
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Figure 25: A flow diagram for the preliminary identification of staphylococci
In view of the importance of the coagulase test in the identification of S. aureus, this test is
described here in detail. Coagulase is an enzyme that causes plasma to clot. Staphylococcal
coagulase exists in two forms: bound coagulase or clumping factor, which is demonstrated in
the slide test, and free coagulase, which is demonstrated in the tube test.
• Slide test. On a clean slide, emulsify one or a few similar colonies of staphylococci in a
drop of saline. The suspension must be fairly thick. Dip a straight wire into plasma and use
this to stir the bacterial suspension.
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Observe for clumping within 10 seconds. False-negative slide tests occur approximately 10%
of the strains of S. aureus. If the slide test is negative for an isolate that seems to be
pathogenic on other grounds (pigment, clinical source), it should be re-examined in the tube
test.
• Tube test. Dispense a few drops (0.5 ml) of plasma into a sterile 12 \ 75 mm tube, and add
two drops of the pure culture in broth. A suspension with an equivalent density may also be
prepared directly from growth on blood agar. Incubate the tube at 35 ∞C for 4–18 hours and
then examine for clotting.
The plasma used in the coagulase test may be fresh human or rabbit plasma obtained with
ethylenediamine tetraacetic acid (EDTA). It should be stored in the refrigerator in small
amounts (1 ml), and its performance checked with cultures of S. aureus and S. epidermidis,
run in parallel.
Antimicrobial susceptibility testing
Prudent use of antimicrobials according to local and national protocols is recommended.
Reporting Procedure
Culture
Report presence or absence of specific pathogens, also report results of supplementary
investigations:
Negatives
"Staphylococcus aureus NOT isolated".
"Lancefield group A streptococcus NOT isolated".
Positives
"Staphylococcus aureus isolated".
"Lancefield group A streptococcus isolated".
6.6 Smart Tests
6.6.1 Helicobacter pylori Serum/Whole Blood
The exact role that H. pylori plays in gastrointestinal disease still needs to be precisely defined
and is the subject of ongoing research. However, the prevalence rates for H. pylori infection as
demonstrated by histological and bacteriological methods can approach 90% in patients who
present clinical symptoms of the gastrointestinal diseases listed below (clinical significant). H.
pylori does not appear to invade the bloodstream since no isolates yet have been detected using
commercial blood culture methods. H. pylori infections occur in human populations throughout
the world. In developed countries, about 50% of the population may have H. pylori infection
by the age of 60 years, while only 10-20% of adults in the third decade of life have it.
In patients who present clinical symptoms relating to the gastrointestinal tract there are two
major methods of investigation: invasive and noninvasive. Invasive methods include culture of
gastric biopsy samples, histological examination of stained biopsy specimens, or direct
detection of the urease activity in the biopsy (CLO test). These methods need to obtain a biopsy
sample by endoscopy, which is expensive, and usually results discomfort and risk to the patient.
Noninvasive techniques include urea breath tests and serological methods. Urea breath test
requires the use of a small amount of radioactivity and a mass spectrometer. Serologic tests are
employed to detect antibodies as human immune response to H. pylori. Two methods appear to
be of great interest regarding their use in H. pylori routine serology, namely the ELISA and the
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Western immune blot because they offer the most versatility in regards to immunoglobulin
specificity and relative ease of use.
This H. pylori rapid test detects IgG antibodies specific to H. pylori infection in patient’s
blood or serum. It is a noninvasive method and does not use radioactive isotopes; the assay
procedures are easy and do not require professional training; it provides a rapid result. It is a
useful onsite aid in the diagnosis of H. pylori infection.
CLINICAL SIGNIFICANT
Helicobacter pylori has been associated with a variety of gastrointestinal diseases including
gastritis, duodenal and gastric ulcer, non-ulcer dyspepsia, gastric adenocarcinoma and
lymphoma.
PRINCIPLE OF THE TEST
This assay is a double antigen chromatographic lateral flow immunoassay. The test strip in
the device includes: 1) a burgundy-colored conjugate pad containing colloidal gold coupled
with H. pylori antigens, and 2) nitrocellulose membrane containing a test line (T line) and a
control line (C line). The T line is coated with H. pylori antigens, and the C line is coated with
goat anti-H. pylori antibody. The antigens used in this device are from H. pylori cell lysate.
When IgG antibodies specific to H. pylori are present in the specimen, the T line will become
a burgundy-colored band. If antibodies to H. pylori are not present or are present below the
detectable level, no T line will develop. The C line should always appear as a burgundycolored band regardless of the presence of antibodies to H. pylori. The C line serves as an
internal qualitative control of the test system to indicate that an adequate volume of specimen
has been applied and the flow occurred.
MATERIALS AND REAGENTS
• Test devices, each sealed in a pouch with a dropper pipette.
• 1 bottle of wash buffer-7 ml PBS diluents with 0.02% sodium azide as a preservative.
• 1 package inserts (Instruction for Use).
• Lancet or other blood collection device (Vacutainer blood collection tubes, sterile needles
and syringes).
• Timer.
Do not expose the kit to temperatures over 30°C (86°F).
SPECIMEN COLLECTION AND STORAGE
1- Serum
• Follow standard laboratory procedures to collect serum specimens.
• Serum specimens can be stored at (9-30) °C for 8 hours, at (2-8) oC for one week, and at (≤ 20) °C or lower for long term storage. Repeatedly frozen and thawed specimens are not
recommended for this assay.
• Any sediment in serum specimens should be removed by centrifugation. Avoid using any
turbid specimens, which may be contaminated by microorganisms.
2- Whole Blood
• Finger stick sampling is recommended for this assay.
• Middle or ring finger is the preferred puncture site.
• Clean patient’s finger with an alcohol swab. Wait until it is dry.
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• Puncture the fingertip with the lancet. Wipe away first sign of blood.
• Gently rub the hand from palm to finger to help form a drop of blood over the punctured
site.
• Use the provided pipette to pick up the blood, and apply one drop of the blood to the sample
well of the device. Then, follow the procedure.
PROCEDURE
1. Refrigerated specimens and other test materials, including devices, must be equilibrated
to room temperature before testing.
2. Remove the device from its wrap pouch prior to performing the assay, label the device with
identification.
3. Add one drop of fresh blood or serum to the sample well marked "S". Allow about 30
seconds for the specimen to be absorbed totally. Discard the first three drops of wash
buffer from the wash buffer squeeze bottle. Then add three drops of wash buffer into the
sample well.
4. Strong positive results may be observed in 2-3 minutes or depending on manufacturers.
Weak positive results may take a longer time, up to 7 minutes. For the whole blood test, a
slight hemolysis might be observed, but it does not interfere with the results.
INTERPRETATION OF RESULTS
IMPORTANT: Interpret the results depending on manufacturers. The T line should
always be interpreted independently of the C line.
Figure 26: Interpretation of Smart Test Results
Positive:
If both the C line and T line appear, the result indicates that the IgG antibodies specific to H.
pylori are detected and the result is positive.
A faint line in test region indicates a borderline specimen, which should be re-tested
using an alternative method for confirmation.
Negative:
If only the C line appears in the control region, the test indicates that no antibodies to H.
pylori are detected and the result is negative.
Invalid:
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When no control line appears within 5 minutes, repeat the test with a new test device.
QUALITY CONTROL PROCEDURE
• Built-in Control Features
This test contains a built-in quality control feature, the C line. The appearance of the
burgundy C line indicates that that an adequate volume of specimen and wash buffer has been
applied and the flow occurred.
• External Quality Control
External controls are recommended, positive and negative, to monitor the performance of the
assay.
6.6.2 Salmonella Typhi Antigen
Immunochromatographic rapid assay for the Detection of Salmonella Typhi Antigen in Human
Stool or Serum Specimens, Typhoid fever is a life threatening illness caused by the bacterium
Salmonella typhi, and was observed by Eberth (1880) in the mesenteric nodes and spleen of
fatal cases of typhoid fever. It is common in developing countries where it affects about 12.5
million persons annually. The infection is acquired typically by ingestion. On reaching the gut,
the bacilli attach themselves to the epithelial cells of the intestinal villi and penetrate to the
lamina and submucosa. They are then phagocytosed there by polymorphs and macrophages.
The ability to resist intracellular killing and to multiply within these cells is a measure of their
virulence. They enter the mesenteric lymph nodes, where they multiply and, via the thoracic
duct, enter the blood stream. A transient bacteremia follows, during which the bacilli are seeded
in the liver, gall bladder, spleen, bone marrow, lymph nodes and kidneys, where further
multiplication takes place. Towards the end of the incubation period, there occurs a massive
bacteremia from these sites, heralding the onset of the clinical symptoms. The diagnosis of
typhoid consists of isolation of the bacilli and the demonstration of antibodies. The isolation of
the bacilli is very time consuming and antibody detection is not very specific. Other tests
include the Widal reaction, has developed a test that takes only 10-20 minutes and requires only
a small quantity of stool or one drop of serum to perform. It is the easiest and most specific
method for detecting S. typhi infection.
PRINCIPLE OF THE TEST
Cortez One Step S. Typhi Antigen test is a qualitative one step immunochromatographic assay.
The test employs a conation of monoclonal antibody/colloidal gold dye conjugate and a
polyclonal antibody immobilized on the solid phase. This will selectively identify S. typhi
antigen associated S. typhi (typhoid) infection with a high degree of sensitivity and specificity.
As the specimen flows through the absorbent pad in the sample well and through the
antibody/colloidal gold complex any S. typhi antigen present in the sample binds to the
conjugate forming an antigen/antibody complex. The sample and dye complex continue to
migrate along the membrane to the immobilized polyclonal antibody. In the presence of S. typhi,
the polyclonal antibody captures the complex. This forms a visible pink/purple band in the (B)
or test area of the card. If no antigen is present, there is no line formation in the (B) area. The
remaining complex continues to migrate to another immobilized antibody on the membrane in
the (C) or Control area of the card, and is captured which then forms a band indicating proper
performance of the test.
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MATERIALS
Each kit contains:
1. Cortez S. typhi Antigen test. Each cassette contains a test strip with S. typhi specific antibody
on the test region of the membrane and colored S. typhi antibody-gold conjugate pad.
2. Fecal sample buffer – 2 bottles, 8 mL each.
3. Instruction for use.
4. Specimen collection container.
5. Timer.
SPECIMEN COLLECTION AND STORAGE
Cortez One Step S. typhi Antigen test can be run on stool or serum samples. The test works best
on fresh samples. If testing cannot be done immediately, they should be stored at (2-8)°C after
collection for up to 3 days. If testing cannot be done within 3 days, serum can be stored frozen
at (–20) °C or colder. Shipment of samples should comply with local regulations for transport
of etiologic agents.
Stool and serum specimens should be collected in containers that do not contain media,
preservatives, animal serum or detergents as any of these additives may interfere with the
Cortez S. typhi Antigen Test.
REAGENT PREPARATION
Bring all reagents, including test device, to room temperature (20-30) oC before use.
STOOL SPECIMEN PREPARATION
Add about 1/4 gram stool to approximately 500μl of the extraction reagent provided (about 12
drops from the dropper vial provided). Mix well and allow to sit for 5 minutes or so to allow
the large particles to settle.(amount of stool, extraction reagent and appropriate wetting time
depending on manufacturer).
Note: Watery or diarrhea specimens are inappropriate for testing.
PROCEDURE
1. Bring all materials and specimens to room temperature (8 – 30) °C.
2. Remove the test card from the sealed foil pouch.
3. For stool samples: use the provided pipette to transfer sample from the upper layer of the
stool extract and add 3 drops to the sample well (marked as “A”).
4. For serum samples: use the provided pipette to transfer the serum sample and add 3 drops
to the sample well (marked as “A”).
5. Read the result at 20 minutes. A strong positive sample may show test band earlier.
However, to confirm a result is negative, it must wait 20 minutes to read the results.
Note: The amount of S. typhi antigens present in serum is typically less than that in
stool. This may decrease the sensitivity of the test when using serum depending how
soon after the onset of the infection the test is performed. Early infection typically
exhibits greater levels of the antigen in the serum than in later infection. To confirm
serum results:
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The use of a stool sample is recommended if serum is used first and a negative result is
obtained and typhoid is still suspected, A second test run on a stool sample should be
performed.
INTERPRETATION OF RESULTS
Positive result: A distinct pink colored band appears on test line regions, in addition to a pink
line on the control line region.
Negative result: No line appears in the test line region. A distinct pink line shows on the
control line Region.
Invalid: The control line next to the test line does not become visible within 20 minutes after
the addition of the sample.
Figure 27: Interpretation of Salmonella Smart Test
QUALITY CONTROL
1. The control band is an internal reagent and procedural control. It will appear if the test has
been performed correctly and the reagents are reactive.
2. Good Laboratory Practice recommends the daily use of control materials to validate the
reliability of the device.
LIMITATIONS
1. The test is for qualitative detection of S. typhi antigen in stool or serum sample and does not
indicate the quantity of the antigens.
2. The test is for in vitro diagnostic use only.
3. For samples that test positive (reactive) by Cortez S. typhi Antigen Test, more specific
confirmatory testing should be done. A definitive clinical diagnosis should only be made by the
physician after all clinical and laboratory finding have been evaluated. The use of a rapid test
alone is not sufficient to diagnose S. typhi infection even if antigen is present. Also, a negative
result does not preclude the possibility of infection with S. typhi.
Specificity
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Negative stool samples from patients in areas where typhoid is relatively rare and would yield
a typical negative population, showed no false positives when the test was read within 20
minutes as specified.
Samples that were positive for s. paratyphi however were also negative as the antibodies used
in the S. typhi rapid test are specific for S. typhi only.
6.6.3 Vibrio Cholerae Rapid Visual Antigen Detection Test
Vibrio cholerae strains belonging to the O1 and O139 serogroups are capable of causing
epidemic and pandemic cholera. The O1 serogroup is subdivided into two serotypes, Ogawa
and Indaba. Serogroup O139, which appeared in India in 1992, has spread rapidly throughout
Asian countries and is considered to be the potential eighth pandemic strain of cholera. Prompt
diagnosis of cholera is of key importance to initiate effective therapy and to institute proper
epidemiological measures. There are definitive indications that the incidence of this serogroup
is on the rise in India and Bangladesh.
Several rapid diagnostic tests for cholera have been described. Some detect the cholera toxin.
The others detect the lipopolysaccharide (LPS) antigen of V. cholerae O1 or O139.
Recently, a multistep colloidal gold-based colorimetric immunoassay known as SMART was
also developed for direct detection of V. cholerae O1 or V. cholerae O139 in stool specimens
and has demonstrated 95% sensitivity and 100% specificity for O1 strains and 100% sensitivity
and 97% specificity for O139 strains.
Clinical Significant
Clinically, cholera may range from asymptomatic colonization to sever diarrhea with massive
fluid loss, leading to dehydration, electrolyte disturbances, and death.
Principle
This test is an immunochromatographic rapid visual antigen detection test for V. cholerae from
stool specimens. The nitrocellulose membrane is coated with monoclonal antibodies to V.
cholerae O1 and O139 lipopolysaccharide (LPS) as two distinct bands. When the test sample
migrates through the nitrocellulose membrane, colloidal gold particles coupled with anti-V.
cholerae O1 and O139 LPS monoclonal antibodies bind to the respective antigens from the test
sample, if present. This antigen-antibody complex moves through the nitrocellulose membrane
and binds to the corresponding immobilized antibodies against V. cholerae O1 and/or O139,
forming a magenta red color band, which confirms positive results. A control test band should
always appear, irrespective of a positive or negative test sample, to validate the test procedure.
Materials and equipment
In countries at risk for epidemics of cholera, the laboratory’s first role is to be prepared for a
possible epidemic. It must have ready or have access to the supplies and equipment necessary
to identify V. cholerae O1 and/or O139 even in remote locations. Contact the local laboratory,
medical center, or field site where testing will be performed in advance to ensure all necessary
supplies, equipment, and personal protective equipment (PPE) will be readily available.
Supplies and equipment necessary to perform the rapid test Included with the rapid test kit:


