|Title of Invention||
THE THROMBOCHEK TEST KIT
|Abstract||The Thrombochek test is a new rapid screening, clinical laboratory test to measure hyperaggregable platelets in whole blood present in the form of 1). circulating platelet aggregates 2). causing spontaneous platelet aggregation and 3).hyper-reacting to a weak agonist of platelet aggregation in low concentration, in order to investigate thrombotic tendencies and to assess the efficacy of antithrombotic therapy,all measured with the help of an automated hematology cell counter and a magnetic stirrer.This is compared to the other existing microcopic method and shows 0.98 %. correlation. A test kit based on the above methodology to be used with the aid of an automated hematology analyser is made available for routine clinical and office laboratory use.|
|Full Text||FORM 2
THE PATENT ACT 1970 (39 of 1970)
The Patents Rules, 2003
COMPLETE SPECIFICATION See Section 10, and rule 13]
TITLE OF INVENTION
A NEW RAPID WHOLE BLOOD SCREENING TEST TO DETECT PLATELET HYPERAGGREGATION IN THE CLINICAL LABORATORY - THE NEW THROMBOCHEK TEST
DILIP SHRINIVAS VELASKAR INDIAN National
SANT JANABAI ROAD,
VILE PARLE (EAST), MUMBAI - 400 057 MAHARASHTRA
PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed : -
A New rapid whole blood screening test to detect platelet hyperaggregation in the Clinical Laboratory -The New Thrombochek test
FIELD OF INVESTIGATION
The present invention relates generally to the methods and measurements for determination of platelet hyperaggrgation in a whole blood sample and for detecting and measuring the efficacy of treatment with antiplatelet drugs or the resistence thereto
BACKGROUND OF THE INVENTION
Many if not most attacks due to thrombosis can be prevented by timely and appropiate antithrombotic therapy and then also the recurrent episodes can be prevented by appropriate secondary therapy (BARBARA MA)
Most venous and 65% arterial thrombosis are associated with either a blood coagulation protein defect or a platelet function defect ,which is either congenital or acquired
With the current boom in medical technology expansion almost all aeitiologies of hypercoagulation and overt thrombosis are clearly emerging The newer developments in antithrombotic therapyfor the treatment of active isorders and for the prophylactic prevention of a thrombotic episode are most promising now than ever before The use of this fresh information added on with regular clinical assessment complimented with wider use of various testing modalities for patient evaluation and the ever increasing influx of newer antithrombotic therapy has lead to conflicting and confusing views amongst the practising physicians and research scientists
There is scanty knowledge regarding "hyperactive" platelets ,more so in that the present literature is one big subject of debate,apart from the single confirmed fact that such a condition definitely exists.
The reason for this lacuna lies mainly in the difficulties involved in setting up,implementing and standardising a proper test method Most results in literature on the hyperactive platelets are derived by platelet aggregometry which in itself is time consuming besides being cumbersome and difficult to standardise Platelet aggregometry was basically devised to assess platelet function by haematologists for their bleeding patients with functional platelet defects.These tests were never invented for the thrombotic patient although a few technical manipulations exist Yet,hyperactive platelets have been widely described by various techniques in a number of disorders like diabetes mellitus leading to its vascular complication (COLWELL JH ET AL),in unstable angina and atrial fibrillation,thrombotic strokes,migraine headaches,anorexia nervosa,mitral valve prolapse ,retinal artery occlusion,pre- eclampsia,arterial thromboembolism and nephrotic syndrome.
