Field of the Invention
The present invention relates to a process of preparation of the Activated
Partial Thromboplastin Time (APTT) reagent which is used for the
determination of Activated partial thromoboplastin time in whole blood or
plasma sample at ambient temperature and to a test device for performing
analysis according to said process.
More specifically, the invention relates to a device and process for the
preparaion of the APTT reagent.
The invention relates to the process of preparation of activated
cephaloplastin reagent.
Background of the invention
Activated Partial Thromboplastin Time (APTT) is a blood test that measures
the time it takes to clot the blood used to screen for bleeding abnormalities.At
least a dozen blood proteins or blood factors are needed to clot blood and
stop bleeding. The Partial thromboplastin time is an important coagulation
test because it measures the time it takes to clot the blood. It is also used to
monitor treatment with medication that prevents the formation of blood clots
and for screening patient's plasma prior to surgery. APTT assays are also
employed for monitoring anticoagulant treatment with Pharmaceuticals,
which affect the intrinsic coagulation pathway. Also it is used to diagnose
liver diseases and coagulation anomalies of blood.
In general, the blood coagulation process depends upon the presence of
interactive blood components, which cause blood to form a gel. The first
stage of coagulation is formation of blood thromboplastin. Several intrinsic
blood constituents interact to form blood thromboplastin, one of which is
platelets. This is followed by the conversion of prothrombin to thrombin, a
process aided by the now present blood thromboplastin. Third, thrombin act
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on fibrinogen present in the blood to form fibrin, an insoluble plasma protein.
Fibrin, by its insoluble nature forms clot.
Diagnos APTT is an activated Cephaloplastin reagent for use in the in vitro
testing of activated Partial Thromboplastin Time by mechanical clot
detection system. This test is used for monitoring heparin therapy, for
diagnosing congential deficiency of factor VII, IX, XI and XII in presurgery
screening. APTT is an important coagulation test because it measures the
presence and activity of four different blood clotting factors (factors VII, IX.XI
and XII).
APTT test is used for taking blood- thinners to determine the most effective
dosage of medication that prevents blood clot. Partial thromboplastin time is
measured along with Prothrombin time to evaluate the bleeding
abnormalities.These two tests together screen for the problems with the
normal blood clotting process and can detect most bleeding clotting
problems caused by abnormal amounts of our abnormal function of
coagulation factors.
In performng a APTT test, sample and liquid Diagnos APTT reagent are
mixed at 37 °C and the time needed for the mixture to clot is determined.
Dignostic assays that employ Diagnos APTT reagent are based on clotting
times have been used extensivley for determining blood coagulation
deficiencies associated with the intrinsic coagulation pathway.
The primary performance criteria for Diagnos APTT reagent for use in APTT
assays are the coagulation time for a normal plasma sample, the sensitivity
of the reagent and the lot- to- lot reproducibility of the reagent.
Prior art
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US Patent No: 5055412: involves a partial thromboplastin time test reagent
which is factor sensitive depending upon the selected reagent constituents.
A method for preparing such reagent is also disclosed. The present
invention further involves a procoagulant precursor reagent and a method of
making it. The factor sensitive reagent is of exceptional stability enabling
more precise partial prothrombin time measurements. The reagents contain
ellagic acid or an ellagic acid salt and divalent metal ions in a specific molar
ratio relative to the ellagic acid or ellagic acid salt.
US Patent No: 6528273 : Coagulation control compositions suitable for use
in connection with PT and/or APTT assays are disclosed along with their
methods of preparation and methods of use. Preferred coagulation controls
comprise plasma and an anticoagulant having activity for enhancing the
activity of antithrombin III (ATIII) or of heparin co-factor II (HCII) against
thrombin or against a clotting factor selected from the group consisting of
factors IXa, Xa and Xla. The anticoagulant is preferably a
glycosaminoglycan such as heparin, a heparin derivative or a heparin
analog. The anticoagulant is preferably combined with (1) an abnormal
plasma (e.g. activated plasma or factor-deficient plasma) and/or (2) a
primate plasma (e.g. human plasma), and a non-primate mammalian plasma
(e.g. bovine plasma). In the latter case, the non-primate mammalian plasma
is preferably present in the coagulation control composition in an amount of
not more than about 12% by volume, relative to total volume.
