TRIPASS XR تري باس

 

5. Avoid exposure of the Uniplastin reagent to elevated

temperatures, contamination and undue stress

due to high and low temperature exposure cycles.

Immediately replace reagent cap after use and store

at recommended temperatures only.

6. On prolonged storage at 2–8°C, the thromboplastin

suspension has a tendency to settle down. Homogenize

the reagent by resuspending before use.

Additional Material Required

12 × 75 mm test tubes (plastic tubes are preferred), 0.1 mL

and 0.2 mL precision pipettes, stopwatch, water bath or

heating block at 37°C, fresh normal plasmas for establishing MNPT.

Sample Collection and Preparation of PPP

Though no special preparation of the patient is required

prior to sample collection by approved techniques, it is

preferable that patients are not heavily exercised before

blood collection. Fasting or only light non-fatty meals prior

to blood collection provide samples with a desirable lower

opacity. Withdraw blood without undue venous stasis or

frothing into a plastic syringe fitted with a short needle of

19 to 20 SWG. The venipuncture must be a ‘clean one’ and,

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%). For

occasional patients with hematocrit less than 20% or

greater than 55%, this ratio must be readjusted to ensure

valid results. Centrifuge immediately for 15 minutes at

1500-2000 rpm (approximately 1500 g) on a laboratory

centrifuge and transfer the plasma into a clean 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 is 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.

Test Procedure

Manual Method

1. Bring the reagent vial to room temperature (20 to

30°C). Mix the contents of the vial to homogenize the

suspension completely.

2. Aspirate from the reagent vial enough reagent for

immediate testing requirements in a thoroughly clean

and dry test tube (plastic test tubes are preferred).

3. Prewarm the reagent and bring to 37°C before use

in test procedure (5-10 minutes may be required

depending on the reagent volume to attain 37°C before

testing).

4. Recap the reagent vial and replace immediately to

2 to 8°C.

5. To a 12 × 75 mm tube add 0.1 mL of plasma (PPP) and

place the tube in a water bath for 3 to 5 minutes at 37°C.

6. To the tube forcibly add 0.2 mL of Uniplastin reagent

(prewarmed at 37oC for at least 3 minutes) and

simultaneously start a stopwatch. Shake the tube

gently to mix contents.

7. 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

‘seconds’.

8. Repeat steps 4 to 6 for a duplicate test on the same

sample.

Clinical Hematology: Bleeding Disorders 287

9. 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.

Calculation of Results

Manual Method

The results may be reported directly in terms of the mean

of the double determination of PT of the test plasma in

‘seconds’.

Or as a ratio ‘R’:

 Mean of the patient plasma PT in seconds

R = _______________________________________

MNPT for the reagent

Or as international normalized ratio (INR), INR = (R)ISI,

where ISI = International sensitivity index of the reagent

(Refer reagent vial label).

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 atleast 20 normal healthy

individuals should be used to establish the MNPT. The

average of such PT results in seconds = MNPT.

Expected Values

Normal values using Uniplastin are between 11–15 seconds.

Between manual and turbodensitometric instrument

results a variation of 1–2 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 Uniplastin.

Oral anticoagulant therapeutic range: INR = 2.0–3.5.

Remarks

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 hemolysed 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 to 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, erythromycin,

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 UNIPLASTIN reagent suspension

before use is important to achieve accurate and

consistent results.

THROMBOPLASTIN REAGENT FOR

PROTHROMBIN TIME (PT) DETERMINATION,

LYOPLASTIN® (LYOPHILIZED REAGENT, ISI=1.0)

(Courtesy: Tulip Group of Companies)

Summary

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

288 Concise Book of Medical Laboratory Technology: Methods and Interpretations 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 anticoagulant therapy.

Reagent

Lyoplastin is a sensitive, lyophilized calcified thromboplastin reagent which is derived from rabbit brain.