each laboratory must calibrate the necessary force and
time required during centrifugation to yield PPP.
2. Incorrect mixture of blood and Profact is a potential
source of error both in coagulation assays and ESR
3. If the reagent vial develops turbidity, do not use the
reagent as this would lead to erroneous results.
Calibration of Instruments/Equipments
¾ Water baths or heating blocks calibrated and preset
at 37 + 0.5°C are an important requirement to achieve
• The whole process of the coagulation tests is based on
a series of enzymatic reactions, which are dependent
on pH, ionic strength and the temperature of the
reaction process. A correct temperature at 37 +
0.5°C is critical as most of the reagent systems
are standardized at this temperature. Day-to-day
shift in reaction temperature of equipment will
introduce uncontrolled variation into test conditions. Therefore, temperature of all equipments must
be calibrated daily and diligently to avoid erroneous
results and ensure accuracy and reproducibility.
• Sample/reagent dispensing mechanisms must be
• Well-calibrated dispensing mechanisms are required
for all coagulation- based tests to accurately dispense
samples as well as reagents. Any shift in ratio or
individual volumes of the sample and/or reagent can
lead to shortening or prolongation of results.
• Straight 0.1 and 0.2 mL glass pipettes are usually
satisfactory, provided they are scrupulously clean
• Automatic micropipettes, which are able to deliver
the required volumes, are replacing the glass pipettes,
provided these pipettes are calibrated frequently.
The use of clean disposable tips places this system
at an advantage over the older mechanisms.
¾ Usually reagent manufacturers recommend aspiration
of adequate reagent for the days use in a thoroughly
clean and dry tube instead of intermittent aspiration
from reagent vials at the time of test.
• Most coagulation reagents are extremely delicate
reagents. For them to maintain their sensitivity and
performance the reagent formulations must maintain
reagent integrity over the usage period. Repeated
intrusions into the reagent vial exponentially
increases the chances of reagent contamination
and destruction of reagent formulations and
integrity. Undried and/or contaminated pipettes,
tips, glassware are usually the main culprits. Such
contaminated reagents perform suboptimally.
• The reagent vials must be immediately stored back
to the recommended storage temperatures after
the aspiration of the day's requirement separately
so that the remaining reagent remains at optimal
temperature for future use. Keeping unused reagents
at higher ambient temperatures during the day
causes steady deterioration of the reagent due to
¾ The recommended storage temperature for reagents
should be strictly complied to:
• Most of the liquid stable or reconstituted reagents
such as PT and APTT are colloidal suspensions of
lipoproteins and/or phospholipids. Subjecting them
detrimental are the effects of freezing (below 2°C).
After freezing the reagent colloidal suspension
undergoes an irreversible change and precipitates
out or present itself as a particulate mass. Such
reagents give erroneous results.
¾ Bringing reagents/samples to room temperature
• When enough reagents are aspirated out for the
days testing as recommended the reagent and
samples stored at 2 to 8°C should be first allowed to
attain room temperature (25 to 30°C) and then they
should be subsequently brought to the optimal test
• When reagent samples from 2 to 8°C are directly
brought to 37°C the required time of 3 to 5 minutes
may not be sufficient for the reagent samples to
attain a homogeneous temperature of 37°C within
the recommended time. This affects the reaction
kinetics leading to erroneous results.
282 Concise Book of Medical Laboratory Technology: Methods and Interpretations End Point Reading
¾ Reading of endpoint of clot-based tests varies from user
• Usually when manual techniques are followed the
definition of “end point” is important. Ideally, the
end point tests should be read “as soon as the first
fibrin strand is visible and the gel clot formation
• When some users use a fully formed gel clot as an
end point, there is a variation of 1 to 3 seconds
between the end points as recorded by the ideal
method and user-based variation.
• It is advisable to have well illuminated background
for reading the clot-based end points. Since user
variations based on proficiency continue to influence
results, it is advisable not to change personnel
involved in coagulation tests off and on.
¾ Manufacturer’s instructions must be followed meticulously when instrument based or automatic clot
• Each clot detection system works on a different
p r i n c i p l e , s u c h a s e l e c t r o m e c h a n i c a l ,
turbidimetric or photo-optical. Each system of
clot detection has its requirements for optimum
functioning. Special care must be taken while
using optical instruments for clot detection since
reagent-induced turbidity can influence the
results dramatically. Usually low turbidity reagents
are preferred for manual as well as instrumentsbased clot detection.
Drug/Clinical Conditions Influencing Patient Results
¾ Drugs/clinical conditions influence results of patients
PT tests are influenced on administration of following
PT may be shortened PT may be prolonged
• Antihistamines • Corticosteroids
• Phenobarbital • Asparaginase
• Oral contraceptives • Erythromycin
APTT tests are influenced on administration of
APTT may be shortened APTT may be prolonged
• Oral contraceptives • Diphenylhydantoin
• Conjugated estrogen • Heparin
Thrombin time test is prolonged in the following clinical
• Normal newborn infant • Hepatic diseases
• Macroglobulinemia • Multiple myeloma
Mean Normal Prothrombin (MNPT) and International
¾ MNPT is a critical requirement in the derivation of INR.
¾ MNPT is a critical requirement in the derivation of
INR. Ideally each laboratory must derive its own MNPT
from 20 or more normal patients for a given PT reagent
and Lot under use. This corrects within laboratory test
variables that influence PT results.
¾ By definition INR represents the PT ratio which would
have been obtained for a particular patient sample as if
the WHO reference thromboplastin itself (ISI=1.0) had
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