Manual Method

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 the reagent to room temperature before

prewarming at 37°C for testing purpose.

3. Recap the reagent vial and replace immediately

2–8°C.

4. 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.

5. To the tube forcibly add 0.2 mL of Lyoplastin reagent

(prewarmed at 37°C for at least 3 minutes) and

simultaneously start a stopwatch. Shake the tube

gently to mix contents.

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

‘seconds’.

7. Repeat steps 4–6 for a duplicate test on the same

sample.

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.

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).

*lt 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.

Expected Values

Normal values using Lyoplastin are between 11-15

seconds. Between manual and turbo densitomeltric

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 Lyoplastin.

Oral anticoagulant therapeutic range: INR = 2.0-3.5.

Remarks

1. lt 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, 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.

290 Concise Book of Medical Laboratory Technology: Methods and Interpretations 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 Lyoplastin reagent suspension

before use is important to achieve accurate and

consistent results.

Clinical Implications

Conditions accompanied by an increased prothrombin

time (PT) include:

¾ Prothrombin deficiency

¾ Vitamin K deficiency

¾ Hemorrhagic disease of the newborn

¾ Liver disease (e.g. alcoholic hepatitis)

¾ Anticoagulant therapy

¾ Biliary obstruction

¾ Salicylate intoxication

¾ Hypervitaminosis A

¾ DIC disease.

Interfering Factors

1. Diet: Excessive amounts of green, leafy vegetables will

increase body’s absorption of vitamin K.

2. Alcohol: PT is increased due to liver disease.

3. Diarrhea and vomiting: These increase PT.

4. Quality of venipuncture: It is important that a clean and

careful venipuncture is done, otherwise the PT can be

shortened.

5. Many drugs can alter PT.

Clinical Alert

1. If PT is excessively prolonged, vitamin K is given

intramuscularly. Ordinarily, intramuscular injections

are contraindicated during anticoagulant therapy

because large painful hematomas may form at the

injection site. As values get into danger zones, assess

carefully for bleeding, including: (i) craniotomy checks,

(ii) lung auscultation (especially of upper lobes), and

(iii) occult blood in the urine.

2. Patients who are being monitored by PT for long-term

anticoagulant therapy should not take any drugs

unless absolutely necessary.

3. When unexpected changes in anticoagulant doses

are needed to maintain a stable PT, or when there is a

consistent change in PT, a drug interaction should be

suspected.

4. Blood for PT should be drawn for a base line and prior

to administration of anticoagulants.

5. Protamine sulphate is the antidote for heparin.

The INR Method of Reporting Results

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 been used in

the PT determination.

INR = [R]ISI

INR = Patient PT in seconds

Mean of the normal range

ISI





 



