1. INTRODUCTION TO
MEDICAL LABORATORY TEST
EP0211 Introduction to Biomedical Science for Healthcare
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Introduction to Medical Laboratory Test
Explain the Workflow in Medical Testing
Practical skills and knowledge in specimen collection,
handling, testing, verification and reporting
Factors affecting integrity in Medical Testing
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What you will learn in this chapter..
■ Describe the various types of medical laboratory tests
■ Explain the workflow in a medical laboratory test (pre-analysis,
analysis and post analysis)
■ Discuss the procedure for safe collection, handling, and
disposal of patients’ specimens (blood and non-blood)
■ Discuss the use of the evacuated collection system and anticoagulants for blood collection
■ Identify variables that can affect sample and test integrity in
medical testing
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A. Workflow in Medical Laboratory Test
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Medical Diagnostic Services
Diagnostic Services
Laboratory Services
Imaging
Physiological tests
Clinical biochemistry
Haematology
Histopathology
Immunology
Microbiology
X-Ray
MRI (Magnetic
Resonance Imaging)
ECG
EEG
Lung function
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Workflow in Medical Laboratory Test
Analysis
Pre-Analysis
Sample
Collection
• Specimen
Collection
• Labelling/
Identification
• Documentation
• Transportation
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Sample
Processing
Sample
Analysis
Centrifuge
Decap
Aliquoting
Reagent
Automated/
Manual
• Measurement
with analysers
• Spectrophoto
metry
• Immunoassay
• Microscopic
• Microbiology
Post-Analysis
Data
Verfication
• QC check
samples
• Internal
validation
• External
validation
Reporting
• Report
generation
• Storage of
specimen
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Workflow in Medical Laboratory Test
Specimen Collection
Labeling &
Identification
Documentation
(Transport)
Request
Pre-analysis
Analysis
Post-analysis
Final Test
Results
Laboratory Analysis
(Sample
Preparation
& Analysis)
Results
Data verification
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Pre-Analytical Phase
■ All the steps involved before a sample can be analysed
■ Pre-analytical factors
o Patient-related variables
o Specimen collection techniques
o Specimen preservatives and anticoagulants
o Specimen transport
o Process and storage
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Pre-Analytical Phase
■ Patient related variables
o Age
o Gender/ Race
o Medication/ Drug
o Diet
o Tobacco smoking
o Exercise
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Pre-Analytical Phase
■ Specimen collection and transportation
o Time of collection (morning/ afternoon)
o Correct tubes and order of draw (blood)
o Addition of preservatives (urine)
o Sample storage before analysis (duration)
o Temperature and light sensitive samples
o Labelling and documentation and samples
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Analytical Phase
■ All the steps involved in analysing the samples
■ Analytical factors
o Centrifuge of samples (blood)
o Decaping and aliquoting
o Reagent type and volume
o Analysers (automated/ modular/ stand-alone)
o Spectrophotometric, light scattering, electrochemistry,
immunoassay, others
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Post-Analytical Phase
■ All the steps involved in the data verification and reporting
■ Post analytical factors
o Internal quality control (QC Check samples)
o External quality control (external lab)
o Instrument maintenance & calibration
o Performance qualification, operational qualification
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B. Specimen Collection and Handling
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Specimen Types
Blood
■ Urine
■ Cerebral Spinal Fluid
■ Faeces
■ Saliva
■ Pleural Fluid
■ Synovial Fluid
■ Gastric Fluid
■ Amniotic Fluid
■
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Precautions for blood sample collection
Centre for Disease Control & Prevention (CDC):
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■
Wear gloves/change gloves between patients
Wash hand as needed
Wear gowns that are impermeable to liquids
Wear mask and protective eyewear
Throw needles and sharps into ‘sharp containers’
immediately after use without recapping
■ Do not break or bend needles
■ Do not handle patients if you have infectious condition
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Blood Specimen Components
Serum – clotted
Gel
Red Blood Cells
White blood cells
Platelets
Clinical
Chemistry/
Immunology
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Professional conduct for specimen collection
■ Confident
■ Professional
■ Polite
■ Calm & Composed
■ Assure the Patient
■ Refer all questions to the patient’s doctor
■ Avoid revealing any test results to the patient or even
