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.

 

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.

 

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

Calibration Curve Methods

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

establishing MNPT.

Test Procedure

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 this reagent to room temperature before

prewarming at 37°C for testing purposes.

3. Recap the reagent vial and replace immediately to

2–8°C.

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

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

samples.

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

Calculation of Results

Manual Method

The result 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 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 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.

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 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

PROTHROMBIN

TIME (PT) DETERMINATION (ISI=1.0)

UNIPLASTIN®

(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 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

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.

286 Concise Book of Medical Laboratory Technology: Methods and Interpretations Reagent Storage and Stability

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.

Principle

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.

Note

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

excluded.

3. Reagent contains 0.01% Thimerosal as preservative.

4. It is very important that scrupulously clean and dry

micropipette tips be used to aspirate/dispense the

reagent.

 

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,

 

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

estimation.

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

accuracy and reproducibility.

• 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

accurate and precise.

• 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

and dry.

• 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.

Storage of Reagents

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

thermal stress.

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

to elevated temperatures through repeated freezethaw cycles stresses the colloidal system. Especially

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

should be a two-step process:

• 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

temperature of 37 + 0.5°C.

• 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

to 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

begins”

• 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

detection systems are used.

• 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

coagulation studies.

PT tests are influenced on administration of following

drugs

PT may be shortened PT may be prolonged

drugs drugs

• Antihistamines • Corticosteroids

• Butabarbital • EDTA

• Phenobarbital • Asparaginase

• Caffeine • Clofibrate

• Oral contraceptives • Erythromycin

• Vitamin K • Ethanol

• Tetracycline

• Aspirin

• Anticoagulants such as

warfarin and heparin

APTT tests are influenced on administration of

following drugs

APTT may be shortened APTT may be prolonged

drugs drugs

• Oral contraceptives • Diphenylhydantoin

• Conjugated estrogen • Heparin

therapy • Warfarin

• Naloxone

• Radiographic agents

Thrombin time test is prolonged in the following clinical

conditions

• Normal newborn infant • Hepatic diseases

• Systemic lupus • Toxemia of

erythematosus pregnancy

• Macroglobulinemia • Multiple myeloma

• Presence of exogenous/

endogenous circulating

anticoagulants

Mean Normal Prothrombin (MNPT) and International

Normalized Ratio (INR)

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

been used in the PT determination.

INR = [R][ISI]

INR = Patient PT in seconds

Mean of the normal range

ISI

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