A PT ratio is obtained by dividing the patient PT in

seconds by the “Mean of the normal range” (MNPT).

This ratio is then “normalized” by raising the results to the

power of the ISI of the PT reagent used.

If “normal control plasmas” are used in place of patient

plasma for arriving at the MNPT it can affect the evaluation

of the patients level of anti-coagulation.

Clinical Hematology: Bleeding Disorders 283

For example:

Reagent ISI=2.5 Test Day 1 Test Day 2 Test Day 3

Patient PT (sec)

Normal Control

(10.4-12.3 sec)

INR Formula

[R]’SI

Resulting INR

16.0

11.5

16.02.5

11.5

2.27

16.0

10.4

16.02.5

10.4

2.89

16.0

12.3

16.02.5

12.3

1.92

If the control time is greater than the mean normal

range (MNPT), the PT ratio for any patient, PT will be

smaller, potentially leading to over coagulation. If the

control time is lesser than MNPT the ratio for any patient

PT will be greater, leading to under coagulation.

On the other hand, MNPT for a particular laboratory

using the same combination of methodology, reagent and

instrument would remain constant.

Quality Control Aspects

¾ Quality of water used for reconstituting lyophilized

coagulation reagents must be good.

• The water used for reconstitution of lyophilized

coagulation reagents should be at least distilled twice

and kept separately labeled for “coagulation studies”.

The reagents employed for coagulation studies

are extremely delicate and inability to use good

quality distilled water could lead to incorporation

of metallic impurities in the reagent formulation as

well as change in pH. Such changes can alter reaction

kinetics and overall stability and performance of

reagents.

¾ Quality assurance for coagulation-based reagents must

be performed preferably on a daily basis.

• Each laboratory should test coagulation reagents with

normal and abnormal control plasma specimens at

the beginning of each day's work to verify instruments, temperature calibration and also reagent

performance.

If the control results fall within the stated limits, the test

results are considered valid.

But if the results fall outside the stated control limits

then the reagents, control and equipments are checked

and the problem should be corrected.

Control results should be recorded and analyzed after

regular intervals to ascertain the long-term validity of

results.

PROTHROMBIN TIME (QUICK ONE-STAGE

METHOD) LIQUIPLASTIN®

(Courtesy: Tulip Group of Companies)

Thromboplastin Reagent for Prothrombin Time (PT)

Determination

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 these factors 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, determination of congenital

deficiency of factors II, V, VII and X and for monitoring

of patients on oral anticoagulant therapy and as a liver

function test.

Clotting mechanism—cascade system

284 Concise Book of Medical Laboratory Technology: Methods and Interpretations Reagent

Liquiplastin is a liquid ready to use calcium

thromboplastin reagent, which is derived from rabbit

brain. Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its sensitivity

and performance.

Reagent Storage and Stability

(a) Store the reagent at 2 to 8°C. Do not freeze.

(b) The shelf-life of reagent is as per the expiry date

mentioned on the reagent vial label. The uncontaminated reagent is stable for: 1 year at 2 to 8°C, 1 week

at 18 to 25°C, 2 days at 37°C.

Principle

Tissue thromboplastin in the presence of calcium activates

the extrinsic pathway of human blood coagulation

mechanism. When Liquiplastin reagent is added to

normal anticoagulated 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 a deficiency of factors/factor activity in the

extrinsic pathway of the coagulation mechanism.

Note

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. Liquiplastin reagent is not from human source hence,

contamination due to HBsAg and HIV is practically

excluded.

3. Liquiplastin reagent contains 0.01% Thimerosal as

preservative.

4. It is very important that clean and dry micropipette

tips be used to dispense the reagent.

5. Avoid exposure of the reagent to elevated temperatures

and contamination. Immediately replace cap after use

and store at recommended temperatures only.

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,