family members (maintain medical confidentiality)
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Blood Specimen Collection Preparation
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Ascertain patient’s identity
Know the tests requested
Ensure patient is fasted if required
Know the site of blood collection
Observe universal precautions
Choose the appropriate equipments
Ensure correct order of draw
Ensure blood drawn is not lysed
Ensure appropriate volume drawn
Correctly label the specimen
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Phlebotomy Supplies
 Phlebotomy trays are used in hospitals and clinics
 Convenient access of tubes, syringes, gauze, torniquets
 Phlebotomy trays should not be placed on patients’ bed
 Change gloves and wash hands between patients
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Blood Collection Needles
 Multi-sample needles (evacuated tube system)
 Hypodermic needles (syringe system)
 Winged infusion (butterfly) needles
 For both evacuated tube & syringe systems
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Blood Collection - Needles
Parts of a hypodermic needle
Lumen
Bevel
Point
Hilt
Shaft
Hub
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Blood Collection - Needles
Parts of a multi-sample needle
Bevel
Shaft
Threaded hub
Rubber sleeve
over needle
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Blood Collection - Needles
■ Gauge (G) size: diameter of the needle
– The smaller the gauge number, the larger the
needle diameter and higher the flow rate.
■ To select needle gauge
– Physical characteristics of the vein
– Location of the vein
– Volume of blood drawn
23G
22G
21G
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Blood Collection - Holders
■ Tube holder for the needle
Tube adapter, which makes
collection of blood sample easier
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Blood Collection - Syringes
Parts of a syringe
■ Sizes ranges from 2 to 20 mL most often used in
venipuncture
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Blood Collection - Tourniquets
■ Provides a barrier to slow down venous flow.
– Distended veins become more visible or palpable
– Should not be applied more than 1 minute
■ Types
– Elastic strap
– Velcro type / buckle type
– Blood pressure cuff (40 mmHg)
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Blood Collection - Venipuncture Chairs
■ Designed for maximum comfort and safety of patients
■ Give ergonomic comfort and easy accessibility to patients
■ Both armrests
– adjustable height
– prevent patients from falling if feeling faint
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Blood Collection – Site of collection
 Artery
 Vein
 Capillary
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Arterial Blood Collection
■ Arterial blood gas (ABG)
only arterial blood accurately reflects the amount of PO2
transferred from the lungs
■ PO2
■ PCO2
■ pH
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Capillary Blood Collection
■ Capillary blood is utilized when:
– sample volume is limited, i.e. paediatrics
– repeated venipuncture have resulted in severe vein damage
– patient has been burned or bandaged
– veins must be preserved for emergency intravenous drug
injection or intravenous chemotherapy
■ Volume must be sufficient for:
ESR, coagulation, blood culture tests
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Puncture Site
Correct
NOT this!
 3rd or 4th finger of the non-dominant hand
 Puncture should be made off-center, perpendicular to the
fingerprint ridges.
– a puncture parallel to the ridges tends to make the blood
run down the ridges and hinder collection
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Puncture Site
Pinky
• Tissues is thinner
Middle & Ring finger
• Preferred site of
puncture
Index Finger
Thumb
• Has a pulse.
• More sensitive or
callused (hardened and
thickened)
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Capillary Blood Collection – Lancets
■ Lancet
a sterile, disposable, sharp pointed
or bladed instrument used to pierce
the skin to obtain blood for testing
■ CLSI guidelines
puncturing deeper than 2.0mm on
the plantar surface of the heel of
small infants may risk bone damage
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Capillary Blood Collection – Lancets
BD Quikheel
Lancet puncture
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Capillary Blood Collection – Lancets
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Capillary Blood Collection – Lancets
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Infant’s Heel Puncture Site
■ Lateral or medial plantar surface of the heel
– Plantar surface medial to a line drawn posteriorly from the
middle of the great toe to the heel, or
– Lateral to a line drawn posteriorly from between the 4th and
5th toes to the heel
■ Heel bone is NOT usually located beneath these areas
■ Do not puncture at the curvature of the heel.