Patients in intensive care units have been described as having hyperaggregable platelets.There are some reports of elevated levels of platelet release proteins such as B-thromboblobulin,platelet factor 4 and thromboxane A2 suggesting that the platelets are activated in vivo. It has been so far difficult to establish whether the platelet hyperactivity has been the cause or the effect Platelet aggregometry has technical difficulties and produces artifacts depending on the aggregometer,technologist and the varried normal ranges for the different agonists(KIRCHOF B ET AL) Probably hyperaggrgability is facilitated by the involvement of receptors on the surface of platelet membranes Association of clinical events and emotional stress is highlighted in many studies and the hyperaggregation response of platelets to adrenaline release or stress is a main point of study espescially since it is known that there are platelets with hyperaggregable platelets whose platelet release proteins are not elevated (ROSWELL HC ET AL)
SUMMARY OF THE INVENTION
Hyperaggregable platelets and their role in thrombogenesis were not well known and little is published on hyperactive platelets, the controversy and lack of information is mainly due to lack of methodology as there is as yet no screening test for hyperaggregable platelets,while aggregometry so far used was basically designed for studying bleeding disorders rather than thrombotic disease (FRENKEL EP ET AL).
The recent flow cytometric studies though adding valuable information can hardly be described as an affordable screening test for hyperactive platelets A first aspect of this invention relates to the methods of demonstrating hyperactive platelets by
1) Measuring circulating platelet aggregates by collecting a blood sample from an individual in K2EDTA anticoagulant vacutainer first and then taking blood in trisodium citrate- anticoagulant vacutainer with the same venipuncture and mixing both the samples with the respective anticoagulants atleast 15 to 20 times by inversion and allowing K2EDTA sample to stand at room temperature for a minimum of 20 minutes and then analysing both the samples on an automated haematology analyser to determine the platelet counts and the difference between K2EDTA platelet count and citrate platelet count is taken as a measure of circulating platelet aggregates ss after working out percentage..
2) Demonstrating spontaneous platelet aggregation by taking a part of the citrated sample into a flat bottomed plastic tube and stirring the same with a magnetic stirrer at room temperature for 3 minutes and analysing the same on automated haematology analyser and the difference observed between the platelet count of the stirred sample and the platelet count of the not stirred citrated sample is taken as a measure of the spontaneous platelet aggregation after working out the percentage
3) Demonstrating increased platelet response to a weak agonist in low concentration by treating the stirred citrated sample with weak agonist in low concentration and countinuos stirring at room temperature and analysing the same on an automated haematology analyser exactly at the end of 30 sec and 180 sec after the addition of the agonist and the difference between the citrated sample count and the agonist treated count is taken as a measure of response to the agonist after calculating the percentage of the same for corresponding 30 sec and 180 seconds
DETAILED DESCRIPTION OF THE INVENTION
The new test relates to measuring platelet hyper-aggregations in the view of investigating thrombotic tendencies and to form its treatment modalities and subsequent followup:-
It can be studied in mammals-primate such as humans ,chimpanzees and dogs,pigs etc. Blood is obtained by venipuncture using prefictibly precisionglyde (Becton Dikinson) it can be collected during a medically invariable procedure ,such as the open heaart procedure Blood sample collected by vaccutainer collection procedure and the vaccutainer connected to veinipuncture needle in the following order.First the plain vaccutainer which is to be discarded then the K2EDTA vaccutainer and thirdly the Na citrate 3.8 % to be connected to the vaccutainer needle.The samples collected in K2EDTA and 3.8% Na citrate anticoagulant vacutainers are immediately thoroughly mixed by inversion (15 to 20 times) and then allowed to stand at room temperature for a minimum period of 20 minutes before starting the test.Twenty minutes is the time given for the K2EDTA to act on existing platelet aggregates to break down andlience give the single platelet count.Heparin,Na Citrate 3.2 % can also be used instead of citrate3.8% as anticoagulant to study platelet hyperaggregation. D-Phe-Pro-Arg chloromethyl ketone dihydrochloride used as an anticoagulant in some studies is not considered here as an anticoagulant for this study because there is substantial disaggregation of platelet aggregation caused by addition of agonist creating the doubt whether D-Phe-Pro-Arg chloromethyl ketone dihydrochloride has antplatelet action.
Analysis of blood sample can be carried out on any haematology analyser preferably automated impedencce type of haematology analyser eg. Coulter counter.This haematology analyser can be used to identify platelet aggregation as a result of changes in the platelet counts caused by either stirring of blood sample by magnetic stirrer or by addition of agonist causing platelet aggregation..The magnetic stirrer and teflon coated magnetic bar used here for stirring is from Spinnot magnetic stirrers.
