Clinical pathology
Dr S Homathy
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Pathology
• The branch of medicine dealing with the
essential nature of disease, especially changes
in body tissues and organs that cause or are
caused by disease.
• The discipline of Pathology includes both
Clinical and Anatomic Pathology.
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• Clinical pathology
– pathology applied to the solution of clinical
problems, especially the use of laboratory methods
in clinical diagnosis.
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• It is vital for educating physicians to order the
correct tests and understand the clinical
significance of test results.
• The clinician depend upon the laboratory for
the diagnosis of diseases
• The laboratory plays a crucial role in the
diagnosis of disease and therapeutic
management of patients.
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• It is the key subject in the studies of
paramedical sciences
• It forms bridge between
– the preclinical sciences of anatomy, physilogy and
biochemistry on one hand and
– clinical branches of medical and surgical
disciplines on the other
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• The diagnosis of a disease involves
examination of specimens collected from the
patients include
– urine,
– stool,
– sputum,
– blood,
– CSF,
– other body fluids and
– biopsy material.
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• The laboratory study of disease by a
pathologist using techniques appropriate to the
specimen being studied.
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Major subspecialty areas in Clinical Pathology are:
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Clinical Chemistry/Toxicology
Coagulation
Cytogenetics
Flow Cytometry
Haematology
Haematopathology
Microbiology
Molecular Diagnostics
Transfusion Medicine
Bacteriology,
Chemistry, and
Serology.
Molecular genetics pathology.
In some countries other subspecialties fall under certified Clinical
Biologists responsibility
– Assisted reproductive technology, Sperm bank and Semen analysis
• Immunopathology
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• What are the sections of the lab and their
functions?
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Tools of Clinical Pathology
• Microscopes, analysers, strips, centrifugal
machines...
Macroscopic examination
• The visual examination of the taken liquid is a
first main indication for the pathologist or the
physician.
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Microscopical examination
• Microscopic analysis is an important activity of the
pathologist and the laboratory assistant.
• They have many different colourings at their disposal
(H&E,Gram, Grocott, Ziehl-Neelsen, …).
• Immunofluorescence, cytochemistry, the
immunocytochemistry and FISH are also used in order
make a correct diagnosis.
• This stage allows the pathologist to determine the
character of the liquid:
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“normal”,
tumoral,
Inflammatory
even infectious.
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Analyzers
• The analysers, by the association of robotics and spectrophotometry,
– allowed these last decades a better reproducibility of the results of
proportionings,
– in particular in medical biochemistry and hematology.
• in -vitro diagnosis include chains of automats,
– i.e. a system allowing the automatic transfer of the tubes towards the
various types of automats of the same mark.
• These systems can include the computer-assisted management of a
serum library.
• These analysers must undergo daily controls to guarantee a result
just possible, one speaks about quality control.
• These analysers must also undergo daily, weekly and monthly
maintenances.
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Cultures
• A big part of the examinations of clinical
pathology, primarily in medical microbiology,
use culture media.
• Those allow, for example, the description of
one or several infectious agents responsible of
the clinical signs.
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• Values known as “normal” or reference
values
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Quality
• Laboratories can only produce quality results
on quality samples
• And on quality requests
• Rubbish In - Rubbish Out
• Ideal sample mimics the in vivo state
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Specimen
• Any biological material taken from a patient for
diagnostic, prognostic or therapeutic monitoring
• Specimens consist
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Urine
Stool
Sputum
Wound drainage
Blood
Biopsy material
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• All samples must be considered to be
infectious
– Use of “ Universal precautions” handling
– Never assume any sample is “ safe”
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Phase of analysis
• Pre – analytical (from the patient to the lab)
• Analytical
• Post – analytical (from the lab to the notes)
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Pre – analytical (From the patient to the
lab)
What can and does go wrong
• Incorrect identification of patient
• Patient preparation
– Fasting, diet, supine, time, drugs
• Sample poorly/ incorrectly taken
• Inaccurate timing
• Wrong type of sample
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Incorrect containers
Under- filling
Mislabelling/ no labelling
Incorrect storage / transport (ice, warm, delay)
Loss, breakage
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The patient
• Do patients always disclose history?
– Confused
– Frightened
– In pain
– Want to help
– Any previous diagnosis available
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Patient preparation
• Prior to each collection,
– review the appropriate test description,
– including the specimen type to be collected,
– the volume,
– the procedure,
– the collection materials, and
– the storage and handling instructions
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• Proper sample collection and handling is an
integral part of obtaining a valid and timely
laboratory test result
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• Specimens must be obtained using
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proper phlebotomy techniques,
collected in the proper container,
correctly labeled (in the presence of the patient) and
promptly transported to the laboratory.
It is the policy of the laboratory to reject samples
when there is failure to follow these guidelines.
• All specimens should be handled with universal
precautions, as if they are hazardous and
infectious.
