THROMBOSIS RESEARCH 62; 625633,1991 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.
THE INFLUENCE OF TIME, TEMPERATURE AND PACKED CELL ON ACTIVATED PARTIAL THROMBOPLASTIN TIME AND PROTHROMBIN TIME
Chao-Hung Ho, and Shwu-Yann Wu Division of Hematology, Department of Internal Medicine, General Hospital, Taipei, Taiwan, R.O.C.
Veterans
(Received 25.12.1990; accepted in revised form 9.3.1991 by Editor A. Takada)
ABSTRACT Activated partial thromboplastin time (APTT) and prothrombin time (PT) were performed in four groups of studies in order to evaluate the influences of time, temperature, and different forms of plasma storage to the result. Different designs for storage of the plasmas were studied, including the plasmas stored either with or without packed cells, the plasmas stored in the cuvette with exact volume for performing the test or in the test tube. The temperatures for store of the plasma were at room temperature, at 4-C and at -70-C. The time for store of the plasma was from 1 hour up to 7 hours. The plasmas included normal pooled plasmas and diseased plasmas. From this study, it is found that the PT test was not easily affected by the temperature, storage time and the form of storage in comparison with the APTT test which was much easily affected by the above conditions. APTT should be done within 2 hours after sampling and the plasma should be stored with the packed cells at 4-C in order to obtain a reliable result. PT could be done within 7 hours without influence to the result if the plasma was stored with the packed cells at 4-C. No significant cold-induced shortening of PT could be noted when the plasma was incubated at 4-C up to 7 hours. In either PT or APTT, the most suitable condition for storing the plasma should be with the packed cells at 4-c. _-__--_--_--_-
Key words: Time, temperature, activated time, prothrombin time, packed cell
625
partial
thromboplastin
626
INFLUENCES ON APTT & PT
Vol. 62, No. 6
INTRODUCTION The activated partial thromboplastin time (APTT) and the prothrombin time (PT) are the most simple and useful procedures for routine screening of coagulation disorders in the intrinsic and extrinsic systems respectively. Many factors can affect the results of these tests (l), of them, the time between sampling and sample performance and the temperature for store of samples were two of the important factors. The time from sample collection to sample performance was advised to be within 2 hours to avoid invalid prolonged results (2-3). The PT performed on blood samples stored at 4'C may give markedly shorter clotting time than those performed on the same samples before storage [l], and this cold-promoted activation of the PT would be prevented by the addition of inhibitors of factor XIIa or factor XII fragments (4). In the present study, it is planned to perform PT and APTT in different time periods with different temperatures, and to store the plasma either with or without the packed cells after centrifugation, in order to find the influence of the time, the temperature and the packed cells to the PT and the APTT, and to find out a minimum influence in spite of delayed handling of the sample. MATERIALS AND METHODS Clottinq Assays After clean venipuncture by a plastic syringe blood was collected into the anticoagulant (l/10 volume of 3.8% sodium citrate), clotting assays were performed using the reagents of Behring, Marburg, BRD. The reagents for PT and APTT were Thromborel and Pathromtin, respectively. The detail procedures followed the instructions of the manufacturer. An automatic coagulometer KC 10 (Heinrich Amelung GmbH, Lemgo, Germany) was used to perform both the PT and APTT. Study Projects and Sample Proceedinq Four different study programs were planned as follows. The first group included 35 normal pooled plasmas, each collected from 5-7 healthy volunteers without a bleeding history. After blood collection, plasma was taken immediately by centrifugation at 1500 g and at room temperature for 15 minutes. After obtaining the pooled plasma by mixing 5-7 normal plasmas, PT and APTT were performed immediately and the result was recorded as "0 hour". The remaining plasma was divided and put into three test tubes with stoppers (i.e. covered with paraffin sheet), one leaving at room temperature, one in a 4-C refrigerator and the other in a 70-C freezer. For the part at -70-C, the plasmas were divided and stored in the cuvette cups supplied with the coagulometer KC 10 with the exact volume ready for performing PT and APTT, i.e. O.lml. And when the tests were to perform, it would be thawed in a 37-C incubator for approximately 3 min. For these 3 parts, PT and APTT were performed 1, 2, 4, 5, 6 and 7 hours after blood collection, and the results were compared.