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Infant’s Heel Puncture Site
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Heel Prick
■ Heel puncture is generally performed in infants less
than one year old
■ For children older than one, the palmar surface of
the finger’s is used
NCCLS H3-A5. Procedures and Devices for the collection of diagnostic capillary blood specimens;
Approved Standard- 5th Edition.
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Capillary Microtainer Tubes
Minimum Volume: 250µl
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Best Sites for Venipuncture
Large and superficial (near to skin
surface) veins
 Median cubital vein (1)
 Cephalic vein (2)
 Basilic vein (3)
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Sites for Venipuncture
 Median cubital vein
– Proximity to skin’s
surface
– Immobility
– Safety
– Comfort
 Median & cephalic veins
be ruled out first before
selecting basilic vein
– Brachial artery
– Median nerve
NCCLS H3-A5. Procedures for the collection of diagnostic blood specimens by venipuncture;
Approved Standard- 5th Edition.
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Sites for Venipuncture
 Veins to the underside of the wrist must not be used
 Arterial punctures are not to be considered an
alternative to venipunctures
 Alternative sites such as ankles or lower extremities
must not be used without the permission of the
doctor, due to possible complication like thrombosis
NCCLS H3-A5. Procedures for the collection of diagnostic blood specimens by venipuncture;
Approved Standard- 5th Edition.
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Sites for Venipuncture
Avoid:
 Arm on side of mastectomy, due to lymphostasis/ lymphedema,
affecting the blood composition
 Edematous area
 Arm in which blood is being transfused
 Collecting from an arm with intravenous line
 Collecting from site that is extensively scarred or with
haematoma
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Sites to Avoid for Venipuncture
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Sites for Venipuncture
 When blood specimen cannot be obtained
– Change the position of the needle
• Pull the needle back a bit
• Advance it farther into vein
• Rotate the needle half a turn
– Manipulation other than that
• Probing / fishing
• Not recommended
 Lateral needle relocation should never be attempted to access
basilic vein
NCCLS H3-A5. Procedures for the collection of diagnostic blood specimens by venipuncture;
Approved Standard- 5th Edition.
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Complications Associated with Venipuncture
Needle Positioning and Failure to Draw Blood
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NEWS
http://news.bbc.co.uk/2/hi/health/7525932.stm
Accessed 27 Mar 2009
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NEWS
http://news.bbc.co.uk/2/hi/health/7525932.stm
Accessed 27 Mar 2009
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Dorsal Hand Vein Procedure
 Infants below 2 year of age
 Use 21 -23 needle, blood collection set (butterfly)
 Position middle and index finger to form a "V" over the vein,
apply pressure.
 Bend baby's wrist over middle finger
 May be used on adult (veins challenging to locate)
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Dorsal Hand Vein Procedure
 Locate the vein, release pressure, cleanse the site
 Insert the needle, when blood appears in the hub collect in
appropriate tubes/ microtainers
 Steady flow of blood is sustained by applying gentle, periodic
pressure
 After collection, remove needle, apply pressure until bleeding stops
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Dorsal Hand Vein Procedure
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Venipuncture – Vacutainer System
 Vacutainer tubes (evacuated tubes)
– minimize exposure to blood
– minimize exposure of blood to the
environment
– ensure correct blood volume
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Syringes and Needles
 NEVER perform this!
 HIGH risk of getting needlestick injuries
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Syringes and Needles
 Dangerous procedure
• use a needle to transfer blood into a blood collection tube
or culture bottle
 Blood Transfer Device
• safety feature: reduces the risk of transfer related injuries
• single-use, sterile device
• maintains specimen integrity and reduces potential
exposure to patient’s blood
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Syringes and Needles
 Rubber stoppers should not be removed from venous blood
collection tubes to transfer blood to multiple tubes
 Activate the safety feature of the needle or winged blood collection
set and apply a safety transfer device
 Stopper is pierced with the needle and tube is allowed to fill (without
applying any pressure to the plunger) until flow ceases
– This technique helps to maintain the correct ratio of blood to additives
if an additive tube is used.
