if there is any difficulty, take a new syringe and needle and
try another vein. Transfer the blood into anticoagulated
tubes, after detaching the needle from the syringe. Do
not delay mixing blood with anticoagulant. Avoid foam
formation during mixing. Mix exactly nine parts of freshly
collected blood with one part of trisodium citrate (0.11
mol/L, 3.2%) or Profact available from Tulip; For occasional
patients with hematocrit less than 20% or greater than
50%, this ratio must be readjusted to ensure valid results.
Centrifuge immediately for 15 minutes at 1500-3000 rpm
(approximately 1500 g) on a laboratory centrifuge and
transfer the plasma into a dean test tube. It should be
ensured that the plasma is free from platelets (PPP). Cap
the test tubes to prevent deterioration of samples. Plasma
must be tested preferably immediately. However if the
specimen are held at 22 to 24°C then they may be tested
within 2 hours and if the specimen is held at 2 to 4°C then
they may be tested within 3 hours.
Additional Material Required for Manual and
12 × 75 mm test tubes (plastic tubes are preferred), 0.1
mL and 0.2 mL precision pipettes, Stop watch, Water
bath or heating block at 37°C, fresh normal plasmas for
1. Aspirate from the reagent vial enough reagent for
immediate testing requirements in a thoroughly clean
and dry test tube (plastic test tubes are preferred).
2. Bring this reagent to room temperature before
prewarming at 37°C for testing purposes.
3. Recap the reagent vial and replace immediately to
4. To a 12 × 75 mm tube add 0.1 mL of plasma and place
the tube in a water bath for 3 to 5 minutes at 37°C.
5. To the tube forcibly add 0.2 mL of Liquiplastin
reagent (prewarmed at 37°C for at least 3 minutes)
and simultaneously start a stopwatch. Shake the tube
6. Gently tilt the tube back and forth and stop the
stopwatch as soon as the first fibrin strand is visible
and the gel/clot formation begins. Record the time in
7. Repeat steps 4-6 for a duplicate test on the same
8. Find the average of the duplicate test values. This is the
prothrombin time (PT). If a coagulation instrument
is being used to perform the tests, the instrument
manufacturer's instructions must be strictly adhered to.
Clinical Hematology: Bleeding Disorders 285
The result may be reported directly in terms of the mean
of the double determination of PT of the test plasma in
Mean of the patient plasma PT in seconds
R = _______________________________________
Or as international normalized ratio (INR), INR = (R)ISI
‘where ISI = International sensitivity index of the reagent
It is recommended by the WHO that MNPT should be
established for each lot of PT reagents by each laboratory,
since PT results are dependent on the combination of
reagent lot, instrument and technique followed at each
laboratory. Usually plasma from at least 20 normal healthy
individuals should be used to establish the MNPT. The
average of such PT results in seconds = MNPT.
Normal values using Liquiplastin are between 10 and
14 seconds. Between manual and Turbodensitometric
instrument results a variation of 1 to 2 seconds may
be expected. For photo-optical instruments, it is
recommended that each laboratory must establish their
own normal range. It is mandatory that each laboratory
must establish its own MNPT for each lot of Liquiplastin.
Oral anticoagulant therapeutic range : INR = 2.0-3.5.
(1) It is recommended that controls with known factor
activity should be run simultaneously with each test
series to validate test run. (2) Incorrect mixture of blood
and Trisodium citrate, insufficient prewarming of plasma
and reagent, contaminated reagents, glassware, etc. are
potential source of errors. (3) Oxalated plasma may induce
prolonged clotting times. (4) Since the PT test functions
correctly only at 37 ± 0.5°C, temperature of all equipment
must be calibrated daily. (5) Clotting time of patients on
anticoagulant therapy depends upon the type and dosage of
anticoagulant and also the time lag between the specimen
collected and the last dose. (6) Turbid , icteric, lipemic
or grossly hemolyzed samples may generate erroneous
PT results. (7) Glasswares and cuvettes used in the test
must be scrupulously clean and free from even traces of
acids/alkalies or detergents. (8) Plasma samples held at
4-8° C may undergo ‘cold activation’ leading to a marked
shortening of the PT. (9) The PT may be shortened during
acute inflammatory conditions, which are accompanied by
increase in Fibrinogen levels and also by agents, such as
antihistamines, butabarbital, phenobarbital, caffeine, oral
contraceptives and vitamin K. The PT may be prolonged by
corticosteroids, EDTA, oral contraceptives, asparaginase,
clofibrate, ethanol, tetracycline, aspirin and anticoagulants
such as heparin and warfarin. (10) It is important that each
laboratory express the results in terms of INR for patients
on oral anticoagulant therapy for the clinician to adjust the
dosage based on INR. (11) Since the test uses platelet poor
plasma, each laboratory must calibrate the necessary force
and time required during centrifugation to yield the PPP.
Contamination of plasma with excess platelets could falsely
elevate levels of some of the factors. (12) Homogenization
of Liquiplastin reagent suspension before use is important
to achieve accurate and consistent results.
SENSITIVE THROMBOPLASTIN REAGENT FOR
TIME (PT) DETERMINATION (ISI=1.0)
(Courtesy: Tulip Group of Companies)
The arrest of bleeding depends upon primary platelet plug
formed along with the formation of a stable fibrin clot.
Formation of this clot involves the sequential interaction of
series of plasma proteins in a highly ordered and complex
manner and also the interaction of these complexes with
blood platelets and materials released from the tissues.
Tissue thromboplastin, in the presence of calcium, is
an activator, which initiates the extrinsic pathway of
coagulation, which includes plasma coagulation factors VII,
X, V, prothrombin and fibrinogen. During oral anticoagulant
therapy most of the vitamin K-dependent factors, such as
II, VII, IX, X, protein C and protein S are depressed, as also
during the deficiencies of clotting factor activity which may
be hereditary or acquired. Prothrombin time determination
is the preferred method for presurgical screening, as a liver
function test, determination of congenital deficiency of
factors II, V, VII and X and for monitoring of patients on oral
Uniplastin is a novel, highly-sensitive, low opacity, ready
to use liquid calcified thromboplastin reagent, which is
derived from rabbit brain. Each batch of reagent undergoes
rigorous quality control at various stages of manufacture
for its sensitivity and performance.
a. Store the reagent at 2 to 8°C. Do not freeze.
b. The shelflife of the reagent is as per the expiry
date mentioned on the reagent vial label. The
uncontaminated reagent is stable for: 1 year at 2–8°C,
1 week at 18–25°C, 2 days at 37°C.
Tissue thromboplastin in the presence of calcium activates
the extrinsic pathway of human blood coagulation
mechanism. When Uniplastin reagent is added to normal
citrated plasma, the clotting mechanism is initiated,
forming a solid gel clot within a specified period. The time
required for clot formation would be prolonged if there is
acquired or congenital deficiency of factors/factor activity
in the extrinsic pathway of the coagulation mechanism or
reduction in the activity of vitamin K-dependent clotting
factors during oral anticoagulant therapy.
1. In vitro diagnostic reagent for laboratory and
professional use only. Not for medicinal use.
2. Uniplastin reagent is not from human source, hence
contamination due to HBsAg and HIV is practically
3. Reagent contains 0.01% Thimerosal as preservative.
4. It is very important that scrupulously clean and dry
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