US Patent No: 7148067: A thromboplastin reagent includes tissue factor,
Factor Vila, and a net negatively charged phospholipid. The thromboplastin
reagent is a synthetic thromboplastin reagent, and is in dried form.
Description of the invention
Activated Partial Thromboplastin Time is a useful and effective method for
screening patients with a bleeding tendency, for evaluating the effect of
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therapy in procoagulant disorders and on the basis for several specific
coagulant factor assay procedures. It is a laboratory diagnostic coagulation
test by which the functionality of the intrinsic coagulation pathway is
assessed. More specifically the object of the present invention is the a
process of preparation of the activated partial thromboplastin reagent.
The test is used for the determination of the blood cloating factors VIII, IX, XI
and XII monitoring anticoagulat therapy for diagnosing acquired or inherited
bleeding disorders.
The present invention relates to the determination of Activated partial
thromboplastin time reagent. The test is used for monotoring heparin
therapy for diagonosing congential deficiency of factor VIII, IX, XI and XII in
presurgery screening.
Diagnos APTT is a liquid cephaloplastin reagent activated with Ellagic acid.
Blood is withdrawn from the human body and centrifuged to remove the
blood cells and platelets. The supernatant plasma is withdrawn and mixed
with a partial thromboplastin time reagent containing an activator and a
platelet substitute. After a period the clotting factors are activated. After the
addition of calcium, the solution is put into a cuvette. Formation of a clot is
observed directly by the human eye.
The method is responsive to coagulation factors of the intrinsic pathway in
plasma in the presence of calcium ions. The deficiency of one or more
clotting factors of the intrinsic pathway and also the presence of heparin
prolongs APTT of the plasma.The time taken for the clot to form is measured
and used to determine the anticoagulant status of the patient.
In an embodiment of the present invention, the sample is collected as
follows:
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Collect nine parts of venous whole blood in a tube having one part of TSC
solution, 3.2 % Tri- sodium citrate solution and immediately mix the blood
with anticoagulant to avoid the foam formation.Centrifuge the sample for 15
minutes at app. 3000 rpm and collect the plasma in a separate tube.
In an embiodiment of the present invention, Diagnos APTT reagent is
prepared as follows:
a. Take 0.5-1.5 gm of rabbit brain Acetone powder and dissolve in 500
ml of chloroform.
b. Shake and stirr for 1-1.5 hr at room temperature.
c. Filter through Whatman No.1 filter paper.
d. Dry above extract with Nitrogen gas.
e. Dissolve in 0.9 % NaCI solution (1 liter).
f. Add 1-5 gm of Phenol and 0.025- 0.045 gm Ellagic acid.
g. Add 0.6- 4.6 gm dry HEPES powder.
h. Adjust the pH with 1 M HCI. Adjust the final pH to 7.2 +1.
In another embodiment of the present invention, the test procedure is as
follows:
a. Shake the Diagnos APTT gently before use.
b. Pipette 100μl of well mixed Diagnos APTT and 0.02- 0.025 mol/L
Calcium Chloride reagent in separate test tubes and incubate in water
bath at 37°C for 3 minutes.
c. Pipette 100 ΜI of patient plasma in to the tube containig pre- warmed
APTT reagent and incubate in water bath at 37°C for 2-3 minutes.
d. After 3 minutes, add 100 μl of well mixed Calcium Chloride reagent pre-
warmed to 37°C to the tube containing Diagnos APTT reagent and
plasma and simultaneously start the stop watch.Mix the contents gently
for 20 seconds at 37 °C in water bath. Incubate it for 20 seconds at 37°C
6

and remove after that and tilt gently and stop the stop watch as soon as
fibrin strand is visible which initiates gel clot formation.
e. Measure the time taken for the clot formation to the earest 0.1 seconds.
This is the activated partial thromboplastin time in seconds.
f. Repeat the same procedure for the same sample and calculate the mean
APTT.
Calculation of results
The results may be interpreted directly in terms of APTT of the test plasma
in seconds or as a ratio 'R'
R = Mean of the plasma APTT in seconds
APTT of FNP in seconds
Normal Values
Each laboaratory must establish their own normal values for photo-optical
systems as it depends on method used, activation time and instrument used.