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Blood collection
• As patients to identify themselves
• When blood taken write all relevant details on
collection tubes immediately
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Other information should include
• Ward
• Date of collection
• Time of collection e.g: cortisol
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Patient identification
• All patients from whom clinical specimens are
obtained must be positively identified prior to
specimen collection.
• Positive identification is the responsibility of
the person collecting the sample.
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• Identify the patient prior to sample
collection, using at least two patient
identifiers.
– Verify the patient’s name,
– unit history number on the identification armband
(inpatients), or
– drivers’ license or other ID (outpatients) with the
information on the requisition.
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SPECIMEN COLLECTION TIMING
• The basal state (the early morning
approximately 12 hours after the last ingestion
of food) is recommended for determining the
concentration of body constituents such as
– glucose,
– cholesterol,
– triglycerides,
– electrolytes, and
– proteins.
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Test request
• The test requisition must include:
– the patient's name
– a unique identification number which assures positive
patient identification (e.g., BHT number)
– the unit / clinic location
– The name(s) and signature of the authorized provider
requesting the test(s)
– the test(s)/procedure(s) being ordered
– the date and time of specimen collection is required on all
requisitions
– name or initials of person collecting specimen
– pager/ phone number of appropriate contact person
– office address of requesting physician
– diagnosis: ICD9 code or narrative description
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Specimen collection
• Prioritize collection
– "STAT" means special turnaround time and must be
collected immediately.
– It may involve a patient whose medical condition has
suddenly become very critical and must be treated as a
medical emergency.
• Fasting: Requests for "fasting" specimens are
performed before routine requests, so patients can
eat meals on schedule.
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Order of draw
• Blood collection tubes must be drawn in a
specific order to avoid cross-contamination of
additives between tubes.
The recommended order of draw for plastic
vacutainer tubes is:
• First – blood culture bottle or tube (yellow or yellowblack top)
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• Second – coagulation tube (light blue top).
– If just a routine coagulation assay is the only test
ordered, then a single light blue top tube may be
drawn.
– If there is a concern regarding contamination by
tissue fluids or thromboplastins, then one may
draw a non-additive tube (red top) first, and then
the light blue top tube.
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• Third – non-additive tube (red top)
• Last - additive tubes in this order:
– SST (red-gray or gold top): Contains a gel separator
and a clot activator.
– Sodium heparin (dark green top)
– PST (light green top): Contains lithium heparin
anticoagulant and a gel separator.
– EDTA (lavender top)
– ACDA or ACDB (pale yellow top): Contains acid
citrate dextrose.
– Oxalate/fluoride (light gray top)
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• Tubes with additives must be filled to their
stated volumes and thoroughly mixed.
• Erroneous test results may be obtained when the
tube is not filled to the stated volume and blood is
not thoroughly mixed with the additive.
• Mix all tubes with anticoagulant by gentle
inversion for 15 seconds.
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Types of blood specimens
• Venous blood
• Capillary blood
– Finger prick specimen
– Heel prick specimen
• Arterial blood
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Types of blood samples
• Non anticoagulated blood samples
– Blood clotting occurs and in 20 min serum can be
separated
• Anticoagulated blood samples
– Na Citrate
– Na oxalate
– Na/K EDTA
– heparin
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Stopper Color (Plastic Tubes) Additive
1. Blood Culture
Broth mixture
2. Light Blue*
Sodium Citrate
3. Red
None
4. SST (Red-Gray, or Gold
stopper)
5. Dark Green
Gel separator + clot activator
6. PST - Light Green
Lithium Heparin + gel separator
7. Lavender
EDTA
8. Pale Yellow (ACDA or
ACDB)
9. Light Gray
Acid citrate dextrose
Sodium Heparin
Oxalate/fluoride
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Red Top/Serum:
Chem: all chemistry panels
• Thyroid: T3, T4, T-Uptake, TSH
• Coronary Risk (CHD): Chol, HDL, LDL
• Tumor Markers: PSA, CEA, B-HCG, AFP
• Hepatitis: HBsAg, HBsAb, IgM anti-HBc, Hepatitis C, Hepatitis A
(HAVAB, HAVAB-M)
• Infectious disease molecular tests for RNA or DNA.
• AIDS Panel: HIV-1 EIA, Western Blot
• Misc: RPR (syphilis), RA screen, nicotine, alcohol, beta-2
microglobulin, CDT
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Lavender/Purple Top/EDTA tube:
• Anticoaqulant of choice for routine
haematologic work
– CBC
– Molecular test for RNA or DNA
– Glycohemoglobin (glycoHB, glycoA1C, Hb A1C)
– Sedimentation Rate (ESR)
– Reticulocyte Count
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• An excess of EDTA affects red cells, white
cells and platelets causing shrinkage and
degenerative changes
• Excess of 2mg/ml may result in significant
– decrease in packed cell volume and
– an increase in mean corpuscular haemoglobin
concentration
• Mix the anticoagulant and blood by repeated
inversion of the container
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Blue Top/Sodium Citrate:
• PT
• APTT
• Fibrinogen
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Gray Top/Sodium Fluoride:
• Glucose, Blood Alcohol
Urine:
• Urinalysis
• Drug Screen
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Heparin
• Used in the osmotic fragility test and for
immuno phenotyping
• Should not be used to prepare blood films due
to faint blue coloration to the background.