Agonist available for inducing platelet aggregation are Adenosine diphosphate (ADP),epinephrine,collagen,thrombin,etc.Epinephrine is the preferred agonist as it is released in stress.
In carrying out the methods of current invention the circulating platelet aggregates are measured
by analysing the K2EDTA sample, after thoroughly mixing by inversion after collection and
allowing to stand at room temperatue for twenty minutes,on haematology
analyser. Simultaneously cirate samples are also analysed on the haematology analyser after thoroughly mixing and the difference between K2EDTA platelet count and citrate sample platelet count devided by the EDTA platelet count multiplied by 100 gives the percentage of circulating platelet aggregates.
In carrying out methods for measurement of spontaneous platelet aggregation by the present invention the blood sample collected in 3.8% Na citrate is thoroughly mixed and part of the sample is transferred in flat bottom plastic tube and stirred with magnetic stirreer after inserting a teflon coated magnetic bar in the blood sample continuosly for three minutes at room temperature at the speed of 1000 rpm and then analysing the sample on the haematology analyser for platelet count.Simultaneously a platelet count measurement is also obtained from the unstirred citrated sample after thorough mixing by inversion..The Percentage of spontaneous platelet aggregation is calculated by noting the difference between the two platelet counts of stirred and not-stirred citrated blood samples and the divided by the initial citrated platelet count and this then is multiplied by 100.
In carrying out the measurement of platelet hyperaggregation by the present invention the citrated blood sample aliquoted into the flat bottomed plastic vial with magnetic teflon coated bar inside is treated with a weak agonist of platelet aggregation and then stirred continuously and then analysed at 30 sec on an automated hematology analyser and again stirred for 150 sec and analysed at the end of this 180 sec for another platelet count measurement on the automated hematology analyser .The percentage of platelet aggregation at 30 sec is calculated from the difference in platelet count between the initial Citrate platelet count and the 30 sec platelet count after exposure to epinephrine with continuous stirring.The percentage of platelet aggregation at 180 sec is determined by calculating the difference of initial Citrate platelet count and the platelet count after 180 sec of continuos stiiring after exposue to agonist.
The tests were carried out on healthy adults (male and female) and the normals were established .The test was carried out on patients of diabetes mellitus and known cases of myocardial infarction. Simultaneously blood smears were prepared at each step and stained and examined to correlate with the microscopic method for platelet aggregation for measurement of hyperactive platelets(VELASKAR DS ET AL).A strong correlation is obtained between the two methods and the present invention is found to be best suited for the demonstration of hyperaggregable platelets in investigating thrombotic tendencies and for its subsequent treatment and followup A group of patients on anti-platelet -Aspirin therapy were tested by new Thrombochek test for assessment of efficasy.
MATERIAL AND METHODS
Four groups of individuals were examined:
Group 1 :twenty healthy adult men and twenty healthy adult women between the ages of 22
to 35 years were tested appropriately as detailed
Group 2 : Twenty cases of diabetes mellitus (fasting plasma glucose levels of more than
1,30 mg/dl were tested appropriately as detailed
Group 3 : Twenty cases of myocardial infarction were tested appropiately as detailed
Group 4 : Twenty patient on Aspirin for minimum period of 3 weeks.
Equipment and Reagents :
B D vacutainers for K2EDTA ,3-8% trisodium citrate and 20 G needles,8/30 mm flat bottomed plastic tubes,Magnetic stirrer (Spinnot),teflon coated magnetic bars,automated hematology analyser (Coulter -Impedence type particle counter),Glass slides,automated Pipettes,stop watch and microscope with oil immersion lens. Reagents used:Epinephrine bitartrate 0.2 uM ,0.5 uM, as agonist of platelet aggregation,alcohol and Wright -Giemsa,s stain. Samples for Analysis
All samples were collected in the mornsing when the subjects were fasting.Blood was collected in Plain vaccutainer then K2EDTA vacutainer by a clean venepuncture using a 20 G needle and then In the 3.8% Na citrate vacutainer.Both these anticoagulated blood samples were thoroughly mixed with respective anticoagulants by inversion (15 to 20 times) and the
K2EDTA sample was made to stand at room temperature for 20 minutes after collection and only the analysed for platelet count measurements on the automated hematology analyser.Both samples were analysed within two hours of collection.