For determination of Heparin concentration, a calibration curve has to be
made.
1. Prepare a pool of fresh normal plasma from atleast 5 normal healthy
individuals.
2. Dilute Heparin with 0.9% normal saline to a concentration of 10 U/ml.
3. Mix 0.2 ml of diluted 10 U/ml Heparin with 1.8 ml of FNP as to give
Heparin concentration of 1 U/ml.
4. Dilute the 1 U/ml Heparin as per the standards of FNP.
5. Pipette 100μl each of the 7 Heparin dilutions in to separate test tubes.
6. Add 100μl Diagnos APTT reagent pre-warmed to 37°C to each test tube.
Mix well and incubate at 37°C for 3 minutes.
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7. Add 100μl Calcium Chloride pre-warmed to 37°C to each test tube, one
by one whilst simultaneously start the stop watch.
8. Mix the reagent properly and tilt the test tube back and forth and stop the
stop watch as soon as fibrin strand is visible which indicates gel clot
formation.
9. Repeat the steps 5-8 for each dilution for duplicate reading and calculate
the mean values.
10. Plot mean test values in seconds against each heparin concentration on
heparin graph paper.
11. Connect the points on the graph and plot clotting time of the test
specimen on the calibration curve.
12. Explorate the heparin concentraion of the sample from the graph.
The Activated Partial Thromboplastin Time Reagent is prepared as follow:-
1. Taking 1 -10 gm of Rabbit Brain Acetone Powder and dissolve in 1 litre of
Chloroform.
2. Shaking and stir for 1 -2 hrs at room temperature
3. Filtering the solution through the filter paper.
4. Drying the above extract with inert Nitrogen gas.
5. After drying the extract, obtain the cephaloplastin pellets.Its concentration should
be maintained between 10-100 mg/ deciliter in final APTT reagent.
6. Cephaloplastin pellet is dissolved in required amount of 0.01 to 1.0 M sodium
hydroxide solution.
7. Adding Phenol in the concentration of 0.01 to 0.1 Mole/ litre.
8. Adding Ellagic acid in the concentration of 0.025 to 0.045 gm/litre.
9. Adding HEPES buffer in the concentration of 0.6 to 4.6 gm/ litre.
10. Continuously stirring the solution at 200- 500 rpm for 4 hrs.
11. Adjusting the pH with 1 M HC1 and set the final pH to 7.2 + 0.1.
12. Adding the triple distilled water to adjust the final volume of the reagent.........
8

13. Adding transition metal ions salt preferred Nickel chloride in the concenrationof
10-50mg/litre.
14. Stirring for 2 hrs at 200-500 rpm.
The invention will be described with reference to the accompanying examples, but not
restricting its scope.
According to the present invention, A process of preparation of the Activated
partial Thromboplastin time reagent comprising the following steps:-
a. Taking 1 gm of rabbit brain acetone powder and dissolving in
5ooml of chloroform.
b. Shaking and stirring for 1 hr at room temperature.
c. Filtering through Whatman No 1 filterpaper the above extract and
drying it with Nitrogen gas.
d. Dissolving the extract in 0.9 % NaCI solution( 1 Itr).
e. Adding 4.0 gm of Phenol.
f. Adding 0.035 gm of Ellagic acid.
g. Adding 2.6 gm of dry HEPES powder.
h. Adjusting the pH with 1M HCI and the pH should be 7.2 + 1, thus
obtaining the reagent.
According to the present invention, a process of determining the Activated
Partial Thromboplastin Time in whole blood or plasma comprising the following
steps:
a. Shaking the Diagnos thrombo gently before use.
b. Pipetting 200μl of well mixed Diagnos Thrombo reagent in to a test tube.
c. Pipetting 100 μl of patient plasma in to another test tube.
d. Incubating the test tubes containing reagent and plasma in water bath at
37°C for 2-3 minutes.
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e. Adding 200μl of well mixed Diagnos Thrombo reagent pre warmed to 37
°C to the tube containing plasma sample and simultaneously start the
stopwatch as soon as fibrin strand is visible which initiates gel formation.
f. Measuring the time taken for clot formation to the nearest 0.1 seconds.