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Disposal of Needles
 Remove needle and activate the safety mechanism
 Safely dispose of the unit into an easily accessible sharps container
 Needles should not be re-sheathed, bent, broken, or cut nor should
they be removed from syringes unless a safety transfer device is
attached
 Place the activated device in a sharps container without
disassembling from the tube holder.
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After the blood is drawn…
 Maintain several minutes of pressure on the puncture site
 Not always effective to have patients bend their arm up as a
substitute for pressure, as it does not apply adequate
pressure on all veins of the antecubital area
 Labeling should be done at patient’s bedside
 Never label before blood collection
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Vacutainer System – Order of Draw
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Clot Formation
Oxalate
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Heparin
EDTA
Citrate
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Vacutainer System – Order of Draw
 Blood collected into a vacutainer tube containing one
anticoagulant should never be transferred into another
tube of another anticoagulant.
– e.g. K+ from EDTA would falsely increase the K+
concentration result.
 When multiple vacutainer tubes are drawn during a
single venipuncture, the order of draw is important.
– A tube without anticoagulant should be drawn before
tubes with anticoagulant to prevent contamination
with an undesirable component, e.g. K+
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Vacutainer Tubes
 Order of draw – before Serum tubes.
 Mixing – 3 to 4 times.
 Fill volume very important.
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Vacutainer Tubes
 Citrate tubes should be filled between +/10% of the stated draw volume of the tube
 NCCLS Guideline H1-A4, Dec ’96
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Vacutainer Tubes- Citrate Tubes
 When the citrate tube is the first or only tube drawn
 A discard tube is required only when a winged blood
collection set is used
– to prevent the air in the tubing from causing the
tube to be under-filled.
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Vacutainer Tubes
■ SST Tube contains clot activator (silica) which
accelerates clotting. No anticoagulant.
■ SST Tube also contains an inert, polymer gel which
becomes layered between the serum & clot on
centrifugation. It helps to eliminate the metabolic
changes that occur when the clot are in direct contact
with the serum, eg. K+ is released when RBCs lysed.
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Vacutainer Tubes – SST Tube
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Vacutainer Tubes
■ Glass
– Without
anticoagulant.
– Recommended for
blood banking
■ Plastic
– Without anticoagulant But
With Clot Activator.
– Not recommended for
blood banking. Lysis of
RBCs does not help in
direct blood grouping.
– Tube inversions ensure
mixing of clot activator with
blood & clotting within 60
minutes.
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Vacutainer Tubes
■ Thrombin accelerates clotting
■ Centrifuge after blood has completely clotted
Serum Tube Type
Clotting Time
Thrombin
< 5 mins
SST
30 mins
Plain Plastic with Clot Activator
60 mins
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Vacutainer Tubes - Heparin
 Most widely used anticoagulant in clinical chemistry
analysis.
 Accelerates the action of anti-thrombin III, which
neutralizes thrombin, thus preventing the formation of
fibrin from fibrinogen.
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Vacutainer Tubes - Heparin
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Vacutainer Tubes
■ K2EDTA preferred choice for haematological study as it does not
distort the morphology of the blood cells.
■ Under-filling causes erroneous low blood counts & haematocrit,
staining alterations, and erroneous changes to RBC
morphology.
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Vacutainer Tubes
■ Blood glucose concentration decreases at about 10mg/dl
(0.56mmol/L) per hour due to glycolysis.
■ Sodium Fluoride inhibits glycolysis.
■ The oxalate inhibit blood coagulation by binding with calcium ions to
form insoluble complexes.
■ Fluoride destroys enzymes like CK, ALT, AST & ALP.
■ Oxalate distorts cellular morphology, hence cannot be used for
haematology study.
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Other Evacuated Blood Collection Tubes
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Urine Collection
 Most commonly collected body fluids for analysis.
 Random
– Collected at any time of the day.