STEP 1 : K2EDTA sample analysed on hematology analyser after repeated mixing by inversion 20 minutes after collection and platelet count recorded
STEP 2 : Citrated sample analysed and platelet count recorded and a smear prepared and marked
STEP 3 : Part of the citrated sample is transferred to a flat bottomed plastic tube (8/30mm) nd then stirred using a magnetic stirrer after insertion of a teflon coated magnetic bar in the blood sample,stiring continuously at room temperature at a speed of 1000 rpm for 3 minutes and then analysing this sample on an automated hematology anlyser and recording the platelet count. Simultaneously a smear is prepared and marked
STEP 4 :To the stirred aliquot of citrated sample in the plastic tube an agonist was added the final concentration being 0.2 uM and the stop watch was started while the sample was stirred continuously for 30 sec and then exactly at the end of 30 sec the sample was analysed for platelet count on hematology analyser and this was recorded,simultaneously a smear was prepared and marked.
STEP 5 : Stirring of the agonist added mixture was continued for anotherl50 sec and exactly at the end of 180 sees the mixture was analysed on hematology analyzer and platelet count was recorded Simultaneously a smear was prepared and marked., tep 4 and 5 are repeated with increased concentration agonist. Group 4 was tested with 0.5 uM concentration of agonisat.
EXAMINATION AND RECORDING:
Percentage of Circulating platelet aggregates was calculated by finding out the difference between EDTA count and citrate count and dividing this by the EDTA count and multiplyiung it by 100 Percentage of spontaneous platelet aggregation was calculated by finding out difference between platelet count of citrate sample-not strirred and platelet count of stirred citrate sample and deviding the same by platelet count of not stirred citrate count and then multiplying by 100.
Percentage of platelet aggregation in responce to weak agonist at 30 seconds was calculated by finding out the difference between platelet count of agonist treated stirred citrate sample at 30 seconds and platelet count of not-stirred citrate sample and dividing it by not-stirred citrate sample platelet count and then multiplying it by 100.
Percentage of platelet aggregation in response to weak agonist at 180 seconds was calculated by finding out the difference between Citrated sample platelet count and platelet count of agonist treated stirred citrate sample analysed at 180 seconds and deviding the difference by Citrated sample platelet count and multiplying by 100.
All the platelet counts where dilutional effect was observed due to anticoagulant or agonist were corrected.
All the subjects were simultaneously examined by MICROSCOPIC METHOD of studying platelet aggregation, as follows:
For measuring Circulating platelet aggregates a smear was prepared from the citrated sample of blood. For measuring Spontaneous aggregation of platelets a smear was prepared from citrated sample stirred for three minutes. For measuring platelet aggregation in responce to weak agonist a smear was prepared from agonist treated stirred citrate sample at 30 seconds.For measuring platelet aggregation at 180 seconds in response to agonist a smear was prepared from agonist treated citrated stirred sample at corresponding time.
All the smears were prepared by taking a drop of blood on slide and making a smear by cover slide method of smear preparation. All the smears were fixed in alcohol after drying and stained with WRIGHT-GEIMSA stain and examined under oil immersion lense.Platelets free and in aggregates were counted and percentage of aggregation was worked out.
Results obtained in all the three groups of subjects for Circulating platelet aggregates
Spontaneous platelet aggregation and Platelet aggregation in response toEpinephrine 0.2 uM at
30 seconds and at 180 seconds showing Mean value and Standerd deviation are depicted in
Comparison of Circulating platelet aggregates obtained by New Thrombochek test and
Microscopic method in Normals,Diabetics and patients of MI are as shown in Fig. 1.
Comparison of Spontaneous Platelet aggregation obtained by new Thrombochek test and
Micrscopic method of Platelet aggregation in Normals,Diabetics and Patients of M I are are
shown in Fig 2.