INFLUENCES ON APTT & PT
Vol. 62, No. 6
627
The second group included 18 normal plasma samples. After performing the 0 hour result as mentioned above, the plasma was put into the -70-C freezer with two different preparations: 1) exactly O.lml of plasma in each cuvette cup supplied by the KClO, and 2) the remaining volume of plasma put into a stoppered test tube. The plasmas were thawed as described above. The APTT was performed 2, 4, 6 and 7 hours after blood collection, each time with two different preparations as mentioned above. The difference of the results was then compared. The third group included 33 subjects without bleeding history. After obtaining and centrifugating the blood samples, PT and APTT were performed (recorded as 0 hr). Thereafter, two preparations for storing the plasma at room temperature and at 4' C were designed: 1) the plasma was stored in a stoppered tube altogether with the separated packed cells, i.e. the plasma at the upper and the packed cells at the bottom. 2) the plasma was separated immediately from the packed cells and stored alone up to the performances of the sample. For storing samples at -70-C, the same preparation described as in group 1 was used. PT and APTT were performed l-2, 4 and 6 hr after blood collection. For each time period, PT and APTT were performed with 2 different storage forms of plasma at room temperature and at 4-C, and with one storage form at -70-C, and the results were then compared. The fourth group included 8 patients with bleeding or administration disorders, such as hemophilia, liver cirrhosis of warfarin. PT and APTT were performed 2, 4, 6 and 7 hours after sample collection at 3 different temperatures as described in group I, and at two storage forms as described in group 3. The results were compared at different conditions. Stastistical
analysis
Paired-t test was used to compare the differences of two variables in the same condition in the first, third and fourth group studies. Two-tail level of significance of Wilcoxon Signed Ranks Test was used to compare the differences between the results performing with the cuvette or the test tube at a fixed time in the Group 2 study. The mathematical means were used to represent the means in all 4 groups of people. Results
RESULTS of the first study group
The means of PT and APTT in different temperatures and times of 35 normal pooled plasmas are shown in Table 1. In either PT or APTT, the 0-hr mean was compared with the means of other different conditions and the p value was calculated by the paired-t test. In the PT tests, significant shortening of PT in several conditions in comparison with the 0-hr hr at room temperature, 1 hr at 4-C, 3,4 & 6 hr significant prolongation of PT could be found in
could be found mean (e.g. l-4 at -70-C). No all conditions
INFLUENCES ON APTT & PT
628
Vol. 62, No. 6
TABLE 1 The Means of PT And APTT At Different Temperatures 35 Normal Pooled Plasmas
And Times
in
___--_-_--_-__-_--_-~--~-~~-~-~~-~----~~---~---------~--~----~--~
Room temperature 4-c -7O'C -_-_----------------------_-------------_----_--PT APTT PT APTT PT APTT Mean + SD Mean + SD Mean + SD (set) (set) (see) -__-_--_-____--_-_--~----~-~~-~----~--~-~--~--~-~~---~--~-~--~-~0 11.13 36.60 20.56 +1.63 1 1o.a7*** 37.01 10.94** 38.00*** 11.10 39.28*** +0.65 21.88 +0.54 +2.50 50.79 +2.52 2 10.99* 37.46*** 11.09 38.78** 11.07 39.78*** +0.63 21.71 +0.64 54.14 +0.57 +3.72 4 10.88*** 38.32*** 11.11 40.32*** 10.96*** 40.10*** +0.44 +2.16 +0.52 25.75 LO.53 +3.80 5 10.89*** 38.84*** 11.13 39.94*** 10.91** 39.74*** 20.50 22.42 20.63 24.27 +0.63 23.73 6 11.03 40.80*** 11.22 41.09*** 11.00 39.52*** +0.54 +5.15 +0.64 +4.88 +0.64 +2.76 7 11.09 41.9a*** 11.25 40.72*** 10.89* 39.04*** +0.50 24.65 +0.57 +0.56 +4.71 +2.81 -------_____________~~~~~~~~~~~~~~~~~~-~~~~-~~~~~~~-~~~~~~~~~-~~~ *p
THE INFLUENCE OF TIME, TEMPERATURE AND PACKED CELL ON ACTIVATED PARTIAL THROMBOPLASTIN TIME AND PROTHROMBIN TIME
Chao-Hung Ho, and Shwu-Yann Wu Division of Hematology, Department of Internal Medicine, General Hospital, Taipei, Taiwan, R.O.C.