Individually packaged dipsticks in aluminum pouch with desiccant Plastic, individual,
single-use 5-mL test tubes for sample.
Disposable transfer pipettes (plastic droppers) for stool.
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







Foam test tube stand.
Sterile normal saline or distilled water Disposable, clean sample containers for stool
specimen collection.
Plastic rack of appropriate size for test tubes (suggested).
Watch or timer.
Biosafety supplies/personal protective equipment (PPE):Gloves Goggles or face shield
Lab coat.
Biohazard receptacle for infectious waste.
Disinfectant.
Storage
The rapid test kit may be stored at ambient temperature (24–26) oC but it is recommended that
the kit be stored at 4oC when not in use for 6 months or more (depending on manufacturer).
Specimen’s collection
Fecal specimens should be collected in the early stages of any enteric illness, when pathogens
are usually present in the stool in highest numbers, and before antibiotic therapy has started.
Collect stool specimens from persons who are suspected of being part of an outbreak and who
meet the following criteria:
1. Currently have “rice water stool.”
2. Had onset of illness less than 4 days before sampling.
3. Have not received antimicrobial treatment for the diarrheal illness.
Collect stools from patients in clean (no disinfectant or detergent residue) containers with
tight-fitting, leak-proof lids. Do not collect specimens from bedpans, as they may contain
residual disinfectant or other contaminants. Stool should be refrigerated if possible and
processed within a maximum of 2 hours after collection.
Suitable specimens:
Liquid fecal specimens, ideal “rice water stool”—clear, watery stools, likened to water from
boiled rice, an appearance classically seen in cholera cases.
Viscous, mucoid, or semisolid stools—acceptable but must be diluted before use with
normal sterile saline or sterile distilled water.
Unacceptable specimens:
Solid stools—not recommended, as particulate matter from these specimens may clog the
nitrocellulose membrane restricting migration of the sample.
Rectal swabs—the dipstick assay has not been evaluated for use with rectal swabs.
Procedure
1. Put on personal protective equipment (PPE): lab
coat, gloves, goggles and face shield. Wear PPE at all
times while handling the specimen
2. Collect stool sample in plastic cup. Label cup with
patient identifier.
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3. Remove 1 test tube from kit and place test tube in
Styrofoam or plastic rack. Label the test tube.
4. Remove 1 plastic dropper from kit.
5. Fill dropper halfway (150–200 µL) with stool
sample, and transfer to test tube. Do not put more
liquid than this amount in the test tube.
6. Open packet with rapid test, Label the dipstick
with patient identifier.
7. Place the dipstick in the test tube with the arrows
facing DOWN. The end of the strip should be
submerged in the stool. The arrows should remain
ABOVE the level of the stool.
8. Wait 15–20 minutes for the test to complete. When
complete, you will see a band near the top of the
dipstick (internal control band).
internal 
control band
9. Review and interpret results within 15–20 minutes:
A. Positive, V. cholerae O1 B. Positive, V. cholerae
O139 C. Positive, both V. cholerae O1 and O139 D.
Negative for V. cholerae O1 and O139 E. Test did
not work correctly (invalid test) Do not interpret
results after 30 minutes.
A
B
C
D
E
10. Place all waste in a double-lined plastic bag
labeled “biohazard.”
Figure28: Rapid Test Instruction Sheet, for a graphic representation of the test
procedure
1. Allow time for the kit to come to room temperature before opening and using.
2. Label a clean, unused test tube with the patient identification code. Transfer 150– 200 L of
the patient’s liquid stool specimen to this test tube.
Note: Viscous, mucoid, or semisolid specimens should be diluted with sterile saline or
distilled water before transfer to the test tube.
3. Tear open aluminum pouch at notched area. Remove the dipstick.
4. Label the dipstick with the patient identification code; take care to not touch the area
marked with an arrow (dipping area).
5. Carefully place the dipstick in the test tube with stool with the arrows facing DOWN. The
end of the strip should be submerged in the stool. Be sure the arrows remain ABOVE the
level of the stool.
6. Leave the dipstick undisturbed for 10–15 minutes or depending on manufacturers.
7. Within 15-20 minutes, a magenta red internal “control” band will appear indicating a valid
test; this line indicates that the reaction is complete. The presence of at least one or two
additional magenta red lines signifies the sample is positive for V. cholerae O1 and/or V.
cholerae O139.
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8. Read and record the result of the test within 15–20 minutes. Do not interpret results after 30
minutes. (Reading & interpretation the result depending on manufacturers).
Interpretation and reporting of results
Interpret test results of each dipstick according to the manufacturer’s instructions.
Guidelines for interpreting test results can be found in Table (1-1).
Record all patient test results in a worksheet or notebook as a permanent record. Record
results as positive, negative, or invalid. If a test needs to be repeated because of an invalid test
result, record the first result (invalid), resolve the problem, and record the repeated result.
Report positive and negative test results to the appropriate person in a timely manner. The
report should include any comments or information the person needs to properly evaluate the
test results.
Table 13: Expected Test Result Outcomes for the Dipstick and Interpretation of Test
Results
Dipstick Test Result
Interpretation
POSITIVE (Figures 3-1 and 3-2, column
A): Appearance of two bands, one for the
control test band and one test band specific
for V. cholerae O1 (Vc O1).
Indicative that the sample is positive only for V.
cholerae O1.
POSITIVE (Figures 3-1 and 3-2, column
B): Appearance of two bands, one for the
control test band and one test band specific
for V. cholerae O139 (Vc O139).
Indicative that the sample is positive only for V.
cholerae O139.
POSITIVE (Figures 3-1 and 3-2, column
C): Appearance of three bands, one for the
control test band, one test band specific for
V. cholerae O1 (Vc O1) and one test band
specific for V. cholerae O139 (Vc O139)
Indicative that the sample is positive for both V.
cholerae O1 and O139.
NEGATIVE (Figures 3-1 and 3-2, column
D): Appearance of only the control test
band.
Indicative that the sample is negative for V. cholerae
O1 and O139.
INVALID (Figures 3-1 and 3-2, column
E): Appearance of no control test band
Indicates a procedural error, deterioration of the
specimen/dipstick or the presence of particulate matter
in the specimen preventing flow of fluid through the
nitrocellulose membrane.
Quality control
Common source of error
Specimens that are not classified as “watery” may clog the nitrocellulose
membrane strip, limit migration of the liquid, and prevent the dipstick
reaction resulting in an invalid test.
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These specimens should be either diluted with sterile saline or distilled
water or rejected as unacceptable specimens for testing.
A
B
C
D
E
Figure 29:Visual Interpretation of Rapid Dipstick Test Results
Precautions
Be sure to run the test exactly as instructed, adhering to all procedures. Use sterile technique
throughout the test procedure. Wear gloves, goggles and face shield, and a lab coat when
handling and testing all clinical specimens.
6.7 Preparation of Media
With just a few diagnostic materials, a laboratory can make an important contribution to
individual patient care through accurate etiological diagnosis.
In most developing countries bacteriological laboratory practice is hampered by a shortage of
culture media and basic reagents, which are very costly to import. However, the number of
culture media and reagents that have to be purchased can be reduced to the essential ones,
through rational selection, as has been the case with essential drug lists. Additionally, some
simple media
and reagents can be produced or prepared locally.
Each batch of medium prepared from individual ingredients or each different manufacturer’s
lot number of commercial dehydrated medium and each batch of medium prepared from
individual ingredients should be quality controlled before use. Immediately after preparation,
each medium should be tested, as appropriate, with a reference strain .
for proper growth characteristics as described for each medium.
A record of all media preparation or purchase dates and quality control test results should be
kept, and any unusual characteristic (e.g., the color of the medium or slow growth of test
bacteria) should be noted. Many media call for the use of defibrinated blood.
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Agar media should be dispensed into 15 x 100-mm or 15 x 150-mm Petri dishes to a uniform
depth of 3–4 mm; approximately 20-ml of liquid agar medium will achieve this depth in a 15
x 100-mm plate. If agar is cooled to 50°C prior to pouring, condensation is minimized. After
pouring, the plates should be kept at room temperature for several hours to prevent excess
condensation from forming on the covers of the dishes. Another means by which
condensation will be reduced is if plates are stacked so that they cool more slowly.
Alternatively, if when
preparing selective media (e.g., MacConkey [MAC], xylose lysine desoxycholate [XLD],
thiosulfate citrate bile salts [TCBS] agar, etc.), conditions are such that there is little chance
that the cooling media will be contaminated, after the agar is poured into the plates, the lids
can be placed on the dish so that a small opening is left to let the heat out, resulting in the
formation of less condensation on the upper lid; the lid should remain slightly open like this
for approximately 30 minutes,
while the agar solidifies. If, however, it is likely that the agar will be contaminated if the lid is
left partly open, the agar should be allowed to solidify with the lid closed.
Note: Covering the agar while it is still hot will allow for the formation of a substantial
amount of condensation on the upper lid. If the plates contain condensation, the plates should
be covered at room temperature for 24 hours to allow the condensation to evaporate. After
condensation has evaporated, the plates should be placed in an inverted position and stored in
a plastic bag in an inverted position at 4°C.
6.7.1 Blood agar: TSA with 5% sheep blood
Sheep blood agar is used as a general blood agar medium, and consists of TSA plus 5%
sheep’s blood. The sheep blood agar plate should appear a bright red color. If the plates
appear dark red, the blood has been added when the agar was too hot; if this happens, the
medium should be discarded and a new batch prepared.
A. Prepare TSA according to the instructions given on the label of the dehydrated
powder. For convenience, 500 ml of molten agar can be prepared in a l-liter flask.
Add 20 g of agar into 500 ml of water. Heat to dissolve.
B. Autoclave at 121°C for 20 minutes. Cool to 60°C.
C. Add 5% sterile, defibrinated sheep blood (i.e., add 25 ml sheep blood to 500 ml of
agar). If a different volume of basal medium is prepared, the amount of blood added
must be adjusted accordingly to 5% (e.g., 50 ml of blood per liter of medium).
D. Dispense 20 ml into 15 x 100-mm Petri dishes. Allow the medium to solidify and dry
out, place in a plastic bag, and store at 4°C.
Quality control: Test each new, freshly prepared or purchased batch of blood agar plates for
growth and hemolytic reaction with a strain of S. pneumoniae. The colonies are small and
should appear grey to grey-green surrounded by a distinct greenish halo in the agar.
6.7.2 Chocolate agar with TSA base and growth supplement
Chocolate agar with growth supplements is a medium that supports the special growth
requirements needed for the isolation of fastidious organisms (when incubated in a 5% CO2
atmosphere). Chocolate agar contains a reduced concentration of agar, which increases the
moisture content of the medium.
Supplemented chocolate agar should support the growth of H. influenzae.
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Chocolate agar slants for transport and short-term storage can be prepared in the same manner
as that described for agar plates, except that the medium is dispensed in 16 x 125-mm screwcap tubes and slanted before solidifying.
A. Use TSA as the basal medium. Prepare double strength (i.e., 20 g in 250 ml distilled
water). Autoclave and then cool to 50°C. Use the thermometer to verify the cooling
temperature.
B. Prepare a solution of 2% hemoglobin (i.e., 5 g in 250 ml distilled water). Mix the
hemoglobin in 5–6 ml of the distilled water to form a smooth paste. Continue mixing
as the rest of the water is added. Autoclave, and cool to 50°C.
C. Add the hemoglobin solution to the double-strength TSA and continue to hold at
50°C.
D. • Alternative to steps a-c: If a hemoglobin solution is unavailable, an alternative is to