Comparison of platelet aggregation in response to epinephrine 0.2 uM at 30 seconds
obtained by New Thrombochek test and Microscopic method of platelet aggregation in
Normals,Diabetics, MI are shown in Fig.3.
Comparison of platelet aggregation in response to epinephrine 0.2 uM at 180 seconds obtained
by new Thrombochek test and Microscopic method of measuring platelet aggregation in
Normals, Diabetics and M I are shown in Fig 4.
Comparison of platelet aggregation in response to epinephrine 0.5 uM at 30 seconds obtained by
new Thrombochek test and Microscopic method of platelet aggregation in Normals ,Diabetics
and MI are shown in Fig.5.
Comparison of platelet aggregation in response to epinephrine 0.5 uM at 180 seconds obtained
by new Thrombochek test and Microscopic method in Normals,Diabetcs and MI in Fig 6.
TWENTY PATIENTS OF MYOCARDIAL INFARCTION ON DAILY ASPIRIN FOR MINIMUM PERIOD OF 3 WEEKS WERE TESTED FOR CIRCULATING PLATELET AGGREGATES, SPONTANEOUS PLATELET AGGREGATION AND FOR PERCENTAGE OF PLATELET AGGREGATION IN RESPONSE TO EPINEPHRINE 0.2 uM AT 30
SECONDS AND AT 180 SECONDS BY NEW THROMBOCHEK TEST AS WELL AS BY MICROSCOPIC METHOD FOR ASSESSMENT OF EFFICACY OF ANTIPLATELET TREATMENT. THE RESULTS ARE AS FOLLOWS: FOUR PATIENTS SHOWED PRESENCE OF CIRCULATING PLATELET AGGREGATES AND THE PERCENTAGE RESPONSE TO AGONIST AT 30 SECONDS AND AT 180 SECONDS WAS ABOVE NORMAL RANGE BY BOTH THE METHODS INDICATING THAT ANTI-PLATELET THERAPY WAS LESS THAN EFFECTIVE IN FOUR OF THEM. Table 2.
1. THE THROMBOCHEK TEST IS BASED IN THE CLINICAL LABORATORY SETTING AND IS AN EFFICIENT AND RAPID SCREENING TEST FOR EARLY DETECTION OF PLATELET HYPERAGGREGATION IN THROMBOTIC DISEASE, THEREFORE CAN BE USED IN PREVENTION , ESPESCIALLY IN DIABETICS PATIENTS WITH MYOCARDIAL INFARCTS AND SMOKERS IT CAN BE USED FOR DOSAGE MONITORING FOR PATIENTS ON ANTIPLATELET THERAPY
2. THE THROMOCHEK TEST COMPARES WELL WITH THE KNOWN AND ESTABLISHED MICROSCOPIC METHOD OF MEASURING PLATELET HYPERAGGREGATION
3. THE THROMBOCHEK TEST CAN EASILY BE USED AT POINT OF CARE WITH APPROPRIATE ANTICOAGULANT FOR BLOOD SAMPLE COLLECTION FOR PATIENTS ON THERAPY,FOR MEDICAL MANAGEMENT OF NON-ST -SEGMENT ELEVATION ACUTE CORONARY SYNDROMES AND ESPESCIALY PATIENTS TREATED WITH PER CUTANEOUS CORONARY INTERVENTION
1).BARBARA MA;SEM HEMAT 2002;39:143-144 2).COLWELL JH ET AL .MED CLIN NORTH AM 1978;62:;753-756 3).FRENKEL EP ET AL :HEMATOL ONCOL CLIN N AM 17 (2003) 63-83 4).ROSWELL H C ET AL BR J HAEMAT 1966;12::66-71S 5).VELASKAR DS ET AL :AM J.CLIN.PATH 1982;77:3:267-274
1. A method of measuring platelet hyperaggregation in the form of circulating platelet aggregates in whole blood comprising of obtaining a smooth flow blood sample from an individual first in a K2EDTA vacutainer and then in a 3.8% trisodium-citrate anticoagulant vacutainer ,in that order and after proper mixing blood with corresponding anticoagulant by inversion 15 to 20 times and allowing both samples to stand at room temperature for a minimum of 20 minutes and then analysing both the samples within 2 hours on haematology analyser after again proper mixing by inversion and calculating the difference between the K2EDTA platelet count and the citrated sample count, to give exact percentage of circulating platelet aggrgates and to obtain blood samples in the sameway from statistically significant number of normal healthy individuals to analyse circulating platelet aggregates in them to establish normal range by calculating its mean and standerd deviation.