Veterans
(Received 25.12.1990; accepted in revised form 9.3.1991 by Editor A. Takada)
ABSTRACT Activated partial thromboplastin time (APTT) and prothrombin time (PT) were performed in four groups of studies in order to evaluate the influences of time, temperature, and different forms of plasma storage to the result. Different designs for storage of the plasmas were studied, including the plasmas stored either with or without packed cells, the plasmas stored in the cuvette with exact volume for performing the test or in the test tube. The temperatures for store of the plasma were at room temperature, at 4-C and at -70-C. The time for store of the plasma was from 1 hour up to 7 hours. The plasmas included normal pooled plasmas and diseased plasmas. From this study, it is found that the PT test was not easily affected by the temperature, storage time and the form of storage in comparison with the APTT test which was much easily affected by the above conditions. APTT should be done within 2 hours after sampling and the plasma should be stored with the packed cells at 4-C in order to obtain a reliable result. PT could be done within 7 hours without influence to the result if the plasma was stored with the packed cells at 4-C. No significant cold-induced shortening of PT could be noted when the plasma was incubated at 4-C up to 7 hours. In either PT or APTT, the most suitable condition for storing the plasma should be with the packed cells at 4-c. _-__--_--_--_-
Key words: Time, temperature, activated time, prothrombin time, packed cell
625
partial
thromboplastin
626
INFLUENCES ON APTT & PT
Vol. 62, No. 6
INTRODUCTION The activated partial thromboplastin time (APTT) and the prothrombin time (PT) are the most simple and useful procedures for routine screening of coagulation disorders in the intrinsic and extrinsic systems respectively. Many factors can affect the results of these tests (l), of them, the time between sampling and sample performance and the temperature for store of samples were two of the important factors. The time from sample collection to sample performance was advised to be within 2 hours to avoid invalid prolonged results (2-3). The PT performed on blood samples stored at 4'C may give markedly shorter clotting time than those performed on the same samples before storage [l], and this cold-promoted activation of the PT would be prevented by the addition of inhibitors of factor XIIa or factor XII fragments (4). In the present study, it is planned to perform PT and APTT in different time periods with different temperatures, and to store the plasma either with or without the packed cells after centrifugation, in order to find the influence of the time, the temperature and the packed cells to the PT and the APTT, and to find out a minimum influence in spite of delayed handling of the sample. MATERIALS AND METHODS Clottinq Assays After clean venipuncture by a plastic syringe blood was collected into the anticoagulant (l/10 volume of 3.8% sodium citrate), clotting assays were performed using the reagents of Behring, Marburg, BRD. The reagents for PT and APTT were Thromborel and Pathromtin, respectively. The detail procedures followed the instructions of the manufacturer. An automatic coagulometer KC 10 (Heinrich Amelung GmbH, Lemgo, Germany) was used to perform both the PT and APTT. Study Projects and Sample Proceedinq Four different study programs were planned as follows. The first group included 35 normal pooled plasmas, each collected from 5-7 healthy volunteers without a bleeding history. After blood collection, plasma was taken immediately by centrifugation at 1500 g and at room temperature for 15 minutes. After obtaining the pooled plasma by mixing 5-7 normal plasmas, PT and APTT were performed immediately and the result was recorded as "0 hour". The remaining plasma was divided and put into three test tubes with stoppers (i.e. covered with paraffin sheet), one leaving at room temperature, one in a 4-C refrigerator and the other in a 70-C freezer. For the part at -70-C, the plasmas were divided and stored in the cuvette cups supplied with the coagulometer KC 10 with the exact volume ready for performing PT and APTT, i.e. O.lml. And when the tests were to perform, it would be thawed in a 37-C incubator for approximately 3 min. For these 3 parts, PT and APTT were performed 1, 2, 4, 5, 6 and 7 hours after blood collection, and the results were compared.
