Critical requirement of MNPT in the derivation of INR

•  MNPT should ideally be derived by each laboratory from 20 or more normal patients for a given PT reagent and lot under use. If “normal

control plasmas” are used in place of patient plasma for arriving at MNPT, it can effect the evaluation of the patients’ level of anticoagulation

ISI value of PT used and method of clot detection:

•  INR looses some precision when comparisons are made with thromboplastins with different ISI values. ISI values should be adapted to the

methods used for clot detection ideally close to 1.0 as possible

INR System

Accuracy of the INR system depends on:

• The INR system effectiveness would still depend on the calibration of the coagulation instruments as well as thromboplastin reagents used

• Derivation of the correct MNPT and use of mean normal range in each laboratory

• Usage of thromboplastin reagents with ISI of preferably 1.0 or as close to 1.0 as possible

• The correct use of formula to compute the INR

• Uniform understanding of the INR system by clinicians as well as laboratarians

PROTHROMBIN TIME

Uniplastin/Liquiplastin/Lyoplastin

Problem: Prolonged Clotting Time

Possible causes Solutions

1. Oxalated plasma may be used Factor V and factor VII are more labile in sodium oxalate. Buffered 3.2% sodium citrate is an

ideal anticoagulant since factor V and factor VII are more stable in citrate. Therefore, citrate

plasma should be used

2. Plasma not tested immediately Plasma must be tested within 3 hours of blood collection

after blood collection

3. Insufficient prewarming of the Plasma should be incubated for 3–5 minutes at 37°C and the reagents should be incubated

plasma and reagents for at least 10 minutes at 37°C before commencing the testing procedure. Do not incubate

the entire vial. Incubate only the requisite quantity

Possible causes Solutions

4. Incorrect mixture of blood and Ensure that nine parts of freshly collected blood are mixed with one part of sodium citrate

sodium citrate. The concentra- for normal hematocrit or PCV. For occasional patients with PCV less than 20%

tion of sodium citrate, which is (e.g. Microcytic hypochromic anemia) and greater than 55% (e.g. Polycythemia vera), the

used for the test, may be anticoagulant to blood ratio must be readjusted using the formula,

 incorrect C = 1.85 × 10-3(100-H)V

Contd...

Contd...

Clinical Hematology: Bleeding Disorders 311

Where,

 C = Volume of sodium citrate in mL

V = Volume of whole blood + sodium citrate in mL

 H = Hemocrit in percentage

5. The manufacturer’s test Adhere to the instrument manufacturer’s instructions and test protocol

 protocol not followed in case

 of automated instruments

6. Temperature of water bath not The temperature of the water bath should be preset at 37 ± 1°C

set correctly

7. Expired reagents are used for testing Check the expiry date of the reagents before use

8. Incorrect addition of reagent The test requires extreme precision, besides the addition of the reagent should be followed

as per the instructions given in the package insert. Exactly 0.1 mL of plasma and 0.2 mL of

liquiplastin/uniplastin reagent prewarmed at 37°C should be used. Well-calibrated micropipettes should be used for dispensing

9. Contaminated reagents and Check the working of the reagents with normal control plasmas. Check the reagents for

glassware used for testing turbidity. Ensure that clean and dry glassware and micropipettes are used

10. Contaminated/wet micro-pipette Ensure that clean and dry tips are used for retrieving the reagent

tips used for retrieving the reagent

11. Clotting times of patients on The history of the patient must be taken into consideration to determine the anticoagulant

anticoagulant therapy depends used as well as the time lag between specimen collection and last dose of anticoagulant

on the type and dosage of anticoagulants and also on the time

 and last dose of anticoagulant

12. Error in reading and interpre- The test protocol should be adhered to. The results should be read and interpreted as

tation of test results: per the test protocol

a. The stopwatch is not The stopwatch should be stopped as soon as the gel clot is seen since there is a

 stopped as soon as the difference of 2–3 seconds between the beginning and end of clot formation

 gel clot is seen

b. Clot formation is observed There should be adequate light while observing for clot formation

 in inadequate light

 c. Water droplets are present Wipe the base of the tube to remove additional water as it hampers reading of

   at the base of the tube the test results.

13. Improper storage condition To maintain the sensitivity and performance of the reagent, avoid thermal stress due to

leading to reagent precipitation exposure to high ambient temperature thereby causing precipitation, ensure that the

reagent is stored at the recommended temperature

14. Improper mixing of the reagent On prolonged storage at 2-8°C, thromboplastin suspension tends to settle down.

after prolonged storage at 2-8°C Homogenize the reagent by resuspending before use

15. In case of automated instru- Only the requisite quantity of the reagent for performing the test should

ments, the entire reagent vial is be incubated

incubated thereby leading to

deterioration of the reagent on

repeated incubation

Contd...

Contd...

312 Concise Book of Medical Laboratory Technology: Methods and Interpretations Problem: Shortened Clotting Time

Possible causes Solutions

1. Broken glassware used allowing Ensure that broken glassware is not used for testing purposes

silica to trigger the clotting reaction

2. Administration of drugs/other PT tests are shortened because of the administration of drugs such as antihistamines,

clinical conditions butabarbital, phenobarbital, caffeine, oral contraceptives and vitamin K. It is therefore essential to know the patient history to determine the type of anticoagulant used and the time

lag between the last dose of the anticoagulant and specimen collection

DPTT/PTTK

Liquicelin-E

Problem: Prolonged Clotting Time

Possible causes Solutions

1. Oxalated plasma may be used Factor V and factor VII are more labile in sodium oxalate. Buffered 3.2% sodium citrate is an

ideal anticoagulant since factor V and factor VII are more stable in citrate. Therefore, citrate

plasma should be used

2. Plasma not tested immediately Plasma must be tested within 3 hours of blood collection

after blood collection

3. Insufficient prewarming of the Plasma should be incubated for 3–5 minutes at 37°C and the reagents should be incubated

plasma and reagents for at least 10 minutes at 37°C. Do not incubate the entire vial. Incubate only the requisite

quantity

4. Incorrect mixture of blood and Ensure that nine parts of freshly collected blood are mixed with one part of sodium citrate

sodium citrate. The concentra- for normal hematocrit or PCV. For occasional patients with PCV less than 20%

tion of sodium citrate, which is (e.g. Microcytic hypochromic anemia) and greater than 55% (e.g. polycythemia vera),

used for the test, may be the anticoagulant to blood ratio must be readjusted using the formula,

 incorrect C = 1.85 × 10–3 (100-H)V.