add 5% sterile defibrinated sheep, rabbit, guinea pig, or horse blood (i.e., 5 ml blood
per 100-ml agar) to full-strength TSA (i.e., 20 g in 500 ml distilled water). DO NOT
use human blood. After the base medium has been autoclaved and cooled to 50°C,
add the blood and place in
E. a hot water bath at no more than 80°C for 15 minutes or until a chocolate color is
achieved. Then cool to 50°C.
F. After the hemoglobin solution or the defibrinated blood has been added to the base
medium and the medium has cooled to 50°C, add growth supplement (e.g.,
IsoVitaleX or Vitox) to a final concentration of 1%.Mix the ingredients by gently
swirling the flask; avoid forming bubbles. Dispense 15–20 ml in each 15 x 100-mm
Petri dish.
Quality control: All freshly prepared or purchased chocolate agar media should be tested to
determine the medium’s capacity to support growth of the bacteria to beisolated, particularly
H. influenzae. If the medium does not support the growth ofone or all of the bacteria, the
medium should be discarded, and a new batch of medium should be prepared or purchased.
• Chocolate agar should look brown to brownish-red in color. N. meningitidis
and H. influenzae should appear as a greyish, almost translucent film on the slant’s surface
with no discoloring of the medium after 24 hours of incubation; S. pneumoniae should appear
as small grey to grey-green colonies with a very distinct greenish discoloring of the medium .
If H. influenzae does not grow, the growth supplement (IsoVitaleX or its equivalent)
may have been inadvertently omitted.C
6.7.3 MacConkey agar (MAC)
MacConkey agar (MAC) is a differential plating medium recommended for use in the
isolation and differentiation of lactose-nonfermenting, gram-negative enteric bacteria from
lactose- ermenting organisms. Colonies of Shigella on MAC appear as convex, colorless
colonies about 2–3 mm in diameter. S. dysenteriae 1 colonies may be smaller. S. Typhi
colonies are flat, colorless and usually 2–3 mm in diameter. Several commercial brands of
MAC are available.Most manufacturers prepare several formulations of MAC, which may
vary in selectivity and thereby affect the isolation of Shigella. For example, some
formulations of MAC do not contain
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crystal violet, a selective agent; these types are not as selective and should not be used for
isolation of Shigella. Oxoid MacConkey Agar No. 3, Difco Bacto MacConkey Agar, and
BBL MacConkey Agar are all suitable.
A. Prepare MAC according to manufacturer’s instructions. [Note: MAC can also be
prepared from individual ingredients, but this produces more lot-to-lot variation than
preparation of a commercially available dehydrated formulation.]
B. Sterilize the medium by autoclaving at 121°C for 15 minutes.
C. Cool to 50°C and pour into Petri plates (to a uniform depth of 3–4 mm).
D. Leave lids ajar for about 20 minutes so that the surface of the agar will dry. Close lids
and store at 4°C for up to 1 month. If plates are to be stored for more than a few days,
put them in a sealed plastic bag to prevent drying.
Quality control: For quality control of MAC, the following organisms should be adequate for
confirmation of selective and inhibitory growth characteristics:
• E. coli should produce pink to red colonies with good to excellent growth; and,
• S. flexneri should produce colorless colonies with fair to good growth, but S. dysenteriae 1
colonies may be smaller.
6.7.4 Mueller-Hinton agar
Mueller-Hinton agar is the NCCLS-recommended medium used for standardized
antimicrobial susceptibility testing of certain bacteria; the organisms in this document for
which it is appropriate to use this formulation of Mueller-Hinton medium (i.e.,
unsupplemented Mueller-Hinton) are S. Typhi, Shigella spp., and V. cholerae.
[Note: Several formulations of Mueller-Hinton agar are commercially available.
This laboratory manual suggests that Mueller-Hinton agar medium should not be prepared
from individual ingredients because this can diminish the quality. Commercial dehydrated
Mueller-Hinton is carefully quality controlled before
being released for sale.]
A. Follow manufacturer’s instructions to prepare medium.
B. After autoclaving, cool medium to 50°C in a water bath.
C. Measure 60–70 ml of medium per plate into 15 x 150-mm plates, or measure 25–30
ml per plate into 15 x 100-mm plates. Agar should be poured into flatbottom glass or
plastic Petri dishes on a level pouring surface to a uniform depth of 3–4 mm. Using
more or less agar will affect the susceptibility results. Agar deeper than 4 mm may
cause false-resistance results, whereas agar less than 4 mm deep may be associated
with a false-susceptibility report.
D. Freshly prepared plates may be used the same day or stored in a refrigerator (at 2°–
8°C) for up to 2 weeks. If plates are not used within 7 days of preparation, they
should be wrapped in plastic to minimize evaporation. Just before use, if excess
moisture is on the surface, plates should be placed in an incubator (35°–37°C) until
the moisture evaporates (usually 10–30 min).
Do not leave lids ajar because the medium is easily contaminated.
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Quality control: Each new lot of Mueller-Hinton agar should be quality controlled before use
by testing the E. coli ATCC 25922 standard strain for antimicrobial susceptibility testing.
(This formulation of Mueller-Hinton agar can also be used for testing of gram-positive
aerobes, in which case S. aureus ATCC 25923 can be used as a quality control strain.) The
pH of each new lot of Mueller-Hinton should be between 7.2 and 7.4; if the pH is outside
this range, the pH of the medium should not be adjusted by the addition of acid or base, i.e.,
the batch of Mueller-Hinton plates should be discarded and a new batch of plates
prepared. If the pH for every batch is too high or low, the entire lot of dehydrated medium
may have to be returned to the manufacturer as unsatisfactory. Inhibition zone sizes / minimal
inhibitory concentration (MIC) values for quality control are included in the antimicrobial
susceptibility testing section of each pathogenspecific chapter.
6.7.5 Cary-Blair medium, Amies medium, and Stuarts medium
Prepare each of these transport media according to the manufacturer’s instructions. [Note:
Several dehydrated formulations of Cary-Blair are commercially available; some require the
addition of calcium chloride and some do not.] These media can also be prepared from
individual ingredients; however, it is very difficult to make a well quality-controlled batch
and so this manual recommends purchasing them from a manufacturer. When the Cary-Blair
medium is prepared, it should be dispensed into containers in sufficient volume so that swabs
will be covered by at least 4 cm of medium. For example, 5- to 6-ml amounts may be
dispensed into 13 x 100-mm screw cap tubes. With the caps loosened, sterilize the medium by
steaming (not by autoclave) at 100°C for 15 minutes. Tighten the caps after sterilization, and
store the medium at 15°–30°C.
These media are quite stable if stored in tightly sealed containers in a cool dark place so that
the medium does not dry out. Each may be used for up to 1 year as long as no loss of volume,
visible contamination (e.g., foreign objects or bacterial growth), or color change is observed.
Prepared Amies medium that has been stored for longer than 9 months, however, should be
freshly steamed and the charcoal re-suspended before use.
6.8 Staining Smears
6.8.1 Gram’s Stain
Organisms are classified according to their Gram staining reaction. Gram positive bacteria
have thicker and denser peptidoglycan layers in their cell walls. Iodine penetrates the cell wall
in these bacteria and alters the blue dye to inhibit its diffusion through the cell wall during
decolourisation. Gram positive bacteria must have an intact cell wall to produce a positive
reaction. Gram negative cells which do not retain the methyl/crystal violet are stained by a
counterstain26. Neutral red, safranin or carbol fuchsin may be used as the counterstain.
Gram ’s Method for examination of smears
1. Prepare a smear and heat gently to fix.
2. Flood the slide with 0.5% methyl/crystal violet and leave for 30 s.
3. Tilt the slide, pour on sufficient (1%) Lugol’s iodine to wash away the stain, cover
with fresh iodine and allow to act for 30 s.
4. Tilt the slide and wash off the iodine with 95 - 100% ethanol or acetone until colour
ceases to run out of the smear.
5. Rinse with water.
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6. Pour on 0.1% counterstain (neutral red, safranin or carbol fuchsin) and leave to act
for about 2 min.
7. Wash with water and blot dry.
Interpretation
Positive Result
Gram positive organisms stain deep blue/purple.
Negative Result
Gram negative organisms stain pink/red.
Quality Control Organisms
A culture containing Gram positive and Gram negative organisms may be used for quality
control.
6.8.2 Ziehl-Neelsen’s stain (for acid fast bacilli)
This staining technique is used to demonstrate the presence of acid and alcohol fast bacilli
(AAFB) which have waxy envelopes that make them difficult to stain and decolourise.
Auramine-phenol staining is more sensitive than Ziehl-Neelsen’s and is thus more suitable for
assessment of smears from clinical specimens. Ziehl-Neelsen’s staining provides
morphological details and is more useful for confirming the presence of AAFB in positive
cultures.
Method
1. Flood the slide with strong carbol fuschin.
2. Heat gently, and once slide is just ‘‘steaming’’ leave for 3-5 min.
3. Rinse well with water.
4. Decolourise for 2-3 min with a (3% v/v) acid-alcohol solution, rinse with water, then
replace with fresh acid-alcohol for 3-4 min until the slide remains a faint pink colour.
5. Rinse well with water.
6. Counter stain with (1% w/v) methylene blue or malachite green for 30 s.
7. Rinse with water and allow to dry.
8. Apply immersion oil and read with a transmitted light microscope.
Note: Follow manufacturer’s procedure if commercial kits are used.
Interpretation
Positive Result
Acid fast bacilli vary from 0.5-10 μm in length16 and stain red. Some may appear beaded.
Negative Result
All other organisms and background material stain green if malachite green counterstain is
used or blue if methylene blue counterstain is used.
Quality control
Positive Control
Mycobacterium species.
Negative Control: A proven negative smear may be used as the negative control.
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7 Parasitology
7.1 Malaria parasite diagnosis
Malaria is a serious, sometimes fatal disease resulting from infection with Plasmodium spp.
transmitted by the bite of Anopheline mosquitoes.. The clinical diagnosis, where malaria is
suspected based on the history, symptoms and clinical findings must always be confirmed by
a laboratory diagnosis.
Laboratory diagnosis of malaria involves identification of malaria parasite or its
antigens/products in the blood of the patient.
Clinical significance
Malaria parasites are usually detected in blood films stained with Field or Geimsa
stains. They may also be detected using an immunological procedure known as a
dipstick test
It is important for the prognosis and treatment of the disease that the species involved
are identified in the laboratory.
Preparation of a thick and a thin blood film on the same slide
There are four species of the genus plasmodium responsible for the malarial parasite
infections that commonly infect man, P.falciparum, P.vivax, P.malariae and
P.ovale. The most important of these is P.falciparum because it can be rapidly fatal
and is responsible for the majority of malaria related deaths
Clinical significance
For routine malaria microscopy, a thin and a thick film are made on the same slide. The thick
film is used for the detection of parasites, while the thin film is used in identifying the species
of parasite
Principal
Blood to be examined for malaria parasites is usually collected at a health centre.
The most suitable time for collection is at the height of an episode of fever, when the parasites
are most numerous in the blood. Blood specimens should always be collected before antimalarial drugs are given Cleaning the finger before collecting a capillary bloodsample
Using a lancet to puncture the ball of the finger
METHOD
1. With the patient’s left hand palm upwards, select the third or fourth finger. (The big toe can
be used with infants. The thumb should never be used for adults or children.) Use cotton wool
lightly soaked in ethanol to clean the finger — using firm strokes to remove dirt and grease
from the ball of the finger, Dry the finger with a clean piece of cotton wool (or lint).
2. With a sterile lancet, puncture the ball of the finger, using a quick rolling action. By
applying gentle pressure to the finger, express the first drop of blood and wipe it away with
dry cotton wool. Make sure that no strands of cotton wool remain on the finger
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3. Working quickly and handling clean slides only by the edges, collect the blood as follows:


Apply gentle pressure to the finger and collect a single small drop of blood,
about this size _, on to the middle of the slide. This is for the thin film.
Apply further pressure to express more blood and collect two or three larger
drops, about this size _, on to the slide about 1 cm from the drop intended for
the thin film Wipe the remaining blood away with cotton wool.
Thin film: Using another clean slide as a “spreader”, and with the slide with the blood
drops resting on a flat, firm surface, touch the small drop with the spreader and allow
the blood to run along its edge. Firmly push the spreader along the slide, away from
the largest drops, keeping the spreader at an angle of 45° Make sure that the spreader
is in even contact with the surface of the slide all the time the blood is being spread.
Thick film: Always handle slides by the edges, or by a corner, to make the thick film
as follows: Using the corner of the spreader, quickly join the larger drops of blood and
spread them to make an even thick film, allow the thick film to dry in a flat, level
position protected from flies, dust and extreme heat. Label the dry film with a grease
pencil by writing across the thicker portion of the thin film the patient’s name or
number and date.
Materials and reagents
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Microscope
Clean glass microscope slides
Sterile blood lancets
Cotton wool
Grease pencil
Methanol
70% Ethanol.
Measuring cylinders, 10, 50 and 100ml
Beakers, 50 and 250ml
Staining troughs
Glass rods
Wash bottle
Slide forceps
Slide racks
Timer
Giemsa stain
Methanol in a drop bottle
Buffered water, pH 7.2 or distilled water
Staining blood films with Giemsa stain
Principle
During staining of the blood film, the haemoglobin in the erythrocytes dissolves(de
haemoglobin ization) and is removed by the water in the staining solution. All that remain are
the parasites and the leukocytes, which can be seen under the microscope.
142 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
First method: Rapid method for staining thick and thin blood films
This method is suitable for rapid staining of thick films when urgent results are required. It
uses much more stain than the regular method
1. Using forceps, place the slides back to back in a staining trough
2- Prepare a 3% Giemsa solution in buffered or distilled water, pH 7.2, in sufficient quantity
to fill the number of staining troughs being used, mix the stain well.
3- Pour the stain gently into the staining trough, until all the slides are totally covered. Stain
for 30–45 minutes out of sunlight.
4. Pour clean water gently into the trough to remove the deposit on the surface of the staining
solution
5. Gently pour off the remaining stain), and rinse again in clean water for a few seconds. Pour
the water off.
Second method
1. Allow the thick film to dry thoroughly; if results are required urgently, drying may be
hastened by fanning, or briefly exposing the slide to gentle heat such as that from a
microscope lamp. Care should be taken to avoid overheating, otherwise the thick film will be
heat-fixed
2. Fix the thin film by adding three drops of methanol, or by dipping it into a container of
ethanol for a few seconds. To permit dehaemoglobinization, the thick film should not be
fixed; therefore avoid exposure of the thick film to methanol or methanol vapour
3. Prepare a 10% Giemsa solution in buffered or distilled water, pH 7.2; if a small quantity is
being used, three drops of stain per ml of buffered water will give the correct concentration of
Giemsa solution. One slide requires about 3ml of madeup stain. Mix the stain well with a
glass rod.
4. Gently pour the stain on to the slides or use a pipette. Stain for 5–10 minutes.
5. Gently flush the stain off the slides by adding drops of clean water. Do not tip off the stain
and then wash, as this will leave a deposit of scum over the smears.
6. Place the slides in the slide rack to drain and dry, film side downwards; making sure that
the film does not touch the slide rack.
Microscopic examination
Examine the slide under the microscope using the ¥100 objective. Malaria parasites found in
the blood are at different stages of development Some malaria parasites have granules of
pigments in their cytoplasm
Thin blood films: In thin blood films, the infected erythrocytes may remain unchanged
or have a different colors or shape, or may contain pink (“Schüffner’s”) or red
(“James”) thin films can be used to identify the species of malaria parasite
Note: In patients who have been suffering from malaria for a long time, monocytes
may be seen in the thin blood film; the cytoplasm often contains brown or greenish
black bodies (siderophils). In patients who have recently received an injection of an
antimalarial drug, the parasites stain poorly and appear distorted and indistinct.
Thick blood films: In thick blood films, the background should be clean and free from
debris, as the infected erythrocytes are lysed. The malaria parasites should have deep red
143 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
chromatin and blue or pale purplish-blue cytoplasm. In thick films stained with Giemsa, the
nuclei of leukocytes should be stained dark purple. Schüffner’s dots may be seen around the
malaria parasites. Thick blood films are used for estimating the parasite density, as described
below.
Reporting results
If the result of the examination of the stained blood films is positive, specify:



the species of parasite found
the stage of development of the parasite
the parasite density.
Blood films containing P. ovale and P. vivax may contain few parasites and therefore
take more time to examine under the microscope. However, it is necessary to
differentiate the two species, since they may reappear in the blood without reinfection.
Patients infected with P. ovale or P. vivax require additional treatment to eradicate
the liver stages of these parasites.
A patient can harbour more than one species of malaria parasite at the same time(e.g.
P. falciparum and P. malariae or P. falciparum and P. vivax). If the result is negative,
report as “no parasites found”.
144 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 30: P. Falciparum
1: Normal red cell , 2-18:Trophozoites (among these) , 2-10:correspond to ring stage
trophozoites , 19-26:Schizonts (26 is ruptured schizont), 27&28: Mature macrogametocytes ,
29&30:Mature microgametocytes (male)
145 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 31: P. Ovale
1: Normal red cell , 2-5: Young trophozoites (Rings) , 6-15:Trophozoites , 1623:Schizonts , 24:Macrogametocytes( female) , 25: Microgametocytes (male)
146 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Figure 32: P. Vivax
1: Normal red cell , 2-6: Young trophozoites (Ring stage parasites) , 718:Trophozoites , 19-27:Schizonts , 28&29:Macrogametocytes( female) , 30:
Microgametocytes (male)
147 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
8. Biochemistry
8.1 Blood glucose
Glucose is the chief source of energy in the body. The levels of this compound are balanced
by digestion and absorption of carbohydrates in the intestine, its storage and release in the
liver and its utilisation in the muscle.
Causes of Hyperglycemia:
 Insulin – dependent type (IDDM)
 Non – insulin dependent type (NIDDM)
 non obese NIDDM
 obese NIDDM
 Gestational diabetes
 Impaired glucose tolerance: Glucose intolerance associated with certain conditions
and syndrome such as (chronic pancreatitis with endocrine and exocrine
insufficiency, cushing syndrome , acromegaly )
Causes of hypoglycemia:
 Deficient glucose production
 Ketotic hypoglycemia
 Glycogen storage disorder
 Deficiency of gluconeogenic enzyme
 Galactosemia
 Hereditary fructose intolerance
 Leucine hypersensitivity
 Endogenous hyperinsulinism
Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The
hydrogen peroxide formed reacts, under catalysis of peroxidase, with phenol and 4aminophenazone to form a red-violet quinoneimine dye. The intensity of the colour produced
is directly proportional to the concentration of glucose in the sample.
Glucose + O2+H2O --------------GOD
H2O2 + Gluconate
2H2O2+ Phenol+ 4-Amino-antipyrine
H2O+ Quinonimine
SPECIMEN
Serum, heparin plasma, or fluoride plasma may be used. Plasma or serum samples without
preservatives should be separated from the cells or clot within a half hour of being drawn.
Glucose in separated, un-hemolyzed serum is stable up to four hours at 25°C and up to 24
hours at 4°C.
REAGENTS AND MATERIALS
1- Full automated auto analyzer or Spectrophotometer
2- Centrifuge
3- Gel tube (clot activator)
4- Spectrophotometer cuvettes
5- Deionized water
6- Pipettes
7- Glucose standards 20 mg/dL 100 mg/dL, 200 mg/dL, 400 mg/dL
8- Heating block or water bath 37°C
9- Timer
148 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
10- Reagents :
Reagent 1
Buffer solution
Reagent 2
tris buffer pH 7
Phenol
glucose oxidase
Peroxidase
4-amino-antipyrine
Reagent 3
standard glucose
Preparation: Dissolve the powder R2 with the Buffer R1
100 mmol/l
0.3 mmol
10 000 u/l
1 000 u/l
2.6 mmol/l
100 mg/dl
PROCEDURE