• Do not use this for WBC as it causes WBC to
clump
• It does not affect RBC size and less likely to
cause haemolysis than EDTA
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Tri sodium citrate
• It is the choice for coagulation studies
• This is the anticoagulant for ESR
– 1 volume of sodium citrate is mixed with 4
volumes of blood
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Difference between serum and
plasma
• Serum is obtained from clotted blood that has not
been mixed with an anticoagulant
• This clotted blood is then centrifuged, yielding
serum
– Which contain two types of protein:- albumin and
globulin
• Plasma is obtained from blood that has been
mixed with an anticoagulant in the collection
tube
– Contain:- albumin, globulin and fibrinogen
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Sampling problems - blood
• Inappropriate site
– Drip arm
– Mastectomy, burns
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Timing
Incorrect use of tourniquet
Wrong container
Incorrect order of draw
Transportation
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Urine collection
• 24 hour sample must include all urine passed
in this period
• If less than 24h, inform the lab
• Mid stream sample – self explanatory
• Early morning sample- often best
• Correct container type
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Urine samples
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Incorrect timing
Inappropriate for test required
Sterility
volume
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Other consideration
• Swabs for culture may need specific transport
media e.g: clamydia
• Blood cultures- special bottles and technique
for taking the blood
• Tissue for histology- fixative
– Extreme care needed when using formalin
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The contribution of the ward staff to
valid laboratory results
• Providing correct and adequate information to
the laboratory staff
• Correct patient preparation for test and
providing correct advice to patients on
preparation
• Collection of specimens by the correct
technique at the appropriate time
• Correct labeling
• Speedy delivery to the lab
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General specimen collection
• Some of the common considerations affecting
all types of specimens include
– Insufficient quantity of specimen to run test
– Failure to use correct container
– Inaccurate patient instruction prior to collection
– Leakage and contamination of specimens
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Other factors affecting laboratory
results
Exercise
• Moderate exercise can cause an increase in
– Blood glucose
– Lactic acid
– Serum protein
– CK
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Emotional / physical stress
Time of day
• Diurnal variations and variations in circadian
rhythm can also affect test results
– GH peaks in the morning before waking and
decreases throughout the day
– Serum iron levels may change as much as 30%
to50% , depending on individual variation, from
morning until evening
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Timed specimens
• Two types of timed specimen
– One is for a single blood specimen ordered to be
drawn at a specific time
– Other is for a test that may require multiple blood
specimens to be collected at several specific times
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• Types of anticoagulants
– EDTA
– Citrate
– Heparin
– Oxalate
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Effects of storage on blood cell morphology
• The changes increase with time
• Minimized by storing samples at 4 degrees
Quantitative effects on storage
• Red cells swell
• PT and osmotic fragility increases
• ESR,WBC, platelet count decreases
• Hb remain unchanged
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Specimen transport and storage
• All laboratory specimens shall be placed in
leakproof containers (i.e., culturettes, vacuum
tubes),
• then bagged in single, biohazard specimen
bags.
• Place the requisition slip in the outside pocket
of the biohazard specimen bag.
• Tubed Specimens: Specimens may be sent
through the tube system
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• To ensure the validity of test results and the
safety of laboratory personnel,
– specimens that leak in transit will be discarded,
– and the sender notified to resend another sample
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Spill Cleanup
• If a pneumatic capsule is received that appears grossly wet
or soiled,
• wear gloves before handling the capsule and removing the
contents.
• Be aware that there may be broken glass or plastic inside!
• Remove sharp objects (broken glass or plastic) using
forceps.
• Discard any wet or soiled padding as infectious waste.
• Clean the inside and outside surfaces of the pneumatic
capsule with a hospital-grade disinfectant.
• Call the physical plant dispatcher and notify them of the
contamination of the tube system
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The Basics of Specimen Collection and
Handling of Urine Testing
Types of Collection
• Laboratory urine specimens are classified
– by the type of collection conducted or
– by the collection procedure used to obtain the
specimen.
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Random Specimen
• This is the specimen most commonly sent to the
laboratory for analysis,
• Primarily, it is the easiest to obtain and is readily
available.
• This specimen is usually submitted for urinalysis
and microscopic analysis,
• although it is not the specimen of choice for either
of these tests.
• Random specimens can sometimes give an
inaccurate view of a patient's health if the
specimen is
– too diluted and
– analyte values are artificially lowered.
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• Pediatric specimens, which routinely undergo
chemistry and microscopic analysis, are generally
of this type.
• As the name implies, the random specimen can be
collected at any time.
• Although there are no specific guidelines for
– how the collection should be conducted,
– avoiding the introduction of contaminants into the
specimen is recommended.