Where,

C = volume of sodium citrate in mL

V = volume of whole blood + sodium citrate in mL

 H = Hemocrit in percentage

5. Molarity of calcium chloride Check the molarity of the calcium chloride being used. It should be 0.025 M

6. Contaminated/wet micropipette Ensure that clean and dry tips are used for retrieving the reagent.

tips used for retrieving the reagent

7. The mixture of plasma and 0.1 mL of plasma and 0.1 mL of Liquicelin-E reagent should be mixed well and

reagent is not incubated for incubated for exactly three minutes at 37°C for effective contact activation and to

minimum time period of 3 minutes obtain accurate results

8. Error in reading and interpre- The test protocol should be adhered to. The results should be read and interpreted as

tation of test results. per the test protocol

a. The stopwatch is not stopped The stopwatch should be stopped as soon as the gel clot is seen since there is a

   as soon as the gel clot is seen difference of 2-3 seconds between the beginning and end of clot formation

Contd...

Contd...

Clinical Hematology: Bleeding Disorders 313

Possible causes Solutions

b. Clot formation is observed There should be adequate light while observing for clot formation

   in inadequate light

c. Water droplets are present Wipe the base of the tube to remove additional water as it hampers reading of the test

   at the base of the tube results

9. Incorrect addition of reagent The test requires extreme precision, besides the addition of the reagents should be as per

the instructions given in the package insert. Exactly 0.1 mL of plasma, 0.1 mL of LiquicelinE reagent and 0.1 mL of calcium chloride prewarmed at 37°C should be used. Well-calibrated micropipettes should be used for dispensing

10. Temperature of water bath not The temperature of the water bath should be set at 37 ± 1°C

set correctly

11. Cotaminated reagents (APTT Check the working of the reagents with normal control plasmas. Check the reagents for

and calcium chloride) and glassware turbidity. Ensure that clean and dry glassware and micropipettes are used

12. Clotting times of patients on The history of the patient must be taken carefully to determine the

anticoagulant therapy depends anticoagulant used as well as the time lag between the specimen

on the type and dosage of anti- collection and the last dose of anticoagulant

coagulants and also on the time

lag between specimen collection

and the last dose of anticoagulant

13. Improper storage condition To maintain the sensitivity and performance of the reagent, avoid thermal stress due to

leading to reagent precipitation exposure to high ambient temperature thereby causing precipitation, ensure that the reagent is stored at the recommended temperature

Problem: Shortened Clotting Time

Possible causes Solutions

1. Broken glassware allowing Ensure that broken glassware is not used for testing purposes

silica to trigger the clotting

 reaction

2. Administration of drugs/other APTT tests are shortened because of the administration of drugs, oral contraceptives or

clinical conditions conjugated estrogen therapy. It is, therefore, essential to know the patient history to determine the type of anticoagulant used and the time lag between the last dose of the anticoagulant and specimen collection

Contd...

314 Concise Book of Medical Laboratory Technology: Methods and Interpretations FIBRINOGEN ESTIMATION

Fibroscreen®/Fibroquant®

Problem: Prolonged Clotting Time

Possible causes Solutions

1. Oxalated plasma may be used Factor V and factor VII are more labile in sodium oxalate. Buffered 3.2% sodium citrate is an

ideal anticoagulant since factor V and factor VII are more stable in citrate. Therefore, citrate

plasma should be used

2. Plasma not tested immediately Plasma must be tested within 3 hours of blood collection.

after blood collection

3. Insufficient prewarming of the Plasma should be incubated for 3–5 minutes at 37°C and the reagents should

plasma and reagents be incubated for at least 10 minutes at 37°C

Do not incubate the entire vial. Incubate only the requisite quantity.

4. Incorrect mixture of blood and Ensure that nine parts of freshly collected blood are mixed with one part of sodium citrate

sodium citrate. The concentra- for normal hematocrit or PCV. For occasional patients with PCV less than 20%

tion of sodium citrate, which is (e.g. Microcytic hypochromic anemia) and greater than 55% (e.g. polycythemia vera),

used for the test, may be the anticoagulant to blood ratio must be readjusted using the formula,

 incorrect C = 1.85 × 10-3 (100-H) V

Where,

C = volume of sodium citrate in mL

V = volume of whole blood-sodium citrate in mL

 H = Hemocrit in percentage

5. Error in reading and interpre- The test protocol should be adhered to. The results should be read and

tation of test results interpreted as per the test protocol

• The stopwatch is not The stopwatch should be stopped as soon as the gel clot is seen since

  stopped as soon as the gel there is a difference of 2–3 seconds between the beginning and end of

 clot is seen clot formation

• Clot formation is observed There should be adequate light while observing for clot formation

  in inadequate light

• Water droplets are present Wipe the base of the tube to remove additional water as it hampers

  at the base of the tube reading of the test results

6. Temperature of water bath not The temperature of the water bath should be set at 37 ± 1°C

set correctly

7. Contaminated reagents and Check the working of the reagents with normal control plasmas. Check the reagents