Errors of the operator can be minimized by the practice
of good technique in the laboratory preparation of the
solutions and in the operation of the instrument. The
operator should be guided in the latter instance by the
instructions provided by the manufacturer. An awareness,
of the sources of error in the preceding categories should
Specimen Collection and Processing
With the exception of glucose, triglycerides and inorganic
phosphorus, most blood chemical constituents reveal no
significant change after a standard breakfast, so it is not
essential for the patient to be in an absolute fasting state
prior to blood specimen collection. However, lipemia
(lactescence), caused by transient rise in triglycerides
as chylomicrons following a meal containing fat may
cause interference with a large number of chemical
determinations because of turbidity. Therefore, blood
is always collected from a patient in the post-absorptive
state. This can be accomplished with an overnight fast
(12–14 hours, especially for lipids), although a 4 to 6 hours
Venipuncture should be performed for obtaining blood.
Disposable needles eliminate the hazard of serum hepatitis
transmission. Heparin is the most ideal anticoagulant for
plasma determination. The cost is quite prohibitive so
others EDTA, and trisodium citrate can be used without
significant alteration in reading and results. For glucose,
oxalate-fluoride mixture is used. Fluoride impairs
glycolysis of the blood cells. Prompt separation of plasma/
serum is essential to yield a proper specimen for most
chemical estimations. Always collect a little more blood
than required so as not to fall short of it subsequently. For
1 mL of serum about 2.5 mL of blood should be withdrawn.
Labelling and identification is important.
Pipettes: For dispensing test materials or reagents, etc. exact
quantities are needed. For volumes till 0.1 mL Borosil’s
pipettes can be used. Otherwise autopipettes for micro to
macro sampling can be used. Dispensing exact amounts
of test samples/reagents is the first step to accurate clinical
If the commercially available kits are being used, follow
the manufacturers guidelines and collect the requisite
466 Concise Book of Medical Laboratory Technology: Methods and Interpretations Hematology (EDTA)
Ideally all measurements should be performed within
1 hour after collection. Tests where proteins are first
precipitated with tungstic acid, trichloroacetic acid or
barium sulfate—samples for these tests can be stored in
a refrigerator at 4–6°C if the interval before the analysis
exceeds 30 minutes. In medical chemistry, plasma can be
used for virtually all measurements (ideal anticoagulant
being heparin), although a few require serum (serum
enzymes and protein electrophoresis), while whole blood
can for all practical purposes be eliminated. Whenever a
delay of more than 1 hour is anticipated, refrigerate the
sample at 4oC. For extracting serum—let the blood clot at
room temperature (takes about 20–30 minutes), loosen the
clot at the top by a stick. Centrifuge blood for 10 minutes
at 3,000 rpm, serum can be removed with the use of a
pasteur pipette. Label and store the serum in a refrigerator
at 4–6°C until analyzed or freeze at –20°C, if the analysis is
to be delayed by more than 4 hours.
While centrifuging the principle of balance must always be
observed. Tubes of equal weight, shape, and size should be
placed in opposing positions in the centrifuge head (using
water filled tubes whenever necessary). Tubes should be
supported by appropriately shaped rubber cushions in the
carrier of the centrifuge head. The speed of the centrifuge
1. All tubes should be chemically clean, i.e. free of actual
or potential organic and/or inorganic constituents that
may alter the result of a chemical analysis. They need
2. Hemolysis: It should always be avoided as release of RBC
contents (e.g. LDH, acid phosphatase and potassium)
or through color change (especially for photometric
measurements using shorter wavelengths of the visible
spectrum 400–500 nm) results may be falsely high.
Hemoglobin interferes with specific chemical reactions
(e.g. diazotization inhibition in bilirubin estimation).
Blood Collection, Precautions and Errors
1. Excessive venous stasis by prolonged application
of tourniquet should be avoided. This would also
raise concentration of certain constituents of blood
hormones, calcium, K+, Lactic acid, etc.).
2. The syringe, needle and the tube should be moisture
3. Blood should be withdrawn by needle of gauge less
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