• This requires explicit instructions to patients so
that they do not touch the inside of the cup or cup
lid
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First Morning Specimen
• This is the specimen of choice for urinalysis and
microscopic analysis,
– since the urine is generally more concentrated (due to
the length of time the urine is allowed to remain in the
bladder) and,
– therefore, contains relatively higher levels of cellular
elements and analytes such as protein, if present.
– Also called an 8-hour specimen, the first morning
specimen is collected when the patient first wakes up
in the morning, having emptied the bladder before
going to sleep.
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– Since the urine can be collected over any eighthour period, collection is practical for patients who
have atypical work/sleep schedules.
– Proper collection practices and accurate recording
of the collection time are important criteria of a
first morning specimen.
– Any urine that is voided from the bladder during
the eight-hour collection period should be pooled
and refrigerated, so that a true 8-hour specimen is
obtained
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Midstream Clean Catch Specimen
• This is the preferred type of specimen for culture and
sensitivity testing
– because of the reduced incidence of cellular and microbial
contamination.
• Patients are required to first cleanse the urethral area.
• The patient should then void the first portion of the urine
stream into the toilet.
• These first steps significantly reduce the opportunities for
contaminants to enter into the urine stream.
• The urine midstream is then collected into a clean container
(any excess urine should be voided into the toilet).
• This method of collection can be conducted at any time of
day or night.
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Timed Collection Specimen
• Among the most commonly performed tests requiring
timed specimens are those measuring
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creatinine,
urine urea nitrogen,
glucose,
sodium,
potassium, or
analytes such as catecholamines and 17-hydroxysteroids
that are affected by diurnal variations.
• A timed specimen is collected to measure the
concentration of these substances in urine over a
specified length of time,
– usually 8 or 24 hours.
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• In this collection method,
– the bladder is emptied prior to beginning the timed
collection.
– Then, for the duration of the designated time period, all
urine is collected and pooled into a collection container,
– with the final collection taking place at the very end of that
period.
– The specimen should be refrigerated during the collection
period, unless otherwise requested by the physician.
– Accurate timing is critical to the calculations that are
conducted to determine analyte concentrations and ratios.
– Interpretations based on faulty calculations can result in
improper diagnoses or medical treatment
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Catheter Collection Specimen
• This assisted procedure is conducted when a patient is
bedridden or cannot urinate independently.
• The healthcare provider inserts a foley catheter into the
bladder through the urethra to collect the urine
specimen.
• Specimens may also be collected through an existing
foley catheter.
• Specimens may be collected directly from a foley into
an evacuated tube or transferred from a syringe into a
tube or cup.
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Suprapubic Aspiration Specimen
• This method is used when a bedridden patient cannot be
catheterized or a sterile specimen is required.
• The urine specimen is collected by needle aspiration
through the abdominal wall into the bladder.
Pediatric Specimen
• For infants and small children, a special urine collection bag
is adhered to the skin surrounding the urethral area.
• Once the collection is completed, the urine is poured into a
collection cup or transferred directly into an evacuated tube
with a transfer straw.
• Urine collected from a diaper is not recommended for
laboratory testing
– since contamination from the diaper material may affect test
results
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Urine Collection Containers (cups for collection and
transport)
• Urine collection container cups come in a variety of
shapes and sizes with lids that are either snap on or
screw on.
• To protect healthcare personnel from exposure to the
specimen and protect the specimen from exposure to
contaminants,
– leak-resistant cups should be utilized.
• Some urine transport cup closures have special access
ports that allow closed-system transfer of urine directly
from the collection device to the tube.
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Urine Collection Containers (24-hour collection)
• Urine collection containers for 24-hour specimens
come in a variety of shapes and colors,
– but most are of 3 liter (L) capacity and
– are amber colored
• to protect light-sensitive analytes such as porphyrins and
urobilinogen.
• Closure types vary and some have a port for ease
of specimen transfer into a tube.
• When a preservative is required, it should be
added to the collection container before the urine
collection begins and warning labels should be
placed on the container.
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• If there is more than one acceptable preservative for the
analyte being tested, the least hazardous one should be
selected.
• A corresponding Material Safety Data Sheet (MSDS)
should be given to the patient, and the healthcare
provider should explain any potential hazards.
• Some common 24-hour preservatives are
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hydrochloric acid,
boric acid,
acetic acid and
toluene
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Urinalysis Tubes
• Urine specimens are poured directly into urinalysis tubes with
screw- or snap-on caps.
• Additionally, there are evacuated tubes similar to those used in
blood collection that are filled by using a straw device, from cups
with integrated transfer devices built into their lid, or from direct
sampling devices that are used to access catheter sampling ports.
• Urinalysis tubes come in an array of tube shapes: conical bottom,
round bottom, or flat bottom.
• Conical bottom tubes provide the best sediment collection for
microscopic analysis.
• Some tubes are specially designed to be used with a pipetter that
allows for standardized sampling.
• The tubes you select should be able to withstand centrifugation and,
• if used with an automated instrument system,
– should be compatible with the corresponding racks and carriers.