2.. The method according to claim 1 of mixing both samples by inversion atleast 15 to 20 times, in their corresponding anticoagulant
3. The method according to claim 1 of letting the K2EDTA sample to stand at room
temperature so as to obtain single platelet counts after allowing K2EDTA, to break existing plaelet aaggregates and allowing the citrated sample to stand at room temperature for the same time
4.. The method according to claim 1 of calculating percentage of circulating platelet aggrgates allowing 0.98% correlation to the existing microscopic method of analysing circulating platelet aggregates.
5.. The method of demonstrating hyperaggregable platelets causing spontaneous platelet aggregation in citrated blood sample comprising of analyzing citrated blood sample on haematology analyzer for platelet count after proper mixing by inversion and transferring part of the citrated blood sample into a flat bottomed non-water-wettable plastic container and stirring the sample at room temperature for 3 minutes at 1000 rpm with a
magnetic stirrer and a magnetic bar also with non-water-wettable surface and then analysing the stirred sample on an automated haematology analyser and then calculating the difference between the first citrated sample platelet count and that of the stirred sample of citrated blood to give exact percentage of spontaneous aggregation of platelets and to analyse blood samples obtained from statistically significant number of normal healthy individuals to establish a normal range for spontaneous platelet aggregation by calculating their mean and standerd deviation.
6 The method according to claim 5 of calculating percentage of spontaneous platelet aggregates allowing 0.98% correlation to the existing microscopic method of analysing spontaneous platelet aggregates.
7 The method of demonstrating hyperactive platelets which react to weak agonist of platelet aggregation in its low concentration for 30 seconds in a citrated sample of blood, comprising of analyzing the blood sample, collected in 3.8 % sodium citrate vacutainer,on a haematology analyzer,after proper mixing by inversion with the anticoagulant, for platelet count and then transferring aliquot of the citrated sample to a flat bottomed non-water-wettable plastic vial and after stirring for two minutes on a magnetic stirrer with a magnetic bar at 1000 rpm at room temparature, exposing the stirred citrated sample to a weak agonist of platelet aggregation with continued stirring , exactly at the end of 30 seconds after addition of agonist, analysing this sample on the automated cell counter and calculating the difference between the Citrate sample platelet count and agonist treated stirred Citrate sample platelet count to give percentage of platelet aggregation at the end of 30 seconds with a weak agonist of platelet aggregation and to analyse blood samples obtained from statistcally significant number of normal healthy individuals in the same manner to establish a normal range for platelet aggregation in response to same weak agonist at 30 seconds by calculating their mean and standerd deviation.
8 The method according to claim 7 of exposing the stirred citrated blood aliquot to the weak agonist of platelet aggregation - Epinephrine bitartrate 0.2 uM final concentration and analysing the sample at exactly 30 sec after addition of agonist.