Wave length ………… 505 nm ( 490 – 550)
Temperature………….37 ċ
Blank
Standard
Sample
Standard
10 µl
Sample
10µl
Working reagent
1 ml
1ml
1ml
 Mix incubate 10 minutes at 37 ċ or 30 minutes at room temperature ( 20 ċ – 30 ċ )
 The color is stable 30 minutes
 Linearity : up to 500 mg / dl
Calculation:
Glucose concentration =O. D. sample / O.D. standard * concentration of standard .
QUALITY CONTROL
Level one and level two serum controls are tested with each patient run. The level one control
range is 70-85 mg/dL and the level two range is 271-306 mg/dL.
RESULTS
Using graph paper, plot the Absorbance on the vertical (y axis) against the concentration on
the horizontal (x axis) for each of the glucose standards.
1- Draw a "best fit line" and use this standard curve to determine the glucose
concentration for the controls and patient specimens.
2- Verify that the control results are acceptable before reporting patient results.
REFERENCE INTERVALS
The reference range for glucose is as follows:
Cord
45-96 mg/dL
Premature 20-60 mg/dL
Newborn
40-60 mg/dL
1 wk
50-80 mg/dL
Child
60-100 mg/dL
149 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Adult
74-100 mg/dL
>60 yr
82-115 mg/dL
>90 yr
75-121 mg/dL
LIMITATIONS
1- Serum and plasma must be separated from the red blood cells promptly to prevent
glycolysis. Glucose will decrease approximately 7% per hour when left in contact
with red cells.
2- Whole blood glucose is 12-15% less than serum glucose.
3- Venous blood glucose is approximately 5 mg/dL less than arterial or capillary blood
glucose.
8.2 Liver Function Test
8.2.1 Alanine Aminotransferase (ALT)
Is found mainly in the liver, but also in smaller amounts in the kidneys , heart , muscles,
and pancreas.
ALT is measured to see if the liver is damaged or diseased. Low levels of ALT are normally
found in the blood. But when the liver is damaged or diseased, it releases ALT into the
bloodstream, which makes ALT levels go up. Most increases in ALT levels are caused by
liver damage.
The ALT test is often done along with other tests that check for liver damage, including
aspartate aminotransferase (AST), alkaline phosphatase, lactate dehydrogenase (LDH),
and bilirubin. Both ALT and AST levels are reliable tests for liver damage.
Causes of high ALT:
 Alcohol abuse
 Medications
 Acute and Chronic hepatitis
Principle:
Kinetic determination of ALT activity , method recommended by IFCC
2-oxoglutarate +l-alanine-----ALT-------- glutamate + pyruvate
Pyruvate + NADH + H+-----------LDH------- l-lactate + NAD+
The rate of NADH consumption is determined photometrically and is direct proportional to
the ALT activity in the sample
SPECIMEN
A. Recommended specimen: 100 μl of serum or heparinised plasma or EDTA plasma.
Collect specimens by standard venipuncture technique. Heparin may be used as an
anticoagulant for plasma specimens. Handle specimens in stoppered containers to
avoid contamination and evaporation. Follow universal precautions when performing
phlebotomy or handling patient specimens, calibrators, or other serum-based
products. Discard contaminated materials with infectious waste.
150 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
B. Remove serum promptly from the clot. Haemolysed specimens should not be used
because ALT contamination from red cells will occur. DO NOT freeze the sample;
this will cause a loss of ALT activity.
C. If concentration is greater than the analyser range, dilute with an equal volume of
isotonic saline and re-analyses. Multiply the result by 2 to obtain the original ALT
activity.
REAGENTS AND MATERIALS
A. full automated autoanalyzer or spectrophotometer
B. Centrifuge
C. Gel tube (clot activator)
D. Reagents :
Reagent 1
Buffer reagent
Reagent 2
Substrate
tris buffer P H 7.5
L-alalnine
NADH
LDH
Oxoglutarate
100mmol/l
500 mmol/l
0.18 mmol/l
1200u/l
15 mmol/l
Preparation:
Dissolve one vial of reagent 2 with 3 ml of R 1 buffer, this working reagent stable 13 days at
2 – 8 ċ or 24 hours at 20 – 25 days.
Procedure:
 Wave length ----------340 nm
 Working reagent 1 ml mixed with sample 100 µl and wait 1 minute
 Measure the extinction decrease per minute for 1 – 3 minutes
Calculation :
Δ OD/min * 1750 = u/l
QUALITY CONTROL
1. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each day. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
2. Variability is expressed as standard deviation (SD) and coefficient of variation CV)
and plotted on a Levy-Jennings graph.
Reference value:
Women up to 31 u/l at 37 ċ
Men up to 40 u/l at 37 ċ
RESULTS
1- Panic values: are double or triple to the normal value.
2- Becareful . Reference value for the end point procedure is differed from the reference
value for the kinetic procedure.
LIMITATIONS
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A. High total protein samples that are predominantly gamma globulins can increase ALT
results. The sample should be diluted with an equal volume of isotonic saline and
then reanalysed.
B. In sera with very high activities, the initial absorbance may be very low because most
of the NADH may have been consumed before the first readings. In this case, return
the sample after dilution as described above.
8.2.2 Aspartate Aminotransferase (AST)
AST (Aspartate transaminase ): AST is normally found in red blood cells, liver, heart,
muscle tissue, pancreas, and kidneys. AST formerly was called serum glutamic oxaloacetic
transaminase.
Low levels of AST are normally found in the blood. When body tissue or an organ such as the
heart or liver is diseased or damaged, additional AST is released into the bloodstream. The
amount of AST in the blood is directly related to the extent of the tissue damage. After severe
damage, AST levels rise in 6 to 10 hours and remain high for about 4 days.
The AST test may be done at the same time as a test for alanine aminotransferase, or ALT.
The ratio of AST to ALT sometimes can help determine whether the liver or another organ
has been damaged. Both ALT and AST levels can test for liver damage.
Causes of increase AST:
 Alcohol abuse .
 viral hepatitis,
 acute MI
Principle:
Kinetic determination of ALT activity , method recommended by IFCC
2-oxoglutarate +l-aspartate-----AST-------- glutamate + oxaloacetate
oxaloacetate + NADH + H+-----------MDH------- malate + NAD+
The rate of NADH consumption is determined photometrically and is direct proportional to
the ALT activity in the sample.
SPECIMEN
A. Recommended specimen: 100 μl of serum or heparinised plasma or EDTA plasma.
Collect specimens by standard venipuncture technique. Heparin may be used as an
anticoagulant for plasma specimens. Handle specimens in stoppered containers to
avoid contamination and evaporation. Follow universal precautions when performing
phlebotomy or handling patient specimens, calibrators, or other serum-based
products. Discard contaminated materials with infectious waste.
B. Remove serum promptly from the clot. Haemolysed specimens should not be used
because AST contamination from red blood cells will occur. Refrigerate specimens if
analysis is not performed immediately. Freeze specimens if analysis is not performed
within 48 hours.
C. If concentration is greater than the analyser range, dilute with an equal volume of
isotonic saline and reanalyse. Multiply the result by 2 to obtain the original AST
activity.
REAGENTS AND MATERIALS
1- full automated auto analyzer or spectrophotometer
2- Gel tube (clot activator)
152 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
3- Centrifuge
4- Reagents :
Reagent 1
Buffer reagent
Reagent 2
Substrate
tris buffer P H 7.8
L-aspartate
NADH
LDH
Oxoglutarate
MDH
80mmol/l
200 mmol/l
0.18 mmol/l
800u/l
12 mmol/l
600 u/l
Preparation:
Dissolve one vial of reagent 2 with 3 ml of R 1 buffer , this working reagent stable 21 days at
2 – 8 ċ or 24 hours at 20 – 25 days .
Procedure:
 Wave length ----------340 nm
 Working reagent 1 ml mixed with sample 100 µl and wait 1 minute
 Measure the extinction decrease per minute for 1 – 3 minutes
Calculation:
Δ OD/min * 1750 = u/l
Reference value:
 Women up to 31 u/l at 37 ċ
 Men up to 38 u/l at 37 ċ
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
LIMITATIONS
A. High total protein samples that are predominantly gamma globulins can increase AST
results. The sample should be diluted with an equal volume of isotonic saline and
reanalysed. Highly active sera can have a very low initial absorbance because much
of the NADH is already consumed prior to measurement. In such cases, dilute sample
as specified.
B. Analyser range: ≤ 150 U/L
8.2.3 Alkaline Phosphatase
Alkaline phosphatase is an enzyme used by the cells to speed up the time of a reaction. It is
found in the bone, placenta, kidneys, small intestines and liver, but is primarily associated
with liver disease and the obstruction of bile flow. Within the liver, alkaline phosphatase is
located near or within the tubes that collect bile.
Principle:
Phenyl phosphate-----------ALP-------- phenol + phosphate
Free phenol liberate by hydrolysis of substrate reacts then with 4-amino-antipyrine in the
presence of alkaline.
153 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Potassium ferricyanide to form a red – colored complex which absorbance measured at 510
nm is directly proportional to ALP activity . sodium arsenate incorporated in the reagent
abolishes further enzyme activity and prevent the dilution of the color inherent in earlier
methods .
SPECIMEN
Recommended specimen: 20 μl of serum or heparinised plasma. Collect specimens by
standard venipuncture technique. Handle specimens in stoppered containers to avoid
contamination and evaporation. Follow universal precautions when performing phlebotomy
or handling patient specimens, calibrators, or other serum-based products. Discard
contaminated materials with infectious waste.
Haemolysed specimens should not be used and samples more than 7 days old should not be
used due to loss of the enzyme activity in 7 days.
If absorbance change per minute exceeds 0.250, dilute 0.1 ml of the sample with 0.5 ml of
normal saline and repeat the assay using this dilution. Multiply the results by 6 to obtain the
alkaline phosphatase activity.
REAGENTS AND MATERIALS
A. full automated autoanalyzer or spectrophotometer .
B. Gel tube (clot activator)
C. Centrifuge
D. Reagents :
reagent 1
Reagent 2
Standard
Reagent 3
Blocking reagent
Reagent 4
Dye reagent
substrate buffer
disodium phenylphosphate
carbonate-biocarbonate buffer
P H 10 stabilizer
phenol
4- aminoantipyrine
sodium arsenate
potassium ferricyanide
5 mmol/l
50 mmol/l
20u/l
60 mmol/l
240 g/l
150 mmol/l
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever [P]. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette.
D. The length of transportation of the air and water will be displayed on the screen.
E. Press [↵] to finish calibration. The program will return to method select menu.
QUALITY CONTROL
154 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
Serum blank
R1
2 ml
Incubate for 5 minutes at 37 ċ
Serum
R2
Incubate for 15 minutes at 37 ċ
R3
0.5 ml
Mix well
R4
0.5 ml
Serum
0.5µl
D.W.
-
Serum sample
2ml
Standard
2ml
Regent blank
2ml
50 µl
-
50 µl
-
0.5 ml
0.5 ml
0.5 ml
0.5 ml
-
0.5 ml
-
0.5 ml
50 µL
Mix well wait for 10 minutes in dark place, read at 510 nm against blank .
Calculation:
Conc.of ALP = O.D of serum / O.D. of standard * concentration of standard
Linearity: up to 285 u /l
Reference value:
children 17 – 142 u / l
Adult 21 – 92 u /l
Conv F.: kau/dl * 7.09 = u/l
8.2.4 Total Serum Bilirubin
bilirubin is formed from the haem fragment of haemoglobin released by damaged or aged
RBC . bone marrow , liver and spleen are the sites of bilirubin production .
Causes for an increase in total bilirubin may be divided into 3 categories:
 Pre- hepatic: resulting from various haemolytic states;
 Hepatic: resulting from hepatitis, cirrhosis, and other causes of hepatic necrosis;\
 Post-hepatic: resulting from an obstruction of the common bile or hepatic duct.
Principle:
Sulfanilic acid react with sodium nitrate to form diazotized sulfanilic acid . in the presence of
dimethyl sulfoxide . total bilirubin react with dizotized sulfanilic acid form azobilirubin .
SPECIMEN
155 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
A. Recommended specimen is 100 μl of serum or heparinised plasma. Collect specimens
by standard venipuncture technique. Handle specimens in stoppered containers to
avoid contamination and evaporation. Follow universal precautions when performing
phlebotomy or handling patient specimens, calibrators, or other serum-based
products. Discard contaminated materials with infectious waste.
B. Protect specimens from light and analyse as soon as possible after collection. Direct
exposure to sunlight is reported to cause as much as 50% loss of bilirubin in one hour,
especially when the specimen is kept in a capillary tube. Exposure to normal room
light can result in a significant loss of serum bilirubin after 2 to 3 hours.
C. If analysis is not performed immediately, samples can be refrigerated for up to 24
hours at (2–5)°C.
D. Samples that come in contact with alcohol from sterile wipes may become
haemolysed, which will increase the value of bilirubin.
E. If concentration is greater than the analyser range, dilute with 5 volumes of normal