– Fill volumes of urinalysis tubes usually range from 8 to 15 mL
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Preservatives for Urinalysis
• NCCLS Guidelines recommend testing urine within
two hours of its collection.
• However, refrigeration or chemical preservation of
urine specimens may be utilized
– if testing or refrigeration within a two-hour window is not
possible
• A variety of urine preservatives (tartaric and boric acids
being the most common) are available that allow urine
to be kept at room temperature while still providing
results comparable to those of refrigerated urine.
• Generally, the length of preservation capacity ranges
from 24 to 72 hours.
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• Claims for the length of specific analyte
preservation should be obtained from the
manufacturer.
• When a specimen is directly transferred from a
collection cup into a preservative tube,
– it provides
– a stable environment for the specimen until testing can
be conducted and
– reduces the risk of bacterial overgrowth or specimen
decomposition.
• Non-additive tubes (those not containing any
chemical preservatives) can be used for urinalysis,
– but must be handled following strict timing and
refrigeration guidelines
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Preservatives for Culture and Sensitivity (C&S)
Testing
• The most common preservative used for culture and
sensitivity is boric acid,
– which comes in tablet, powder or lyophilized form.
• There is clinical evidence to suggest that non-buffered
boric acid may be harmful to certain organisms and
– that buffered boric acid preservatives can reduce the
harmful effects of the preservative on the organisms.
– Preserved urine specimens can be stored at room
temperature until time of testing.
– Product claims regarding duration of preservative potency
should be obtained from the particular manufacturer
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Measuring chemical properties of
urine=Urinalysis
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Glucose
Ketones
Protein
Blood- haematuria
pH
Specific gravity
Microscopic examination
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Serum
Collection
Venous blood in sterile test tube
• let clot for 20 - 30 minutes at ambient temperature
• glass better than plastic
Handling
Place at 4-8oC for clot retraction
for at least 1-2 hours
Centrifuge at 1 500 RPM for 5-10 min
• separates serum from the clot
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• Transport
– 4-8oC if transport lasts less than 10 days
– Freeze at -20oC if storage for weeks or months
before processing and shipment to reference
laboratory
• Avoid repeated freeze-thaw cycles
• destroys IgM
• To avoid hemolysis: do not freeze
unseparated blood
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Specimen Collection and Transport Guidelines
• As with any type of laboratory specimen, there are
certain criteria that need to be met for proper collection
and transportation of urine specimens.
• This will ensure proper stability of the specimen and
more accurate test results.
• All urine collection and/or transport containers should
be clean and free of particles or interfering substances.
• The collection and/or transport container should have a
secure lid and be leak-resistent.
• Leak-resistent containers reduce specimen loss and
healthcare worker exposure to the specimen while also
protecting the specimen from contaminants.
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• It is good practice to use containers that are made of
break-resistant plastic, which is safer than glass.
• The container material should not leach interfering
substances into the specimen.
• Specimen containers should not be reused.
• The NCCLS guidelines for urine, GP-16A2,
recommend the use of a primary collection container
that holds at least 50 mL, has a wide base and an
opening of at least 4 cm.
• The wide base prevents spillage and a 4 cm opening is
an adequate target for urine collection.
• The 24-hour containers should hold up to 3L.
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• The NCCLS guidelines recommend sterile collection containers for
microbiology specimens.
•
• The containers should have secure closures to prevent specimen loss and to
protect the specimen from contaminants.
• Transport tubes should be compatible with automated systems and
instruments used by the lab.
• Collection containers and/or transport tubes should be compatible with the
pneumatic tube system if one is used for urine specimen transport in the
facility.
• A leak-proof device in this situation is critical.
• NCCLS recommends the use of an amber colored container for specimens
being assayed for light sensitive analytes, such as urobilinogen and
porphyrins.
• The colorant prevents the degradation of certain analytes.
• Proper labeling should be applied to the collection container or tubes
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Phlebotomy
1) Wash hands thoroughly before beginning any
phlebotomy procedure.
• Be sure to check expiration dates on tubes before
proceeding.
• DO NOT USE EXPIRED TUBES.
2) Confirm the identity of the patient by checking at least
two identifiers before collecting the specimen(s).
• This can be done by asking the patient to state their full
name and requesting to see the patient's driver's license
to verify picture, name, date of birth and/or drivers
license number and documenting the information on the
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consent/chain of custody form.
3) Explain the procedure, including small risk of hematoma,
slight pain, and some lightheadedness.
• Inquire whether the patient has a history of fainting or
dizziness with phlebotomy procedures so that ammonia
inhalants can be obtained if necessary.
• Explain that loss of vacuum or a collapsed vein may
necessitate another draw.
4) On a table or desk, assemble all necessary equipment:
cotton balls and/or gauze, tubes, safety needle, alcohol
swab, tourniquet, gloves, and bandaid.
• Wearing safety gloves is MANDATORY.
• Wear additional protective equipment if contamination is
expected.