– Problem with concentration of analytes in urine, as the
volume of urine changes as a result of water intake.
– Example: Drug of Abuse, eg. Cocaine.
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Urine Collection
24 Hour Period
 Urine collected over a 24-hour period.
 Result expressed as Total excreted/24 hours
(concentration divided by volume in litres).
 Problems caused by circadian rhythm and water
input or output are eliminated.
 e.g. Catecholamines (epinephrine,
noreponephrine) in diagnosis of
pheochromocytoma (tumor of adrenal gland)
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Urine Collection
 Early morning Urine
– Most concentrated urine of the day
– Most useful for simple qualitative test
– Example: Pregnancy and Ovulation tests
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Urine Collection
 Mid-stream urine
– Fraction of urine collected in the middle of micturition
– First fraction of urine would wash out the urethra of dead cells
and external bacteria
– Last fraction would contain any leftover deposits of the bladder
– Thus, mid fraction regarded as more representative of urine
leaving the kidneys
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Urine Storage
 When urine cannot be analyzed immediately
o Refrigerate at 2 to 8°C or frozen at –24 to –16°C.
o Preservative like boric acid should be added to urine
sample like the 24-hour urine sample.
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Urine Transport
Urine sometimes leaks
out of urine containers
during transport.
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Urine Transport
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Stool Collection
 Collected in a wide-mouth, sterile
container. Spatula attached to cover
helps to scoop the stool into the
container
 Random
– suitable for majority of tests
 Three-day
– performed to ‘even-out’ any dietary
variation. Eg. Malabsorption. Fat in
stool (steatorrhea). Sudan staining
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C. Sample and Test Integrity in Medical Testing
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Pre-analytical phase
 Greatest potential for Error lies in the
Pre-Analytical Phase
 Controllable
Sample Collection
Sample Transport
Sample Storage
 Uncontrollable
Physiological
Environmental
Biological
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Pre-Analysis Phase: Specimen collection
 Correct tube and inversion of tubes
 Correct order of draw
 Appropriate blood volume
 Haemolysis
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Preanalytical phase: sample storage & transportation
• Blood gases
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Pre-analytical phase: sample preparation & storage
o Centrifuge samples to separate blood cells from plasma and serum
o Red blood cells rupture (lyse) when freezed and thawed
o Avoid repeat freezing and thawing of serum and plasma
o Serum and plasma can be stored up to 3 days at 2 to 8 °C
o Blood should be stored at -20 °C for longer period of storage
o Glucose level decreases during storage (affect the result)
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Haemolysis
 Haemolysis increases plasma potassium, magnesium &
phosphate as they are released by the lysed RBCs
 Serum shows visual evidence of haemolysis when
Haemoglobin concentration exceeds 20 mg/dl
 Haemolysis may affect many laboratory tests and patients’
sample need to be retaken
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Difference in Blood Composition
 Serum has higher concentration for the following analytes
compared to plasma
o Glucose
o Phosphate
o Potassium
5.1%
7.0%
8.4%
 The clotting process causes lysis of red blood cells and the above
compounds are released.
 Venous blood glucose concentration is as much as 7
mg/dl (0.39 mmol/L) less than capillary blood glucose
due to result of tissues utilization
Am J Clin Pathol 1974: 62.
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Pre-analytical phase
What will occur when samples are centrifuged before the clotting
process finishes?
 Clotting continues within the serum and fibrin formed would
obstruct the sample aspiration probes of automated analyzers.
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Analytical phase
 Manual entry into analyzer (key-in wrong information)
 Automated analyzer:
– Samples are matched using bar codes
– Less human error but reliant on the system integrity
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Post Analytical Phase: Verification and Reporting
 LIS
o Laboratory Information System
o Utilize computers to connect lab instruments and computers to
process data associated with requesting tests and reporting results
 HIS
o Hospital Information System
o Utilize computer to manage data collected and generated by various
services, laboratories, and facilities served by a hospital
 Possible transcription error when doing manual entry or system not
connected to LIS and HIS
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End of Chapter. Thank You!
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