Specimen Collection and

Handling for Biochemical

Analysis

Zakayo Thaimuta
Human Pathology
School of Medicine
Why are laboratory tests ordered

• Diagnosis • To check the

• Monitor progression of
disease accuracy of an
• Monitor effectiveness of unexpected data
treatment • To conduct research
• Screening population for
diseases • To prevent
• To identify complications malpractice
of treatment
• For predicting • For educating
survivability, employability residents
• To assess nutritional
status and health of
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2

• Responding to total
 The Test
Measuring an analyte as a Marker to
distinguish health and disease
Ideal Marker
• Absolutely specific for a specific disease
• Easily measurable
• Quantity reflective of severity of disease
• Early detection following onset of disease
• Not affected by other biological
disturbances

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 The Test
Highly Specific marker:
Troponin I. It is a marker of Myocardial infarction (Heart
Attack)
Found predominately in Cardiac Tissue
Released into the blood stream following cell
death
Non specific marker: low blood pH (acidosis)
Very important to know but can be caused by a
hosts of events
Drugs
Respiratory problems
Renal problems
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 Biological Specimens
• Blood Comprise the majority of all
• Urine specimens analyzed

• Cerebrospinal Fluid
• Amniotic Fluid
• Duodenal Aspirate
• Gastric Juice
• Gall stone
• Kidney Stone

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 Biological Specimens

• Stools
• Saliva
• Synovial Fluid
• Tissue Specimen
• Choice of specimen type depends on
– Analyte to be measured
– Ease of collection

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 Blood Composition
Plasma is fluid component of blood.
Comprises ~55% of total volume of
whole blood. Contains proteins,
sugars, vitamins,minerals, lipids,
Plasma lipoproteins and clotting factors.
95% of plasma is water

White Blood cells (WBC)

& Platelets Cellular
Red Blood cells (RBC) Components

Whole Blood Whole Blood after centrifugation

Note: clotting has been prevented
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 Blood Composition

If blood is collected and allowed to stand it will clot. Formation

of an insoluble fibrin clot. If blood is then centrifuged the
fluid portion is known as SERUM
Plasma is fluid component of blood.
Comprises ~55% of total volume of
whole blood. Contains proteins, sugars,
Serum vitamins,minerals, lipids, lipoproteins
No clotting factors
95% of plasma is water

Blood Clot
-comprised of clotting factors (Fibrin,platets etc)
-RBCs

Whole Blood Whole Blood after clotting and centrifugation

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 Blood Analysis
• Source • Collection Method
– Veins – Syringe
– Arteries
– Skin puncture-capillary blood
– Evacuated tube
• Additives
• Separator gel
– Intravenous lines
• Factors affecting choice of Blood Source and Collection
Method
– Analyte under investigation
– Patient
• vascular status
• ease of collection

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 Blood Analysis

• Testing can be done on whole blood, serum

or plasma. Choice depends on a number of
factors
• Analyte to be measured
– Most hematology tests requires whole
blood
• Instrumentation used for analysis
– Most automated instruments are not set up
for whole blood analysis

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 Blood Analysis

• The way the test was developed.

– Tests are often only validated on either
plasma or serum
• Turn around time
– Analysis of whole blood is the quickest. No
waiting for clot or spinning
– Plasma requires centrifugation prior to
analysis
– With serum, the blood must clot then you
have to centrifuge
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 Blood specimen
• Blood can be analysed:
– Whole blood
– Serum
– plasma
• Whole blood for determination blood
gases, ammonia, trace elements,
glucose, urea nitrogen and lactate

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 Blood specimen
• Serum is a specimen of choice for
electrophoresis.
• Plasma forms fibrin clots when it is
stored. This can pose interpretation
difficulties on electrophoretic patterns
• Serum is useful in analyte which have
carrier proteins. Examples of carrier
proteins are thyroid binding globulin,
cortisol binding globulin, albumin
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 Blood specimen
• For example, heparin affect binding of
T3 and T4 to their carrier protein (TBG)
thus producing higher free
concentration of the free component

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 Blood specimen
• Anticoagulants and preservatives of
blood:
– Heparin
– EDTA
– Fluoride
– Citrate
– Oxalate
– Iodoacetate

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 Heparin
• Causes least interference with the tests
• Contains antithrombin which prevents
transformation of prothrombin to
thrombin thus formation of fibrin from
fibrinogen
• Demerits include:
– High cost and temporary action
– Inhibition of acid phosphatase activity
– Inactivation of hydroxybutyrate DH and
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LDH 16
 EDTA
• Useful in hematological examinations
because it preserves the cellular
components of blood
• Inhibits activities of: creatine kinase,
leucine aminopeptidase, alkaline
phosphatase
• Chelating agent for divalent cations e.g.
calcium, magnesium