• Safety needles should always be used; the only exception is
if the patient is very hard to draw then a butterfly needle set
may be used.
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5) Position the patient so that they are seated comfortably in a
chair with their arm extended on an armrest, desk, or table to
form a straight line from the shoulder to the wrist.
• The patient’s arm and elbow should be firmly supported, and
not bent at the elbow.
6) Check both arms to select the larger and fuller veins.
• Palpate and trace the path of the veins several times with your
index finger.
• Tap the vein at the site of the draw with your index finger and
second finger.
• This will cause the vein to dilate.
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The following factors should be considered in site
selection:
• i) Extensive scarring.
– Healed burn areas or scar tissue should be avoided.
• ii) Specimens collected from an area with a
hematoma may yield erroneous test results.
– If another vein site is not available, the specimen
should be collected distal to the hematoma.
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7) Apply the tourniquet.
8) Ask the patient to open and close his/her fist
so their veins become prominent.
• Vigorous hand pumping is not necessary to
activate blood flow and should be avoided.
9) Clean the venipuncture site with the alcohol
swab in a circular motion from the center of
the area to the outside.
– Allow the area to air-dry to prevent haemolysis
and a burning sensation to the patient.
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10) Insert the stopper of the first tube to be drawn into the
adaptor.
• Do not push too far to avoid premature loss of vacuum
via puncture of the needle.
• The recommended order of draw when drawing more
than one tube is as follows:
♦ Non additive tube (red stopper)
♦ Coagulation tube (light blue stopper)
♦ Serum separator tube (SST) or serum tube
♦ Additive tube (lavender stopper, green stopper, etc)
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11) Insert the needle into the vein with the bevel facing
upward.
– Puncture the stopper on the tube by pushing it onto the
end of the needle, and grasp the edge of the adaptor to
provide stability once the blood flow has begun.
• Have the patient open his/her fist.
12) Fill the tube until the vacuum is exhausted.
• Remove the tube from the adaptor and insert
subsequent tubes.
• Be sure that all tubes are completely filled to ensure
sufficient blood sample for laboratory analysis.
.
91
13) Place a cotton ball or 2 x 2 square piece of gauze over the
site.
– All used needles must be disposed of in a puncture proof
biohazard receptacle.
– Never recap a needle.
–
–
–
–
–
Recapping,
purposeful bending,
breaking,
removing from disposable syringes, or
other manual manipulations of needles
• is prohibited.
• Apply pressure to the site for 2-5 minutes.
• Place a bandaid over the puncture site.
14) Again verify that the information on the sample tubes
match the consent/requisition form
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15) Remove gloves and dispose of in a properly identified
biohazard bag or container.
– Wash hands thoroughly after phlebotomy.
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Additional venipuncture consideration
1) Prevention of Hematoma:
• a) Puncture only the uppermost wall of the vein
• b) Release the tourniquet before removing the needle from
the vein.
• c) Use only major veins; not superficial veins
• d) Make sure that the needle fully penetrates the uppermost
wall of the vein.
• Partial penetration may allow blood to leak into the soft
tissue surrounding the vein by way of the needle bevel.
• e) Apply a small amount of pressure to the area with the
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2) Prevention of Hemolysis:
• a) Mix anticoagulated specimens thoroughly
by inverting each tube gently 8 to 10 times.
– Do not shake.
– Vigorous mixing may cause hemolysis.
• b) Avoid drawing blood from an area with a
hematoma.
• c) Ascertain that the venipuncture site is dry
without touching it.
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3) If a Blood Sample is Unobtainable
• a) Change the position of the needle.
– If the needle has penetrated too far into the vein, pull it back slightly.
– If it has not penetrated far enough, advance it farther into the vein.
– Rotate the needle a half-turn.
• b) Try another tube; the tube may not have sufficient vacuum.
• c) Loosen the tourniquet.
– It may be applied too tightly, thereby stopping the blood flow.
– Reapply the tourniquet loosely.
– This procedure can be accomplished easily when using the velcro-type
tourniquet by releasing it and quickly pressing it together again.
• d) Probing for the vein is NOT recommended as it is painful to the
patient. In most cases, another puncture in a site below the first site
is advised
• e) Never attempt a venipuncture more than twice.
– Have another person attempt to draw the specimen
96
Blood for smears
Collection
Capillary blood from finger prick
• make smear
• fix with methanol or other fixative
Handling and transport
Transport slides within 24 hours
Do not refrigerate (can alter cell morphology)
97
Fingerstick Collection
Instructions
• 1) Have the applicant wash his/her hands in warm, soapy
water.
– Rinse and dry completely.
• 2) Thoroughly clean site of skin puncture with alcohol swab
and allow finger to air dry.
– Remove collection card from envelope.
• 3) Puncture finger firmly with lancet near the tip but slightly to
the side.
– It is important to obtain a free flow of blood without
excessive squeezing, as this may dilute the blood with
tissue fluid.