saline and reanalyse. Multiply the result by 6 to obtain the original total bilirubin
concentration. The results may show a positive bias of up to 30%.
REAGENTS AND MATERIALS
A. spectrophotometer and bilirubimnometer for neonate .
B. Centrifuge
C. Gel tube (clot activator)
D. Reagents :
Reagent 1
sulfanilic acid
Hydrochloric acid
Dimethylsulfoxide
Reagent 3
Reagent 4
sodium nitrate
Standard
30 mmol/l
150 mmol/l
7 mol/l
20 mmol/l
Procedure:
Working reagent:
Mix
Reagent 1
20 volumes
Reagent 3
1 volume
Stability in absence of light
6 hours at 20-25 ċ
2 days at + 4 ċ
Wave length -------------- 555nm
Temperature ------------- 37 ċ
Read against R1 sample blank
Read against R1 + R3 (working reagent ) sample and standard
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever [P]. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette.
D. The length of transportation of the air and water will be displayed on the screen.
E. Press [↵] to finish calibration. The program will return to method select menu.
156 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
CALCULATIONS
Sample
Calibrator(R4)
Reagent(R1)
Working
reagent
Sample blank
50 µl
1 ml
-
Sample
50 µl
1 ml
Standard blank
50 µl
1 ml
-
Standard
50 µl
1 ml
Mix and read the extinction after incubation for 5 minutes at 37 ċ
Stability of color is 1 hour ( ovoid direct sunlight )
Calculation:
( O.D sample – O. D. sample blank ) / ( O. D. calibrator – O. D. calibrator blank ) * n
N = calibrator concentration
Linearity: linear up to 20 mg / dl , 340 µmol/l
Reference value:
 Serum total bilirubin: < 1 mg/dl, < 17 µmol / l
 Conv. F. : mg / dl * 17.1 = µmol/l
LIMITATIONS
A. Specimens from haemodialysis patients should not be analysed for total bilirubin.
B. Haemoglobin affects bilirubin results.
C. Compounds that discolour serum, such as 4-aminosalicylic acid and phenazopyridine,
may falsely increase bilirubin results.
D. Results for predominantly unconjugated bilirubin (e.g., for neonates) may be up to
10% higher than the reference method.
8.3 Renal Function test
8.3.1 Blood Urea
The liver produces urea in the urea cycle as a waste product of the digestion of protein ,
Elevated levels of blood urea are most commonly associated with renal disease, but may also
result from dehydration, a high-protein diet, excess destruction of body proteins, and
gastrointestinal diseases, especially with intestinal obstruction.
Principle:
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Urea + H2O --------urease------ CO2 + 2NH3
Salicylate and hypochlorite in the reagent react with the ammonium ions to form
2.2dicarboxy –indophenol . the quantity of green compound is proportional to the urea
concentration.
SPECIMEN
A. Recommended specimen is 10 μl of serum or EDTA plasma. Collect specimens by
standard venipuncture technique. Heparin may be used as an anticoagulant for plasma
specimens. Handle specimens in stoppered containers to avoid contamination and
evaporation. Follow universal precautions when performing phlebotomy or handling
patient specimens, calibrators, or other serum-based products. Discard contaminated
materials with infectious waste.
B. Refrigerate specimens up to 3 days at 4°C if analysis is not performed immediately.
C. If concentration is greater than the analyser range, dilute with 9 volumes of isotonic
saline or distilled water and reanalyse. Multiply the result by 10 to obtain the original
urea nitrogen concentration.
REAGENTS AND MATERIALS
A. full automated auto analyzer or spectrophotometer
B. Gel tube (clot activator)
C. Centrifuge
D. Reagent :
R1a
R1b
R2
CAL
urease
phosphate buffer
Sodium salicylate
Sodium nitroprusside
EDTA
sodium hypochloride
Sodium hydroxide
standard
>5000 u/l
120 mmol/l p H 7
63.4 mmol/l
5 mmol/l
1.5 mmol/l
18 mmol/l
750 mmol/l
8.05 mmol/l
Preparation:
Add 1 vial of of urease R1a to one bottle of phosphate buffer R1b
Procedure :
Wave length …….600 nm
Temperature ……25 ċ – 37 ċ
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever [P]. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette.
D. The length of transportation of the air and water will be displayed on the screen.
158 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
E. Press [↵] to finish calibration. The program will return to method select menu.
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
Blank
Standard
standard
10µl
sample
Working reagent
1 ml
1 ml
Mixture incubated for at least 3 minutes at 37 ċ or 5 min. at 20-25 ċ
sample
10µl
1 ml
Sodium
200 µl
200 µl
200 µl
hypochloride
mix. incubate for at least 5 min. at 37 ċ or 10 min. at 20-25 ċ , measurement is against reagent
blank .
Calculation:
O.D.sample / O.D. standard * concentration of standard
CALCULATIONS
The results are reported in mmol/L. No further calculation is necessary unless the specimen
has been diluted.
Conversion
Urea = 2.14 × BUN
BUN = 0.466 × Urea
RESULTS
A. Expected values: (urea) 1.7–8.3 mmol/L
B. Panic values: <1.07 mmol/L and >37.1 mmol/L
C. Each laboratory should confirm these values.
LIMITATIONS
A. Do not use plasma collected with sodium fluoride.
B. Specimens that contain haemoglobin increase urea nitrogen. Haemoglobin of 50
mg/dL (slightly haemolysed) increases BUN levels below 28 mg/dL by 1 mg/dL.
C. Ammonium ions have shown an increase in urea nitrogen equivalent to their nitrogen
content.
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8.3.2 Serum Creatinine
Creatinine measurement is used in the diagnosis and treatment of renal diseases and in
monitoring patients on renal dialysis.
Analysis of creatinine activity in 100 μl of serum or plasma is based on an enzymatic method
that produces a coloured product.
A. The 100 μl of patient sample mixed with picrate in alkaline medium produces a
coloured complex with creatinine. The rate of formation of the complex is then
measured.
B. The creatinine present in the sample reacts with the picrate at 37°C, and the rate of
complex formation is measured and is proportional to the creatinine concentration.
SPECIMEN
A. Recommended specimen is 100 μl of serum or heparinized plasma. Collect specimens
by standard venipuncture technique. Heparin may be used as an anticoagulant for
plasma specimens. Handle specimens in stoppered containers to avoid contamination
and evaporation. Follow universal precautions when performing phlebotomy or
handling patient specimens, calibrators, or other serum-based products. Discard
contaminated infectious waste.
B. Refrigerate specimens at 4°C for 24 hours if analysis is not done immediately.
REAGENTS AND MATERIALS
A. Photometer
B. Gel tube (clot activator)
C. Centrifuge
D. Picric acid 26 mmol/L
E. NaOH 1.6 mmol/L
F. Creatinine 2 mg/dL or 176.8 μmol/L
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever [P]. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette.
D. The length of transportation of the air and water will be displayed on the screen.
E. Press [↵] to finish calibration. The program will return to method select menu.
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
A. Mix reagents according to manufacturer’s recommendations.
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B. Label 2 tubes: in tube or standard tube pipette 1,000 μl of reagents solution and 100
μl of creatinine standard reagent; and in tube 2 or sample tube pipette 1,000 μl of
reagents solution and 100 μl of sample.
C. Mix them well and measure them immediately as indicated.
D. Press ZERO and then push sip by pressing lever [P] to sip water and drain the
system.
E. When the screen displays MEASURE STANDARD, place cuvette with the standard
solution into cuvette holder and aspirate the standard solution by pushing lever [P].
F. When the screen displays MEASURE SAMPLE, place cuvette with the sample
solution into cuvette holder and aspirate the sample solution by pushing lever [P].
G. Press [RESULT] and await possible delay while measuring the sample solution. The
result will be displayed on top line.
H. Print or write out the results. Press [WASH] to sip rinse solution or press
[MEASURE] to measure the next sample by pushing sip lever [P].
CALCULATIONS
The results are reported in μmol/L. No further calculation is necessary unless the specimen
has been diluted.
RESULTS
A. Expected values:
 Males: 53–97 μmol/L
 Females: 44–80 μmol/L
B. Panic values: < 27 μmol/L and > 663 μmol/L
C. Each laboratory should confirm these values.
LIMITATIONS
A. EDTA and fluoride/oxalate anticoagulants will cause low creatinine results.
B. Lidocaine: Patients on long-term lidocaine therapy may show an increase of up to 1.0
mg/dL. Ninety percent of patients receiving intravenous lidocaine will show less than
a 0.3 mg/dL increase.
C. Proline: Patients receiving hyperalimentation fluid that contains proline may show an
increase of up to 2.0 mg/dL.
D. TRIS buffer (control fluid) causes an approximate 50% decrease in results.
E. Dipyrone (Metamizol) at 40 mg/dL shows a –0.6 mg/dL bias at a creatinine
concentration of 1.0 mg/dL.
F. N-acetylcysteine: Patients receiving N-acetylcysteine (Fluimucil, Mucomyst)
intravenously have been reported to show a large negative bias.
8.3.3 Uric acid
Uric acid is a chemical created when the body breaks down substances called purines.
Purines are found in some foods and drinks ,Most uric acid dissolves in blood and travels
to the kidneys, where it passes out in urine. High levels of uric acid in the body is called
hyperuricemia.
Principle :
Uric acid +O2 +H2O---------uricase -------- allantoine + CO2 + H2O2
161 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
2H2O2 +4-aminophenazone+ 2-4dichloro- phenol-sulfonate -----------peroxidase--------
quinonemine
Reagents:
Reagent 1
phosphate buffer P H 7.4
50mmol/l
Buffer solution
2-4 DCPS
4 mmol/l
Reagent 2
uricase
7o u/l
Vial of enzyme
peroxidase
660 u/l
4-aminophenazone
1 mmol/l
uric acid
6 mg/dl
Reagent 3
Standard
Preparation :
Dissolve the content of one bottle R 2 with the content of one bottle buffer R 1
Procedure :
Wave length---------------------510 nm (490 – 550)
Blank
Standard
Sample
Working reagent
1 ml
1 ml
1 ml
Standard
-
20µl
-
Sample
-
-
20 µl
mix. incubate 5 minutes at 37 ċ or 10 minutes at 20-25 ċ the colour is stable for 30
minutes .
calculation :
concentration= O.D.sample / O.D. standard * concentration of standard
reference value : women 2.5 – 6 mg/dl,148 – 357 µmol / l
men 3.4 – 7 mg/dl , 200 – 416 µmol / l
Conv. F. : mg/dl * 60 = µmol / l
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8.4 Lipid Profile
8.4.1 Total Cholesterol
Cholesterol is a waxy, fat-like substance that occurs naturally in all parts of the body. Your
body needs some cholesterol to work properly. But if you have too much in your blood, it can
stick to the walls of your arteries. This is called plaque. Plaque can narrow your arteries or
even block them.
High levels of cholesterol in the blood can increase your risk of heart disease. Your
cholesterol levels tend to rise as you get older. There are usually no signs or symptoms that
you have high blood cholesterol, but it can be detected with a blood test. You are likely to
have high cholesterol if members of your family have it, if you are overweight or if you eat a
lot of fatty foods.
You can lower your cholesterol by exercising more and eating more fruits and vegetables.
You also may need to take medicine to lower your cholesterol.
Causes of hypercholesterolemia:
1- familial hypercholesterolaemia (FH)
2- reduced metabolism due to thyroid problems(hypothyroidism)
3-kidney diseases
4- diabetes particularly when poorly controlled
5- alcohol abuse
6- being overweight – this is probably the commonest cause of high cholesterol levels.
Causes of hypocholesterolemia :
1-Hyperthyroidism
2-Liver disease
3-Malnutrition
4-Cancer
5-Chronic infections or inflammation
Principle:
Esters of cholesterol +H2O -------chloesterol esterase-------> cholesterol + fatty acids
Cholesterol +O2 ---------cholesterol oxidase -----------> cholest-4-en-one +H2O2
H2O2 +phenol+4-aminophenazone ------------peroxidase ------------> quinonimine
SPECIMEN
A. Recommended specimen 10 μl of serum, heparinised or EDTA plasma. Collect
specimens by standard venipuncture technique. Heparin may be used as an
anticoagulant for plasma specimens. Handle specimens in stoppered containers to
avoid contamination and evaporation. Follow universal precautions when performing
phlebotomy or handling patient specimens, calibrators, or other serum-based
products. Discard contaminated materials with infectious waste.
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B. If the result is over 19.3 mmol/L, dilute the sample before precipitation 1:2 with
normal saline and multiply the results by 3 to obtain the original cholesterol
concentration.
C. Cholesterol remains stable in serum for 6 days at 4–25°C and up to 4 months at –
20°C.
D. Lipemic specimens generate turbidity of the sample/reagent mixture, which leads to
falsely elevated results.
REAGENTS AND MATERIALS
A. spectrophotometer
B. Gel tube (clot activator)
C. Centrifuge
D. Reagents :
Reagent 1
pipes p H 6.9
Buffer solution
Phenol
Reagent 2
cholesterol oxidase
Vial of two enzymes
Peroxidase
Cholesterol oxidase
4-aminophenazone
Reagent 3
standard
90 mmol/l
26 mmol/l
399 u/l
1250 u/l
300 u/l
0.4 mmol/l
200 mg/dl
Preparation:

Dissolve the contents of one bottle R2 to the contents of one bottle buffer reagent R1
Sample: serum , heparinized plasma
Procedure:


Wave length …………… 505nm ( 500 550)
Temperature …………... 37 ċ
standard
sample
Working reagent

Blank
1 ml
Standard
10µl
1 ml
sample
10µl
1 ml
Mix. incubate 5 minutes at 37 ċ ,the colour is stable for 30 min.
Calculation:
Cholesterol concentration = O.D.sample / O.D. standard * concentration of standard
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
CALCULATIONS
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The results are reported in mmol/L. No further calculation is necessary unless the specimen
has been diluted.
RESULTS
A. Expected values: 3.6–5.2 mmol/L.
B. Each laboratory should confirm these values.
LIMITATIONS
A. Bilirubin concentrations above 4 mg/dl interferes with tests
B. If results are over 25.9mmol/l dilute the sample and repeat measument
C. High concentration of ascorbic acid (> 2.5 mg/dL) will give lower values.
D. Haemoglobin levels higher than 200 mg/dL do not interfere with the test.
8.4.2 Triglycerides
Triglycerides protein is essential for growth, the production of new tissue, and the repair of
injured tissue. An increase in triglycerides levels may be the result of nephrosis, cholestasis,
pancreatitis, cirrhosis, diabetes mellitus, and hepatitis. Adecrease is seen with malnutrition.
The analysis is based on the enzymatic hydrolysis of triglycerides with lipases in a
quinoneimine formed from hydrogen peroxide used as a coloured indicator. The intensity of
the colour is proportional to the amount of triglycerides in the sample. 10 μl of patient sample
is mixed and incubated with 1,000 μl of solution containing 4- aminoantipyrine and 4chlorophenol in presence of hydrogen peroxide. After incubation for 10 minutes, a reaction
between 4-aminoantipyrine and 4-chlorophenol and hydrogen peroxide takes place, forming
quinoneimine, which is a coloured
product. The amount of coloured compound formed is proportional to the amount of
triglycerides in the specimen.
SPECIMEN
A. Recommended specimen 10 μl of serum, heparinised or EDTA plasma. Collect
specimens by standard venipuncture technique. Heparin may be used as an
anticoagulant for plasma specimens. Handle specimens in stoppered containers to
avoid contamination and evaporation. Follow universal precautions when performing
phlebotomy or handling patient specimens, calibrators, or other serum-based
products. Discard contaminated materials with infectious waste.
B. Avoid haemolysed specimens. If analysis is not performed immediately, freeze
specimens at −25oC for up to 4 months or refrigerate at 2–8oC for up to 3 days.
C. If concentration is greater than the analyser range, dilute 1 part of sample with 4 parts
of isotonic saline or distilled water and reanalyse. Multiply the result by 5 to obtain
the original triglycerides concentration.
REAGENTS AND MATERIALS
A. Photometer
B. Gel tube (clot activator)
C. Centrifuge
D. Reagent PIPES buffer (pH 7.5)
 4-chlorophenol
 4-aminoantipyrine
 Magnesium ion
50 mmol/L
5 mmol/L
0.25 mmol/L
4.5 mmol/L
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





ATP
Lipases
Peroxidase
Glycerol kinase
Glycerol-3-phpsphate oxidase
Standard Triglyceride
2 mmol/L
≥1.3 U/ml
≥0.5 U/ml
≥0.4 U/ml
≥1.5 U/ml
2.28 mmol/L
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever [P]. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette.
D. The length of transportation of the air and water will be displayed on the screen.
E. Press [↵] to finish calibration. The program will return to method select menu.
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and highcontrols)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
A. Mix 2 tubes: one blank with 1,000 μl of standard reagent, and tube 2 with 10 μl of
serum or plasma and 1,000 μl of reagent solution.
B. Mix each well and incubate for 5 minutes at 37oC, and measure the absorbance of the
standard reagent and sample immediately as indicated.
C. Press ZERO and then push sip by pressing lever [P] to sip water and drain the
system.
D. When the screen displays MEASURE RB, place cuvette with the blank solution into
cuvette holder and aspirate the blank solution by pushing lever [P].
E. When the screen displays MEASURE SAMPLE, place cuvette with the sample
solution into cuvette holder and aspirate the sample solution by pushing lever [P].
F. Press [RESULT] and await possible delay while measuring the sample solution. The
result will be displayed on top line.
G. Print or write out the results. Press [WASH] to sip rinse solution or press
[MEASURE] to measure the next sample by pushing sip lever [P].
CALCULATIONS
The results are reported in mmol/L. No further calculation is necessary unless the specimen
has been diluted.
RESULTS
A. Expected values: 0.11–2.15 mmol/L.
B. Each laboratory should confirm these values.
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LIMITATIONS
A. Lipemic specimen usually generates turbidity of the sample reagent mixture, which
leads to falsely elevated results.
B. Ascorbate gives falsely low values.
8.4.3 HDL Cholesterol
High density lipoprotein (HDL) is lipoprotein which is involved in the transport of cholesterol
from the cells to the liver. An increase in HDL levels may be the result of nephrosis,
cholestasis, pancreatitis, cirrhosis, diabetes mellitus, and hepatitis. A decrease is seen with
malnutrition.
The analysis is based on the precipitation of chylomicrons, very low-density lipoprotein
(VLDL), and low density lipoprotein (LDL) upon addition of phospshotungstic acid and
magnesium chloride to the sample. The HDL is contained in the supernatant, which can be
enzymatically assayed after centrifugation of the supernatant. The absorbance of the sample
and the standard reagent is measured against blank reagent.
When 200 μl of test sample is mixed with 500 μl of precipitant reagent containing
phosphotungstic acid and magnesium chloride at room temperature for 10 minutes
chylomicrons, VLDL, and LDL are precipitated . Centrifugation at 10,000 g for 2 minutes
leaves only the HDL in the supernatant, where the concentration of HDL in the sample is
determined enzymatically.
SPECIMEN
A. Recommended specimen 200 μl of serum, heparinised or EDTA plasma. Collect
specimens by standard venipuncture technique. Heparin may be used as an
anticoagulant for plasma specimens. Handle specimens in stoppered containers
toavoid contamination and evaporation. Follow universal precautions when
performing phlebotomy or handling patient specimens, calibrators, or other-based
products. Discard contaminated materials with infectious waste.
B. If the supernatant is not clear, dilute the sample before precipitation 1:1 with normal
saline and multiply the results by 2 to obtain the original HDL concentration.
C. HDL cholesterol remains stable in serum for 6 days at 4–25°C and up to 4 months at
–20°C. Serum must be separated from the blood clot as rapidly as possible.
REAGENTS AND MATERIALS
A. Photometer
B. Gel tube (clot activator)
C. Centrifuge
D. Reagent (Precipitant)
Phosphotungstic acid
0.55 mmol/L
Magnesium chloride
25 mmol/L
Standard Cholesterol
1.29 mmol/L
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
167 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever “P”. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette.
D. The length of transportation of the air and water will be displayed on the screen.
E. Press [↵] to finish calibration. The program will return to method select menu.
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
A. Into a glass centrifuge tube mix 200 μl of test sample (serum or plasma) 500 μl of
precipitant reagent. (Dilute precipitant reagent with distilled water 4 in 1.)
B. Mix them well and incubate for 10 minutes at room temperature.
C. Centrifuge at 4000 rpm for 10 minutes or 10,000 rpm for 2 minutes.
D. After centrifugation remove the clear supernatant and into tube 1 (sample) add 100 μl
of supernatant and 1,000 μl of total cholesterol reagent. In tube 2 (reagent blank) add
100 μl of distilled and 1,000 μl of cholesterol reagent.
E. Mix them well and incubate for 10 minutes at 20–25°C or 5 minutes at 37°C and
measure their absorbance.
F. When the screen displays MEASURE BLANK, press ZERO and then push sip by
pressing lever [P] to sip water and drain the system.
G. When the screen displays MEASURE RB, place cuvette with the blank solution into
cuvette holder and aspirate the blank solution by pushing lever [P].
H. When the screen displays MEASURE SAMPLE, place cuvette with the sample
solution into cuvette holder and aspirate the sample solution by pushing lever [P].
I. Press [RESULT] and await possible delay while measuring the sample solution. The
result will be displayed on top line.
J. Print or write out the results. Press [WASH] to sip rinse solution or press [measure] to
measure the next sample by pushing sip lever [P].
CALCULATIONS
The results are reported in mmol/L. No further calculation is necessary unless the specimen
has been diluted.
RESULTS
A. Expected values: 0–0.9 mmol/L.
B. Each laboratory should confirm these values.
LIMITATIONS
A. High triglyceride level interferes with HDL measure; dilute the sample before
precipitation 1:1 with 0.9% saline and multiply the results by 2.
B. High concentration of ascorbic acid (> 2.5 mg/dL) will give lower values.
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C. Haemoglobin levels higher than 100 mg/dL and bilirubin levels higher than 10 mg/dL
interfere with the test.
8.4.4 LDL Cholesterol
Low density lipoprotein (LDL) is lipoprotein which is involved in the transport of cholesterol
to the cells. An increase in LDL levels may be the result of altered diet and vascular diseases.
A decrease is seen in AIDS, haematological malignancies such as acute myeloid leukaemia
(AML) or chronic myeloid leukaemia (CML), and disorders involving splenomegaly.
The analysis is based on direct homogenous enzymatic assay for quantitative determination of
LDL cholesterol combining two steps. The analysis involves removal of chylomicrons, very
low density lipoprotein (VLDL), and high density lipoprotein (HDL), followed by enzymatic
determination of LDL in the sample.
10 μl of patient sample is mixed and incubated at 37oC for 5 minutes with 750 μl of enzyme
solution containing cholesterol esterase and cholesterol oxidase, which allows the removal of
chylomicrons, VLDL, and HDL from the specimen by formation of cholestenone and
hydrogen peroxide. The LDL present in the sample is converted to cholestenone and
hydrogen peroxide in the presence of cholesterol esterase, cholesterol oxidase, and specific
surfactant. The chromogen in presence of hydrogen peroxide peroxidase is converted to a
coloured dye (quinine) whose concentration is measured by colorimetric method.
SPECIMEN
A. Recommended specimen 10 μl of serum or plasma. Collect specimens by standard
venipuncture technique. Heparin may be used as an anticoagulant for plasma
specimens. Handle specimens in stoppered containers to avoid contamination and
evaporation. Follow universal precautions when performing phlebotomy or handling
patient specimens, calibrators, or other serum-based products. Discard contaminated
materials with infectious waste.
B. If the serum concentration of LDL exceeds the measuring range, dilute the sample
before precipitation 1:1 with normal saline, repeat the test, and multiply the results by
2 to obtain the original LDL concentration.
C. LDL cholesterol remains stable in serum for 6 days at 4–25°C and up to 4 months at –
20°C.
D. Serum must be separated from the blood clot as rapidly as possible.
REAGENTS AND MATERIALS
A. Photometer
B. Gel tube (clot activator)
C. Centrifuge
D. Reagent (enzymes)
 Good’s buffer (pH 7.0)
50 mmol/L
 Cholesterol esterase
600 U/L
 Cholesterol esterase
500 U/L
 Catalase
600 kU/L
 TOOS
2.0 mmol/L
 Detergents
0.3% w/v
 Preservatives
<0.1% w/v
 Substrate Peroxidase
4000 U/L
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




