Reagent

Liquicelin-E is a liquid ready to use activated cephaloplastin reagent for the determination of activated partial

thromboplastin time. It is a phospholipid preparation

derived from rabbit brain with ellagic acid as an activator.

Each batch of reagent undergoes rigorous quality

control at various stages of manufacture for its sensitivity

and performance.

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

a. Store the reagent at 2-8°C. Do not freeze.

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

mentioned on the reagent vial label. The reagent is

stable for: 1 year at 2–8°C, 1 week at 18–25°C, 2 days

at 37°C.

Principle

Cephaloplastin activates the coagulation factors of the

intrinsic pathway of the coagulation mechanism in

the presence of calcium ions. APTT is prolonged by a

deficiency of one or more of these clotting factors of the

intrinsic pathway and in the presence of coagulation;

inhibitors like heparin.

Note

1. In vitro diagnostic reagent for laboratory and

professional use only. Not for medicinal use.

2. Liquicelin-E, 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 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

No special preparation of the patient is required prior to

sample collection by approved techniques. Withdraw

blood without undue venous stasis and without 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 tubes, after detaching

the needle from the syringe.

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; Centrifuge immediately

for 15 minutes at 3000 rpm (approximately 2000 g) and

transfer the plasma into a clean test tube. Plasma must be

tested within three hours of blood collection. For heparin

determination, platelet deficient plasma should be used,

hence higher centrifugation time is required.

FNP Collection

Prepare a plasma pool (FNP) of freshly collected blood

from at least five normal healthy donors and process as

above. Plasma must be tested within three hours of blood

collection.

Additional Material Required*

12 × 75 mm test tubes; 0.1 mL, 0.2 mL and 2.0 mL precision

pipettes; Stopwatch; Water bath or heating block 37°C;

Fresh normal pooled plasma; CaCl2 (0.02 mol/L).

*Available from Tulip Diagnostics.

Test Procedure

Manual Method

1. Before use, the reagent should be mixed well by gentle

swirling. Do not shake.

2. Aspirate from the reagent vial enough reagent for

the immediate testing requirement in a thoroughly

clean and dry test tube. Bring this reagents to room

temperature before prewarming at 37°C for testing

purposes.

3. Separate test tubes containing Liquicelin-E and

Tulip’s calcium chloride solution should be brought

to 37°C (depending on volume, approximately 5 to 10

minutes required). Do not incubate the test plasma.

4. To a 12 × 75 mm test tube, add 0.1 mL test plasma

and 0.1 mL Liquicelin-E. Shake tube briefly to mix

the reagent and plasma, place tube at 37°C for 3 to 5

minutes.

5. Following incubation period, add forcibly 0.1

mL prewarmed calcium chloride into the plasma

and Liquicelin-E mixture, simultaneously start a

stopwatch. Shake tube briefly to mix contents, keep

at 37°C for 20 seconds.

6. Following 20 seconds incubation, remove the tube,

gently tilt back and forth until a gel clot forms, stop

the watch, record time.

7. Repeat steps 2–4 for a duplicate test using the same

test plasma.

8. Find the average from the duplicate test values. This

is the activated partial thromboplastin time (APTT of

patient plasma).

9. Similarly repeat steps 2-4 twice, and record duplicate

values using FNP in place of test plasma (APTT of

FNP).

If a coagulation instrument is being used to perform the

tests, the instrument manufacturer's instructions must be

strictly adhered to.

Calibration Curve Method (For determination of

heparin concentration):

1. Dilute heparin (as used for treatment) with

physiological saline to a concentration of 10 U/mL.

2. Mix 0.2 mL of 10 U/mL diluted heparin with 1.8 mL

of FNP to give a heparin standard of 1 U/mL concentration.

3. Dilute the heparin standard as prepared above

(1 U/mL) with FNP as follows :

Clinical Hematology: Bleeding Disorders 295

Test tube No. 1 2 3 4 5 6 7

Heparin standard

(1 U/mL) in mL

0.5 0.4 0.3 0.2 0.1 0.1 -

FNP in mL - 0.1 0.2 0.3 0.4 0.9 0.5

Heparin concentration (U/mL)

1 0.8 0.6 0.4 0.2 0.1 0.0

4. Pipette 0.1 mL each of the seven heparin dilutions into

clean test tubes.

5. Add 0.1 mL Liquicelin-E reagent to each test tube.

6. Mix well and incubate each test tube at 37°C for exactly

3 minutes before testing.

7. Forcibly add 0.1 mL calcium chloride (prewarmed at

37°C) to each test tube, one by one and simultaneously

start the stopwatch.

8. Gently tilt the tube back and forth and stop the

stopwatch as the first fibrin strand is visible and the

gel/clot formation begins. Record the time in seconds.

9. Repeat steps 4–8 for each dilution for duplicate test,

and find the average of the duplicate test values.

10. Plot the mean of the double determination in ‘seconds’,

against each heparin concentration using Liquicelin-E

graph paper.

11. Clotting times (APTT) of test specimens can be

interpolated against the heparin concentration to

determine the heparin concentration of the sample in

U/mL.

Calculation and Reporting of Results

Manual Method

a. The result may be reported directly in terms of the

mean of the double determination of the APTT of the

test plasma.

OR

b. As a ratio R as follows:

APTT of patient plasma (in seconds)

R = _________________________________

APTT of FNP (in seconds)