This is the prothrombin time in seconds.
g. Repeating the steps for the same sample and calculate the mean PT.
Recording the patient INR using the ISI and MNPT supplied with the reagent.
Example 1
1. Taking 1-10 gm, preferred 5gm/litre of Rabbit Brain Acetone Powder and
dissolving in 1 litre of Chloroform.
2. Shaking and stirring for 1-2 hrs at room temperature
3. Filtering the solution through the filter paper.
4. Drying the above extract with inert Nitrogen gas.
5. After drying the extract, obtain the cephaloplastin pellets. Its concentration should
be maintained between 10-100 mg/ deciliter in final APTT reagent.
6. Cephaloplastin pellet is dissolved in required amount of 0.01 to 1.0 M preferably
0.1 mole/litre sodium hydroxide solution.
7. Adding Phenol in the concentration of 0.01 to 0.1 Mole/ litre preferably
0.05mole/litre.
8. Adding Ellagic acid in the concentration of 0.025 to 0.045 gm/litre preferably
0.035gm/litre.
9. Adding HEPES buffer in the concentration of 0.6 to 4.6 gm/ litre preferably
2.6gm/litre.
10. Continuously stirring the solution at 200- 500 rpm for 4 hrs.
11. Adjusting the pH with 1 M HC1 and set the final pH to 7.2 ±0.1.
12. Adding the triple distilled water to adjust the final volume of the reagent.........
13. Adding transition metal ions salt preferred Nickel chloride in the concenrationof
10-50 mg/litre preferably 25gm/litre.
10

14. Stirring for 2 hrs at 200-500 rpm.
Example 2
1. Taking 1.25 gm of Rabbit Brain Acetone Powder and dissolving in 250ml of
Chloroform.
2. Shaking and stirring for 1 -2 hrs at room temperature
3. Filtering the solution through the filter paper.
4. Drying the above extract with inert Nitrogen gas.
5. After drying the extract, obtain the cephaloplastin pellets. Its concentration should
be maintained between 10-100 mg/ deciliter in final APTT reagent
6. Cephaloplastin pellet is dissolved in required amount of 200ml sodium hydroxide
solution.
7. Adding Phenol in the concentration of 1.17gm.
8. Adding 8.7mg of Ellagic acid to the above solution.
9. Adding HEPES buffer in the concentration of 0.65gm.
10. Continuously stirring the solution at 200- 500 rpm for 4 hrs.
11. Adjust the pH with 1 M HC1 and set the final pH to 7.2 ± 0.1.
12. Adding the triple distilled water to adjust the final volume of the reagent 250ml.
13. Adding transition metal ions salt preferred Nickel chloride in the concenrationof
6.25mg
14. Stirring for 2 hrs at 200-500 rpm.
Example 3
1. Taking 2.5 gm of Rabbit Brain Acetone Powder and dissolving in 500ml of
Chloroform.
2. Shaking and stirring for 1-2 hrs at room temperature
3. Filtering the solution through the filter paper.
4. Drying the above extract with inert Nitrogen gas.
5. After drying the extract, obtain the cephaloplastin pellets. Its concentration should
be maintained between 10-100 mg/ deciliter in final APTT reagent
6. Cephaloplastin pellet is dissolved in required amount of 450ml sodium hydroxide
solution.
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7. Adding Phenol in the concentration of 2.35gm.
8. Adding 17.5 mg of Ellagic acid to the above solution.
9. Adding HEPES buffer in the concentration of 1.3gm.
10. Continuously stir the solution at 200- 500 rpm for 4 hrs.
11. Adjusting the pH with 1 M HC1 and set the final pH to 7.2 ± 0.1.
12. Adding the triple distilled water to adjust the final volume of the reagent 500ml.
13. Adding transition metal ions salt preferred Nickel chloride in the concentration
12.5mg
14. Stirring for 2 hrs at 200-500 rpm.
Example 4
1. Taking 3.75 gm of Rabbit Brain Acetone Powder and dissolve in 750ml of
Chloroform.