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 Fluoride
• Preserves blood glucose
• Acts as a weak anticoagulant
• Preserves by inhibiting glycolytic enzymes
• Floride is a potent inhibitor of many serum
enzymes. High floride concentration affect urease-
used to measure urea nitrogen
• Preserves at 25oc for 24hrs and 48hrs at 4oc.
• Rate of glucose consumption is high in neonates
and leukemic patients because of high metabolic
rates in rbc and wbc respectively

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 Blood Analysis in the Chemistry
• Since most tests in the chemistry lab involve analytes
that are dissolved in the fluid portion of blood, serum
or plasma are the specimens of choice.
• Important exceptions include
– Hemoglobin, Red blood cell (RBC) Folate
– Blood gases
• Protein electrophoresis was developed based on the
analysis of serum. Not done on plasma because of
the presence of the protein fibrinogen which distorts
the electrophoretic pattern.
• Many tests can use either serum or plasma

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 Collection Tubes
• The most widely used tubes for blood collection are
evacuated tubes (Vacutainers)
– Negative pressure facilitates collection
– Easy to use
– Sterile
– Universally used colour-coded rubber stoppers to
denote tube type.
– Tubes can contain various anticoagulants for the
collection of whole blood or plasma.
– Tubes can have additives for specific tests
(glucose, metals)

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 Collection Tubes
(Vacutainers)

Separator Gel

Serum
Separator Gel

Serum Separator Tube (SST) Clot

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 Collection tubes

• Red-top tubes contain no anticoagulants

or preservatives
• Red-top tubes are used for collecting
serum
– 10-15 minutes is required to allow blood to clot
before centrifuging
– Used for blood bank specimens, some
chemistries
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 Collection tubes

• Gold (and “tiger”) top tubes contain a

gel that forms a physical barrier
between the serum and cells after
centrifugation
• No other additives are present
• Gel barrier may affect some lab tests
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 Collection tubes

• Used for Glucose measurement.

• After blood collection, glucose concentration decreases
significantly because of cellular metabolism
• Gray-top tubes contain either:
– Sodium fluoride and potassium oxalate, or
– Sodium iodoacetate
• Both preservatives stabilize glucose in plasma by
inhibiting enzymes of the glycolytic pathway
– NaF/oxalate inhibits enolase
– Iodoacetate inhibits glucose-3-phosphate dehydrogenase
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 Collection tubes

• Green-top tubes contain either the Na, K, or lithium

(Li) salt of heparin. Most widely used anticoagulant
for chemistry tests.
– Should not be used for Na, K or Li measurement
– Can effect the size and integrity of cellular blood
components and not recommended for hematology studies
• Heparin accelerates the action of antithrombin III,
which inhibits thrombin, so blood does not clot
(plasma)
• The advantage of plasma is that no time is wasted
waiting for the specimen to clot
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 Collection tubes

• Lavender-top tubes contain the K salt of

ethylenediaminetetraacetic acid (EDTA),
which chelates calcium (essential for clot
formation) and inhibits coagulation
• Used for hematology, and some
chemistries
• Cannot be used for K or Ca tests
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 Collection tubes

• Blue-top tubes contain sodium citrate,

which chelates calcium and inhibits
coagulation
• Used for coagulation studies because it
is easily reversible.

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 Collection tubes

• Brown and Royal Blue top tubes are

specially cleaned for trace metal studies
– Brown-top tubes are used for lead (Pb)
analysis
– Royal blue-top tubes are used for other
trace element studies (acid washed)
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 Urine Specimen
• Urine to be collected is dictated by the
tests to be done
• Urine specimen collected can be:
– Early morning urine-test concentration of many
metabolic constituents and microscopic
examinations
– Random urine-urinalysis
– Catheter urine-for microscopic examinations in
critically ill patients or those with urinary
obstructions

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 Collection of urine
• Early morning sample-qualitative
• Random sample- routine
• 24hrs sample- quantitative
• Midstream sample-UTI

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 24 hour urine sample
1. For quantitative estimation of proteins
2. Creatinine clearance test
3. For estimation of vanillyl mandelic
acid, 5-hydroxyindole acetic acid,
metanephrines
4. For detection of AFB (acid fast
bacillus) in urine
5. For detection of micro-albuminuria
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 Urine Specimen
• Provide instructions before specimen
collection
• Issue instructions on food and drug
ingestion; e.g. test for 5-hydroxyindole
acetic acid advice against consumption
of avocado, banana, plums, pineapples,
acetaminophen & cough syrup
• Collect mid-stream urine
• Cleanliness
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 stool
• Presence of occult blood-clues to
bleeding ulcer or malignant disease in
GIT
• Faeces from children screen tryptia
activity to detect cystic fibrosis
• Fecal nitrogen and fecal fats in 72hr
specimen is used to assess severity of
malabsorption, measurement of fecal
porphyrins is occasionally required to
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 Spinal fluid
• From lumbar region
• Answer questions of cerebrovascular
defect, meningitis, demyelinating
disease or meningeal involvement in
malignant disease
• Antiglycolytic agents are added for
glucose tests, rapid processing is a
requirement for tests on spinal fluid to
limit glucose metabolism
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 Synovial fluid
• Through athrocentesis
• Drawn from joints
• Aid characterisation of the type of
athritis and to differentiate non-
inflammatory effussions from
flammatory ones