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• 4) With the applicant’s/donor’s arm
slanting downward, palm upright, add
drops of blood to the small rectangular
section of the card until the strip turns
red at the first (dark) line.
– Do not touch the collection strip in
the middle of the card.
– Lay the card on a clean, dry surface
while placing blood on the card
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• 5) When the card is filled per above, apply pressure to
fingertip with alcohol swab until bleeding stops.
• 6) Place adhesive bandage over puncture site.
• 7) Do not attempt to re-cap lancet.
– Place used lancet in lancet disposal tube, dispose of lancet
– NEVER RETURN A USED LANCET!
• 8) Allow blood on card to air dry for at least 20-30 minutes.
Place card into Blood Sample
– Return Bag and seal.
– Place the signed security seal from the consent over sealed end
of Sample Return Bag.
• 9) PLEASE BE SURE TO INCLUDE THE SIGNED
AUTHORIZATION WHEN SENDING SPECIMENS TO
THE LAB.
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101
Bacteriology specimen collection
Collection of Specimens for Culture:
General Information
• 1) Labeling.
– Appropriate information is critical to proper
processing of test requests.
• Although pertinent clinical information is highly
desirable, if it is not available, please provide at
least the following information
–
–
–
–
–
a) Patient’s name and second unique patient identifier
b) Source of specimen or collection site
c) Date
d) Specimen
e) Test desired
102
• 2) Obtain specimen correctly.
– a) Explain completely to the patient.
– b) Use a sterile container
– c) Label correctly and send the specimen to the
laboratory promptly
– d) Avoid contamination of the container
103
• 3) Timing of collection
– a) Sputum, urine, stool, etc. are best collected in
early morning and sent to the laboratory the same
day.
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b) Blood
Collection
Venous blood
• infants: 0.5 – 2 ml
• children: 2 – 5 ml
• adults: 5 – 10 ml
Requires aseptic technique
Collect within 10 minutes of fever
• if suspect bacterial endocarditis: 3 sets of
blood culture
105
• i) A blood culture requires two bottles of blood —
one for aerobic and one for anaerobic culture.
• ii) Collect blood specimens before treatment is
initiated, if possible.
• iii) Collect two or three sets early in the illness;
repeat if they are negative after 48 hours of
growth
– Each blood culture should be collected from a separate
venipuncture
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• iv) Organisms are continuously shed during
intravascular infections, such as
– endocarditis, but they are intermittently shed during
occult infections.
– In some instances of occult infection, there is a
predictable fever pattern.
– If this is the case, the blood for culture is best
collected 30 minutes prior to the fever spike.
• v) The yield beyond three or four cultures is
minimal in most circumstances, and collection of
more than this is discouraged.
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Handling and Transport
Collect into bottles with infusion broth
• change needle to inoculate the broth
Transport upright with cushion
• prevents hemolysis
Wrap tubes with absorbent cotton
Travel at ambient temperature
Store at 4oC if can’t reach laboratory in 24h
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Obtaining culture specimens from the
nasopharyngeal area and the throat.
• 1) A nasopharyngeal culture is obtained by
inserting a thin sterile swab gently through the
nose to touch the pharynx; gently rotate and
remove.
• 2) A throat culture is obtained by introducing a
sterile swab into the mouth.
• Use a tongue blade to avoid contaminating the
specimen with oral secretions.
• Firmly swab both tonsilar fossae, posterior
pharynx, and any inflamed or ulcerated areas.
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Lower Respiratory Tract Sputum.
• sputum cultures,
• including such alternatives as
– induced sputum, tracheal aspiration, and bronchial
washings.
1) Rinsing the mouth with saline or water (but not
mouthwash) may reduce contamination with normal
oropharyngeal flora.
2) Encourage deep cough with expectoration of the
sputum into a sterile specimen collection cup that is
labeled with the patient's name and second patient
identifier.
3) Do not send saliva (spit) for culture
110
4) When the patient is unable to cough
productively, notify the physician.
– An alternative method may be ordered, such as:
a) Induced sputum.
– This is done by a respiratory therapist on the
orders of the physician.
– Involuntary deep coughing is induced by irritation.
b) Tracheal aspiration.
– The trachea is gently irritated with a small lumen
suction catheter,
• which causes deep, productive coughing.
– Also, the specimen may be aspirated with a
syringe.
111
c) Bronchial washings.
– These are done by the physician in the operating
room at the time of bronchoscopic examination.
5) A small amount of sputum is all that is
required, but it must be sputum and not oral
secretions.
112
Specimens of Wound Exudate.
• Follow these steps for using a sterile transport swab in
collecting wound exudate specimens.
1) Gently cleanse the area, using dry, sterile gauze to
remove any contaminants.
2) Using a sterile red-stopper swab culture collection
system, introduce deeply enough to obtain a moist
specimen; replace the swab in the container.
• Do not break the container.
• 3) Store at room temperature.
113
Stool for Culture.
When collecting stool specimens, follow these
guidelines.
1) A small amount is all that is required, about
the size of a walnut.