2. Shaking and stir for 1-2 hrs at room temperature
3. Filtering the solution through the filter paper.
4. Drying the above extract with inert Nitrogen gas.
5. After drying the extract, obtain the cephaloplastin pellets. Its concentration should
be maintained between 10-100 mg/ deciliter in final APTT reagent
6. Cephaloplastin pellet is dissolved in required amount of 700ml sodium hydroxide
solution.
7. Adding Phenol in the concentration of 3.52gm.
8. Adding 26.1mg of Ellagic acid to the above solution.
9. Adding HEPES buffer in the concentration of 1.95gm.
10. Continuously stir the solution at 200- 500 rpm for 4 hrs.
11. Adjusting the pH with 1 M HC1 and set the final pH to 7.2 ± 0.1.
12. Adding the triple distilled water to adjust the final volume of the reagent 750ml.
13. Adding transition metal ions salt preferred Nickel chloride in the concentration
18.75mg
14. Stirring for 2 hrs at 200-500 rpm.
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I Claim:-
1.A process of preparation of the Activated partial Thromboplastin time
reagent comprising the following steps:-
i. Taking 1 gm of rabbit brain acetone powder and dissolving in
5ooml of chloroform.
j. Shaking and stirring for 1 hr at room temperature.
k. Filtering through Whatman No 1 filterpaper the above extract and
drying it with Nitrogen gas.
I. Dissolving the extract in 0.9 % NaCI solution( 1 Itr).
m. Adding 4.0 gm of Phenol.
n. Adding 0.035 gm of Ellagic acid.
o. Adding 2.6 gm of dry HEPES powder.
p. Adjusting the pH with 1M HCI and the pH should be 7.2 ± 1, thus
obtaining the reagent.
2. A process of determining the Activated Partial Thromboplastin Time in
whole blood or plasma comprising the following steps:
a. Shaking the Diagnos thrombo gently before use.
b. Pipetting 200ΜI of well mixed Diagnos Thrombo reagent in to a
test tube.
c. Pipetting 100 μl of patient plasma in to another test tube.
d. Incubating the test tubes containing reagent and plasma in water
bath at 37°C for 2-3 minutes.
e. Adding 200μl of well mixed Diagnos Thrombo reagent pre warmed
to 37 °C to the tube containing plasma sample and simultaneously
start the stopwatch as soon as fibrin strand is visible which
initiates gel formation.
f. Measuring the time taken for clot formation to the nearest 0.1
seconds. This is the prothrombin time in seconds.
13

g. Repeating the steps for the same sample and calculate the mean
PT.
h. Recording the patient INR using the ISI and MNPT supplied with
the reagent.
3. A process as claimed in claim 1 wherein the Diagnos APTT reagent is
Cephalloplastin reagent activated with Ellagic acid.
4. A process as calimed in claim 2 wherein the sample is collected in a
tube containing 1 part TSC ( Tri- Sodium Citrate) solution and 9 part
should be blood. Immediately mix with anticoagulant to avoid foam
formation. Centrifugate the sample for 15 minutes at approximately 3000
rpm and collect the plasma in a separate tube.
5. A process as claimed in claim 2 wherein the reagent and the plasma is to
be incubated in a water bath at 37°C for 3 minutes.
6. A process as claimed in claim 2 wherein the diagnos thrombo reagent is
to be pre-warmed to 37 °C.
7. A test device for performing the analysis according to the claim 2
comprising the diagnos thrombo reagent and TSC ( Tri Sodium Citrate
Solution) solution.
8. A test device as claimed in claim 7 wherein the reagent should be in
liquid form
9. A process of determining the Activated Partial Thromboplastin Time in
whole blood or plasma substantially as hereinbefore described with
reference to the examples.
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10.A test device for performing the analysis substantially as hereinbefore
described with reference to the examples
Dated this 4th day of May 2007.

15

The present invention describes a method of preparation of the partial
thromboplastin time reagent (APTT) which is used for the determination of
activated partial thromboplastin time in a whole blood or plasma at 37°C. A
test kit is also described for analysis of APTT which comprises of Diagnos
Thrombo reagent and TSC solution ( Tri - sodium Citrate solution) . The
reagent is Cephaloplastin reagent activated with Ellagic acid which is in
liquid form.