INFLUENCES ON APTT & PT
Vol. 62, No. 6
627
The second group included 18 normal plasma samples. After performing the 0 hour result as mentioned above, the plasma was put into the -70-C freezer with two different preparations: 1) exactly O.lml of plasma in each cuvette cup supplied by the KClO, and 2) the remaining volume of plasma put into a stoppered test tube. The plasmas were thawed as described above. The APTT was performed 2, 4, 6 and 7 hours after blood collection, each time with two different preparations as mentioned above. The difference of the results was then compared. The third group included 33 subjects without bleeding history. After obtaining and centrifugating the blood samples, PT and APTT were performed (recorded as 0 hr). Thereafter, two preparations for storing the plasma at room temperature and at 4' C were designed: 1) the plasma was stored in a stoppered tube altogether with the separated packed cells, i.e. the plasma at the upper and the packed cells at the bottom. 2) the plasma was separated immediately from the packed cells and stored alone up to the performances of the sample. For storing samples at -70-C, the same preparation described as in group 1 was used. PT and APTT were performed l-2, 4 and 6 hr after blood collection. For each time period, PT and APTT were performed with 2 different storage forms of plasma at room temperature and at 4-C, and with one storage form at -70-C, and the results were then compared. The fourth group included 8 patients with bleeding or administration disorders, such as hemophilia, liver cirrhosis of warfarin. PT and APTT were performed 2, 4, 6 and 7 hours after sample collection at 3 different temperatures as described in group I, and at two storage forms as described in group 3. The results were compared at different conditions. Stastistical
analysis
Paired-t test was used to compare the differences of two variables in the same condition in the first, third and fourth group studies. Two-tail level of significance of Wilcoxon Signed Ranks Test was used to compare the differences between the results performing with the cuvette or the test tube at a fixed time in the Group 2 study. The mathematical means were used to represent the means in all 4 groups of people. Results
RESULTS of the first study group
The means of PT and APTT in different temperatures and times of 35 normal pooled plasmas are shown in Table 1. In either PT or APTT, the 0-hr mean was compared with the means of other different conditions and the p value was calculated by the paired-t test. In the PT tests, significant shortening of PT in several conditions in comparison with the 0-hr hr at room temperature, 1 hr at 4-C, 3,4 & 6 hr significant prolongation of PT could be found in
could be found mean (e.g. l-4 at -70-C). No all conditions
INFLUENCES ON APTT & PT
628
Vol. 62, No. 6
TABLE 1 The Means of PT And APTT At Different Temperatures 35 Normal Pooled Plasmas
And Times
in
___--_-_--_-__-_--_-~--~-~~-~-~~-~----~~---~---------~--~----~--~
Room temperature 4-c -7O'C -_-_----------------------_-------------_----_--PT APTT PT APTT PT APTT Mean + SD Mean + SD Mean + SD (set) (set) (see) -__-_--_-____--_-_--~----~-~~-~----~--~-~--~--~-~~---~--~-~--~-~0 11.13 36.60 20.56 +1.63 1 1o.a7*** 37.01 10.94** 38.00*** 11.10 39.28*** +0.65 21.88 +0.54 +2.50 50.79 +2.52 2 10.99* 37.46*** 11.09 38.78** 11.07 39.78*** +0.63 21.71 +0.64 54.14 +0.57 +3.72 4 10.88*** 38.32*** 11.11 40.32*** 10.96*** 40.10*** +0.44 +2.16 +0.52 25.75 LO.53 +3.80 5 10.89*** 38.84*** 11.13 39.94*** 10.91** 39.74*** 20.50 22.42 20.63 24.27 +0.63 23.73 6 11.03 40.80*** 11.22 41.09*** 11.00 39.52*** +0.54 +5.15 +0.64 +4.88 +0.64 +2.76 7 11.09 41.9a*** 11.25 40.72*** 10.89* 39.04*** +0.50 24.65 +0.57 +0.56 +4.71 +2.81 -------_____________~~~~~~~~~~~~~~~~~~-~~~~-~~~~~~~-~~~~~~~~~-~~~ *p