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 Amniocentesis
• Prenatal diagnosis of cogenital
disorders,
• To assess fetal maturation
• Look for rhesus iso-immunization
• Intrauterine infection
• Collected with aid of ultra sound to aid
location of placenta and fetal
presentation
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Pleural, pericardial, Ascitic
fluid
• These cavities contain small amount of
serous fluid that lubricate the opposing
parietal and visceral membrane
surfaces
• Inflammation or infection affecting the
cavities cause fluid to accumulate.
• This fluid is removed to determine if it is
an effusion or an exudate-by protein or
enzyme analysis
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• Collected through paracentesis
 Stone
• Only specimen from surgery
• Analysed to know chemical content and
its source
• Physical analysis includes: texture,
colour, shape and size

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 Test results
Variations, Errors, Interferences
• Variations
• Clinical variations within an individual and
between individuals
• Analytical variations-no test is perfect. All tests
have some degree of variations for repeated
measurements of the same sample.
• The final test result is affected by factors that
occur
– Pre-analytically
– At the time of the test
– After the test is completed
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Steps in obtaining a laboratory test
• Test is requested by physician and ordered on
the computer. Barcode or label is generated
• Specimen is collected
• Specimen and order are transported to the lab
• The specimen is accessioned in the lab
• The specimen is processed
• The specimen is analyzed
• The results are reviewed and verified by an
technologists
• The results are released to the patient’s record

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 Why Analytical Results Vary
Inter-individual Variation Pre-analytical Variation
• Age •Transport
• Sex •Exposure to UV light
• Race •Standing time before separation of cells
• Genetics •Centrifugation time
• Long term health status •Storage conditions
Intra-individual Variation Analytical Variation
•Diet
•Random errors
•Exercise
•Systematic errors
•Drugs
•Sleep pattern Post-analytical
•Posture •Transcriptions errors
•Time of venipucture •Results reported to wrong patient
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•Length of time tourniquet is applied
 Pre-analytical errors
• Collection
– Was the right tube used?
– Was venipuncture performed correctly?
– Was the specimen properly stored?
• Identification
– Was the blood collected from the correct
patient?
– Was the blood correctly labeled?
• Patient name, ID, date, time of collection,
phlebotomist
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 Specimen identification
• One of the common sources of
erroneous lab results is misidentified
specimens
• The lab is required to have a clear and
rational policy for identifying specimens,
and handling misidentified specimens

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 Prolonged venous stasis
Blocking the flow of blood with the
tourniquet with eventually lead to a
sieving effect. Small molecules, water
and ions are forced out blood vessels
and larger molecules are concentrated
• Increases Total Protein, proteins, iron
(Fe), cholesterol, bilirubin
• Decreases potassium

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Supine vs. sitting or standing
• Going from lying (supine) to upright reduces
total blood volume by about 700 ml
• The following may decrease by 5-15% in the
supine patient:
– Total protein
– Albumin
– Lipids
– Iron
– Calcium
– Enzymes

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 Specimens requiring special
handling
• Should be placed immediately on ice
– Lactate
– Ammonia
– Acid phosphatase
– Plasma catecholamines

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 Significantly affected by
hemolysis:
• Hemolysis-rupture of red blood cell
– Can be due to improper collection
– End result is dumping cellular contents into
blood. Mild dilution effect in some analytes
• Significant increase in potassium,
magnesium, phosphorous

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 Interferences
• Hemolysis
– The release of hemoglobin into blood can effect
the reactions comprising specific tests
– Causes serum or plasma to be red and can effect
tests that are colorimetric
• Lipemia (lots of fats) and proteinemia (lots of protein)
– Causes serum or plasma to be become turbid.
This can effect colorimetric and turbidometric
based tests
– Also can cause a dilution effect. Fats and proteins
are large and displace water in plasma. Can give
falsely low results especially for Na

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 Interferences
• Human Anti Animal Antibodies.
– Occurs in individual that have been exposed to
foreign immunoglobins
– Can significantly increase or decrease
immunoassay based tests since all utilize animal
antibodies, particularly mouse. Referred to as
Human Anti Mouse Antibodies (HAMA)
– Tests usually contain reagent to clear HAMA
– Technicians performs a dilution test to determine if
HAMA are present
– Generally have to send to another lab to test by
alternate method or different antibody

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Thank You