4-Aminoantipyrin
Good’s buffer (pH 7.0)
Sodium azide
Detergents
Preservatives
4 mmol/L
50 mmol/L
0.05%
1% w/v
<0.1% w/v
CALIBRATION
A. Empty and wash flow-through cuvette by pressing [WASH] on the LCD display. An
automatic return to the select menu will occur.
B. Push sip by pressing lever [P] to drain the tube system. Do not sip any solution.
C. Measure exactly 1,000 μl of distilled water into a sample cup and sip distilled water
by pushing sip-press lever [P]. The sipping volume of 1,000 μl is automatically
measured in the flow-through cuvette. The length of transportation of the air and
water will be displayed on the screen.
D. Press [↵] to finish calibration. The program will return to method select menu.
QUALITY CONTROL
A. Run quality control materials in normal and abnormal ranges (low and high controls)
at the beginning of each shift. Always run a set of controls after calibrating the
analyser. If the controls are out of range, do not report patient results until the
problem is resolved and the patient samples are repeated with quality control samples
within acceptable range.
B. Variability is expressed as standard deviation (SD) and coefficient of variation (CV)
and plotted on a Levy-Jennings graph.
PROCEDURE
A. Label 2 tubes and pipette 10 μl water and 750 μl of enzyme solution into tube 1
(blank), and into tube 2 pipette 10 μl of test sample and 750 μl of enzyme solution.
Mix gently and incubate at 37oC for 5 minutes.
B. Into each tube add 250 μl of substrate solution and mix them well and incubate for 5
minutes at 37°C, then measure absorbance of sample against reagent blank.
C. When the screen displays MEASURE BLANK, press ZERO and then push sip by
pressing lever [P] to sip water and drain the system.
D. When the screen displays MEASURE RB, place cuvette with the blank solution into
cuvette holder and aspirate the blank solution by pushing lever [P].
E. When the screen displays MEASURE SAMPLE, place cuvette with the sample
solution into cuvette holder and aspirate the sample solution by pushing lever [P].
F. Press [RESULT] and await possible delay while measuring the sample solution. The
result will be displayed on top line.
G. Print or write out the results. Press [WASH] to sip rinse solution or press
[MEASURE] to measure the next sample by pushing sip lever [P].
CALCULATIONS
The results are reported in mmol/L. No further calculation is necessary unless the specimen
has been diluted.
RESULTS
A. Expected values: 0–0.9 mmol/L.
B. Each laboratory should confirm these values.
170 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
NOTE: Direct measurement of LDL is required instead of the Friedwald, or “calculated”,
method whenever triglyceride values exceed 400 mg/dL. The Friedwald or “calculated”
method of LDL determination is determined by subtracting HDL and VLDL from the total
cholesterol concentration. Total cholesterol = HDL + LDL + VLDL. VLDL is generally
estimated by the triglyceride concentration divided by 5, but is inaccurate when triglyceride
values exceed 400 mg/dL.
LIMITATIONS
A. High triglyceride level interferes with LDL measure; dilute the sample before
precipitation 1:1 with 0.9% saline and multiply the results by 2.
B. High concentration of ascorbic acid (> 2.5 mg/dL) will give lower values.
C. Haemoglobin levels higher than 100 mg/dL and bilirubin levels higher than 10 mg/dL
interfere with the test.
171 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
Annex 1
TABLE 1 - Outline of the properties of heat decontamination methods. For everyday
laboratory purposes, autoclaving is the preferred method, unless the item cannot withstand
the heat and/or moisture of autoclaving.
Principle/Conditions
Advantages
Disadvantages
Uses
Dry Heat
Thermal inactivation:
destroys by oxidation
Noncorrosive
Simple
design and
principle
Less effective
than moist heat;
requires longer
times and/or
higher
temperatures
Materials that are
damaged by, or
are impenetrable
to, moist heat
Hot Air Oven
 160-180?C for 2-4
hours
 penetrates
waterinsoluble
materials
(e.g., grease
and oil)
 slow
diffusion,
penetration
 anhydrous
materials, such as
oils, greases and
powders
 less
corrosive to
metals and
sharp
instruments
than steam
Red-heat
Flame
 oxidation to ashes
(burning)
 rapid
 loading,
packing critical
to performance
 not suitable
for reusable
plastics
 laboratory
glassware,
instruments
 closed
containers
 initial contact  inoculating
with flame can loops, needles
produce a
viable aerosol
 possibility of
accidental fire
Incineration
 oxidation to ashes
(burning)
 1-60 minutes:
temperatures may
exceed 1000?C
 reduces
volume of
waste by up
to 95%
 improper use
may lead to
emission of
pathogens in
smoke
 for
decontamination
of waste items
prior to disposal
in landfill
172 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
 requires
transport of
infectious waste
 excess plastic
(>20%) content
reduces
combustibility
Moist Heat
Irreversible
coagulation of
(microbial) proteins
More rapid
and more
effective
than dry heat
Pasteurization  heating to below
boiling point
(generally 77?C) for
up to 30 minutes
 can be
used on heat
sensitive
liquids and
medical
devices
 not reliably
sporicidal
 milk and dairy
products
 some heatsensitive medical
equipment
 low cost
Tyndallization  heating to 80-100?C
(Fractional
for 30 mins on
Sterilization) successive days, with
incubation periods in
between
Boiling
 resistant
spores
germinate
and are
killed on the
second and
third days
 maximum
 minimal
temperature obtainable equipment
is approximately
required
100?C 10-30 mins
 time
consuming
 not reliably
sporicidal
 heat sensitive
materials such as
bacteriologic
media, solutions
of chemicals,
biological
materials
 cumbersome:  small
not practical for instruments and
everyday lab
equipment
use
 not reliably
sporicidal
autoclaving
 steam under
pressure
 121?C/15 psi for
15-90 mins (gravity
displacement
autoclave)
 minimal
time
required
 loading and
packing critical
to performance
 penetration of
sterile glassware,
media and
instruments
 most
dependable
 shielding dirt
must first be

removed
decontamination
173 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
 132?C/27 psi for 420 minutes (prevacuum autoclave)
sterilant for
lab use
 maintenance of reusable
supplies and
and quality
control essential equipment
 damages
heat-sensitive
itmes

decontamination
of infectious
waste
TABLE 2 - Summary of concentrations used, contact times, advantages and disadvantages
and uses of some of the halogen-releasing chemical germicides. The wide ranges of
effective concentrations and contact times cited are due to a number of factors, including the
interdependence of time and concentration, the variability in resistance of different
microorganisms, the amount of organic material present and the desired effect (e.g., lowlevel vs high-level disinfection)
Effective
Concentrations, Advantages
Contact Times
Chlorine
Compounds:
Sodium hypochlorite
solution 1 (liquid
bleach)
 100-10,000
ppm (.01-1%)
free chlorine
 broad
spectrum

 10-60 minutes inexpensive
(>= 3,000 ppm
 widely
for broad
available
spectrum)

bactericidal
at low
temperature
Examples of
Uses
Disadvantages
 toxic,
 general
corrosive to
disinfectant
skin and metals
 waste liquids
 unstable at
 surface
optimum
effective pH of decontamination
6
 emergency
spill clean up
 inactivated
by organic
matter
 instrument
disinfection
 deteriorates
under light and
heat: shelf life
of dilutions is
less than 1
week
Calcium
hypochlorite2
granules, powder,
tablets
 as for liquid
bleach
 as for
liquid bleach
but more
stable
 as for liquid
bleach above,
except shelf
life is longer
 as for liquid
bleach
174 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
NaDCC3 (Sodium
 as for liquid
dichloroisocyanurate) bleach
powder, granules,
tablets
Chloramine-T4
(Sodium
tosylchloramide)
powder or tablets
 as for liquid
bleach
 more
 toxic,
stable than
corrosive
hypochlorites
 inactivated
 stable at
by organic
pH 6.0
matter
 more
stable, less
affected by
organic
matter than
hypochlorites
 as for liquid
bleach
 deteriorates  as for liquid
under
bleach
humidity, light
and heat
 longer
activity than
hypochlorites
Chlorine dioxide5
 demandrelease of
chlorine dioxide
in situ
 longer
activity than
other
chlorine
compounds
 aqueous
solutions
decompose
under light
 instrument
disinfection
 not
consistently
sporicidal
 germicidal
soaps and
antiseptics
 efficacy
reduced by
organic matter
 surface
decontamination
 less
corrosive,
less toxic
than other
chlorine
compounds
 gas
sterilization of
germ-free
animal
chambers
 effective at
pH 6-10
Iodine
Preparations:
Iodophors6
 30-1,000 ppm  broad
(.003-.1%) free spectrum
iodine
 germicidal
 10-30 minutes over a wide
pH range
 generally
nonstaining,
less toxic and
less irritating
than aqueous
or alcoholic
 work surface
wipedown
 some
iodophor
 instrument
solutions
support growth disinfection
175 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
iodine
solutions
of
Pseudomonas7
TABLE 3A Summary of recommended concentrations, contact times, advantages and
disadvantages of non-halogen chemical germicides. The wide ranges of effective
concentrations and contact times cited reflect the interdependence of time and concentration
as well as factors such as resistance of the particular class or strain of target
microorganism(s) and desired effect. Also, some germicides are available in combinations
(e.g., glutaraldehyde/phenol or peracetic acid/alcohol mixtures) which are synergistic
whereby the components in combination produce a greater antimicrobial effect than the sum
of their individual effects.
Effective
Concentrations
and Contact
Times
Alcohols
 70-80%
ethanol
 60-95%
isopropanol
 10-30
minutes
Advantages
 low toxicity
 rapid action
 low residue
Examples of
Laboratory
Uses
Disadvantages
 rapid
evaporation
limits contact
time
 flammable,
 non-corrosive
eye irratant
 skin
disinfectant
(antiseptic)
 surface
decontamination
 benchtop,
 may damage cabinet
rubber, plastic, wipedown
shellac
 ineffective
against
bacterial
spores
Phenolic
Compounds
 400-50,000
 tolerant of
ppm (.05-1.5%) organic load,
"hard" dilution
 10-30
water
minutes
 leaves an
active residue
(may be
desirable on
some surfaces)
 pungent
odour,
corrosive,
some forms
toxic
 not
sporicidal;
limited activity
against viruses
 instruments
and equipment
disinfection
 disinfection
of floors and
other surfaces
176 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e

biodegradable
 leaves a
 antiseptic
residual film
soaps and
(undesirable in lotions
culture
systems)
 may support
growth of
bacteria1
Quaternary
Ammonium
Compounds
 500-15,000
 combined
ppm (.05-1.5%) detergent and
germicidal
 10-30
activity
minutes
 stable
 working
dilutions have
low toxicity
Hydrogen Peroxide  3-30%
aqueous
solution
 10-60
minutes
 6% for 30
minutes may
kill spores
 rapid action
 no residue
 low toxicity
 non
sporicidal,
limited activity
against viruses,
mycobacteria
 surface
decontamination
 most
formulations
not readily
biodegradable
 antiseptic
formulations
available
 limited
sporicidal
activity
 surface
decontamination
 equipment
wipedown
 floors and
 may support walls
growth of
bacteria2
 corrosive to
some metals
 instruments
and equipment

environmentally  potentially
safe
explosive at
high
concentrations
 stock
solutions
irritating to
skin and eyes
Peracetic Acid
(PAA)
 .001-.3%
aqueous
solution
 broad
spectrum
 pungent
odour
 instruments
and equipment
corrosive to
some metals
 gas phase
sterilization of
177 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
 gas phase: 24%
 5-120
minutes
 sporicidal at
low
temperatures
 shelf life of chambers for
dilutions is less germ-free
animals
than 1 week
 can tolerate
organic load
 stock
solutions
irritating to
skin and eyes
 rapid action
 nontoxic
decomposition
products
 leaves no
residue
 stock must
be protected
from heat,
light
 gas phase:
respiratory
irritant, fire
hazard above
55?C
Table 3B - Summary of recommended concentrations, contact times, advantages and
disadvantages of non-halogen chemical germicides. The wide ranges of effective
concentrations and contact times cited reflect the interdependence of time and concentration
as well as factors such as resistance of the particular class or strain of target
microorganism(s) and desired effect. Also, some germicides are available in combinations
(e.g., glutaraldehyde/phenol or peracetic acid/alcohol mixtures) which are synergistic
whereby the components in combination produce a greater antimicrobial effect than the sum
of their individual effects.
Effective
Concentrations
Advantages Disadvantages
and Contact
Times
Examples of
Laboratory
Uses
Aldehydes:
Glutaraldehyde
 0.5-2.5%
alkalinized
aqueous
solution
 broad
spectrum
 does not
corrode
 2-30 mins; up metal
to 12 hours to
kill all spores
 expensive
 pH,
temperature
dependent
 cold sterilant
and fixative
 surface
decontamination
 pungent
odour
178 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
 can
 toxic: skin,
tolerate
eye,
organic load respiratory
tract irritant
 instruments,
equipment,
glassware
 activated
solutions have
less than 2week shelf life
Formalin (37%
aqueous
formaldehyde)
 3-27%
formalin (110%
formaldehyde)
in 70-90%
alcohol
 10-30
minutes
Formaldehyde (gas)
Ethylene Oxide Gas
 1-3 hours
 50-1200
mg/L
 1-12 hours
 broad
spectrum
 pungent
odour
 cold sterilant
and fixative
 skin, eye

inexpensive and respiratory
tract irritant
 does not
 potential
corrode
metal
carcinogen
(animal
 can
studies)
tolerate
organic load  may require
24 hrs or more
to kill all
spores
 surface
decontamination
 as for
formalin
 as for
formulin
 effective
penetration
 flammable
 on site
decontamination
of biological
safety cabinet
HEPA filters
 broad
spectrum
 porr
penetration of
covered
surfaces
 instruments
and equipment
 enclosed
areas
 flammable,
reactive
 heat or
moisture
sensitive
 no heat or  toxic:
supplies,
moisture
potential
instruments and
evolved
carcinogen and equipment
mutagen

 some
penetrates
sterilized items
may need more
179 | P a g e P H C P I L a b o r a t o r y S t a n d a r d s G u i d e l i n e
packaging
materials
than 24 hours
for outgassing
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