– If several different types of cultures are requested,
submit a walnut-sized sample for each.
– Place the specimen in transport medium or in a
sterile leakproof container.
2) When stool cultures are not readily obtainable,
rectal swabs are acceptable;
– however, it must be indicated whether the
specimen is a stool or a rectal swab.
114
Use of Sterile Swab (Red-Stopper) Collection Kit
• The swab system is guaranteed sterile until the seal is
broken.
• Directions for use:
1) Peel open and remove the swab from the package.
2) Remove the cap/swab stick from the tube.
3) Collect the appropriate specimen and put the cap/swab into
the tube. Push the cap to bring the swab into contact with
the transport medium.
4) Print the patient's name, second patient identifier and the
culture site on the specimen tube.
5) Place the specimen in a zip-lock bag and put the completed
test request form in the side pouch
6) Store it at room temperature.
7) Send specimen to the laboratory
115
116
Stool Specimen
Collection:
Freshly passed stool samples
• avoid specimens from a bed pan
Use sterile or clean container
• do not clean with disinfectant
During an outbreak - collect from 10-20
patients
117
Stool Specimen
Analysis of fecal material can detect pathological
conditions ie: tumors, haemorrhage, infection
• Tests
– Occult blood
– Pus
– Ova & Parasites
118
• ? Chemical preservatives
• Medical aseptic technique
• To lab on time
• Labelling
• Documentation
Guaiac Test
Colorectal cancer screening test
FOBT
Hemoccult slide test
119
• Color
– melena
•
•
•
•
•
•
Odor
Consistency
Frequency
Amount
Shape
Constituents
120
Guaiac Test
• Single positive test result does not confirm
bleeding or colorectal cancer.
• Repeat test 3X
• Meat free, high residue diet
121
Stool samples for viruses
Timing
– within 48 hours of onset
Sample amount
– 5-10 ml fresh stool from patients (and controls)
Methods
– fresh stool unmixed with urine in clean, dry and sterile container
Storage
– refrigerate at 4oC; do not freeze
– store at -15oC - for Ag detection,polymerase chain reaction
(PCR)
Transport
– 4oC (do not freeze); dry ice for (Ag detection and PCR)
122
Stool samples for bacteria
Timing
– during active phase
Sample amount and size
– fresh sample and two swabs from patients,
controls and
carriers (if indicated)
Method
– Cary-Blair medium
– For Ag detection/PCR – no transport medium
Storage
– refrigerate at 4oC if testing within 48 hours, -70oC if longer; store at 15oC for Ag detection and PCR
Transport
– 4oC (do not freeze); dry ice for Ag, PCR detection
123
Stool samples for parasites
Timing
– as soon as possible after onset
Sample amount and size
– at least 3 x 5-10 ml fresh stool from patients and controls
Method
– mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1
part preservative
– unpreserved samples for Ag detection and PCR
Storage
– refrigerate at 4oC; store at -15oC for Ag detection and PCR
Transport
– 4oC (do not freeze); dry ice for antigen detection and PCR
124
Rectal swabs
Advantage
– convenient
– adapted to small children, debilitated patients and
other situations where voided stool sample not
feasible
Drawbacks
– no macroscopic assessment possible
– less material available
– not recommended for viruses
125
Post-mortem samples
Collection
Biopsy relevant tissues
• place in formalin for histopathology
• place in transport medium for microbiological testing
• place in sterile saline for isolation of viral pathogens
126
Handling and transportation
Fixed specimens can be transported at ambient
temperatures
• transport specimens in transport media within 24h at ambient
temperature
• transport specimens in sterile saline at 4-8oC within 48h
127
Biosafety: protect the patient
Use single use equipment
Disinfect
Work in a clean, dedicated area
128
Biosafety: protect yourself
Use personal protective equipment
– disposable gloves
– laboratory coats / gown
– mask
– protective eyewear / face shields if procedure is likely
to generate aerosols
If no sharps container: collect sharps immediately to prevent needlestick injury
Have first aid kit readily accessible
Do not reuse contaminated equipment
129
Biosafety: protect others, the
environment
Package samples appropriately for transport
Decontaminate spills - 10% bleach after wiping the surface clean
Disinfect working areas for future use - 1% household bleach daily
Soak contaminated non-disposable equipment/material in 1%
household bleach for 5 minutes
– wash in soapy water before re-use, sterilize if
necessary
Place waste in leak-proof biohazard bags - ensure safe final
management of waste
Protect cleaning/decontamination personnel with protective coat, thick
rubber gloves
130
Cerebrospinal fluid (CSF)
Collection
Lumbar puncture
Sterile tubes
Aseptic conditions
Trained person
131
Handling and transportation
Bacteria
• preferably in trans-isolate medium,
• pre-warmed to 25-37°C before inoculation
OR
• transport at ambient temperature (relevant pathogens do not
survive at low temperatures)
Viruses
• transport at 4-8oC (if up to 48hrs or -70oC for longer
duration)
132