9 The method according to claim 7 of calculating percentage of platelet aggregation at 30 sec with exposure to weak agonist by calculating the difference between Citrate sample platelet count and agonist treated stirred citrated sample platelet count at 30 sec allowing 0.98% correlation to the existing microscopic method of platelet aggrgation
10 The method of demonstrating hyperactive platelets which react to weak agonist of platelet aggregation in its low concentration for 180 seconds in a citrated sample of blood comprising of analyzing blood sample collected in 3.8 % trisodium citrate vacutainer after proper mixing by inversion on haematology analyzer for platelet count and then transferring aliquot of same citrated blood sample to flat bottomed non-water-wettable plastic vial and after stirring for two minutes on magnetic stirrer with magnetic bar with non-water-wettable surface at 1000 rpm at room temparature , exposing the stirred citrated sample to weak agonist of platelet aggregation with continued stirring, and exactly at the end of 180 seconds after addition of agonist analyzing it on automated haematology analyzer for platelet count and calculating the difference between the citrate sample platelet count and agonist treated stirred citrate sample platelet count to give percentage of platelet aggregation at the end of 180 seconds with a weak agonist of platelet aggregation and to analyse blood samples obtained from statistically significant number of healthy individuals in the same manner to establish normal range of platelet aggregation in response to weak agonist of platelet aggregation at 180 seconds by calculating their mean and standerd deviation..
11 The method according to claim 10 of exposing the stirred citrated blood aliquot to the weak agonist of platelet aggregation -Epinephrine bitartate at low concentration- 0.2 uM final concentration and analysing the sample at exactly 180 sec
12 The method according to claim 10 of calculating percentage of platelet aggregation at 180sec with exposure to weak agonist by calculating the difference between Citrated platelet count and agonist treated stirred citrated sample platelet count at 180 sec allowing 0.98% correlation to the existing microscopic method of platelet aggrgation
13. The method determining or monitoring the efficacy of antiplatelet therapy comprising of obtaining a blood sample from an individual treated with an anti-platelet therapy in K2EDTA vaccutainer and in trisodium Citrate 3.8 % vaccutainer and analysing the same for Circulating platelet aggregates, for demonstrating Spontaneous platelet aggregation and measuring platelet response to agonist of platelet aggregation at 30 seconds and at 180 seconds on an automated haematology analyser with the help of magnetic stirrer to determine the platelet aggregation inhibition wherein presence of Circulating platelet aggregates or Sponteneous platelet aggregation or percentage of platelet aggregation in respnse to agonist at 30 seconds and 180 seconds is at or above normal range indicating that antiplatelet therapy is less than effective.
14. The method according to claim 13 wherein anti-platelet therapy comprises Cyclo-oxygenage inhibitor.
15. The metod according to claim 13 wherein Cyclo-oxygenage inhibitor is aspirin.
16 The metod accorcing to claim 13 wherein the anticoagulants are K2EDTA and Na2 Citrate 3.8 %.
17. The THROMBOCHEK TEST kit based on the thrombochek test methodology described in the above claims comprising of platelet agonist reagent,a diluting fluid for the reagent and a flat bottomed plastic tube containing a magnetic bar with non water wettable surface alongwith instruction for application to measure platelet hyperaggregation in the form of l).circulating platelet aggregates 2).causing spontaneous platelet aggregation and 3).hyper-reacting to a weak agonist of platelet aggregation,all to be measured with the help of an automated hematology analyser and a magnetic stirrer
Dated this 26th day of June, 2006
HIRAL CHANDRAKANT JOSHI
DILIP SHRINIVAS VELASKAR
The Thrombochek test is a new rapid screening ,clinical laboratory test to measure hyperaggregable platelets in whole blood present in the form of 1). circulating platelet aggregates 2). causing spontaneous platelet aggregation and 3).hyper-reacting to a weak agonist of platelet aggregation in low concentration, in order to investigate thrombotic tendencies and to assess the efficacy of antithrombotic therapy,all measured with the help of an automated hematology cell counter and a magnetic stirrer.This is compared to the other existing microcopic method and shows 0.98 %. correlation . A test kit based on the above methodology to be used with the aid of an automated hematology analyser is made available for routine clinical and office laboratory use.
The Controller of Patents,
The Patent Office,
|Indian Patent Application Number||1012/MUM/2006|
|PG Journal Number||42/2011|
|Date of Filing||27-Jun-2006|
|Name of Patentee||DILIP SHRINIVAS VELASKAR|
|Applicant Address||20/PUJA BHAVAN SANT JANABAI ROAD, VILE PARLE (EAST), MUMBAI - 400 057,|
|PCT International Classification Number||G01N33/53|
|PCT International Application Number||N/A|
|PCT International Filing date|