Stability of Plasma for Add-On PT and APTT Tests

Transcription

1 COAGULATION AND TRANSFUSION MEDICINE Original Article Stability of Plasma for Add-On PT and APTT Tests DEMETRA NEOFOTISTOS, MT, MARIA OROPEZA, MT(ASCP), AND CHUNG-HSIN TS'AO, PhD We conducted studies to determine at what time point an add-on prothrombin time (PT) or activated partial thromboplastin time (APTT) test can be honored on specimens that have been received in the laboratory hours earlier without yielding results with clinically significant differences from those if the test had been performed on the original unstored plasma. PT and APTT tests were performed on blood samples from 20 healthy subjects, 30 patients receiving warfarin, and 30 patients receiving heparin anticoagulation therapy. The tests were performed on plasma prepared initially after the samples were obtained. The same tests were assayed on plasma that had been left on spun-down blood cells at room temperature for 2, 4, and 8 hours. We found that the PT of the majority of plasma samples from healthy subjects and from patients receiving oral anticoagulant therapy tended to become shorter on storage. However, the difference in PT values was small and had no clinical significance. In most cases, the APTT values for the stored plasma from healthy subjects tended to increase with time. Except in one specimen in which the 8-hour add-on APTT was 1.2 seconds longer than the APTT result for the original sample, all others had APTT results less than 1.2 seconds longer than the original values. In patients receiving heparin, the differences in APTT values between the initial and add-on tests were larger than those observed for healthy subjects. However, those differences are not beyond what we would accept for duplicate checks for heparinized samples with high APTT values. Unlike samples from healthy subjects, there was no obvious trend of time-related prolongation of the APTT in heparinized plasma. These results led us to believe that within an 8-hour period and with plasma on spun-down cells at room temperature, add-on tests for PT and APTT could be performed with results similar to what would be obtained from testing unstored samples. (Key words: Plasma stability; Prothrombin time [PT]; Activated partial thromboplastin time [APTT]; Add-on tests) Am J Clin Pathol 1998;109: Recently, two articles 1,2 and a letter 3 appeared in the literature addressing the issue of validity of prothrombin time (PT) and activated partial thromboplastin time (APTT) testing on the first tube of blood drawn. An important reason for the studies is resource utilization and cost containment. These studies indicate that the first tube of blood drawn by experienced phlebotomists would yield valid PT and APTT results. Therefore, to not draw the first tube of blood for coagulation testing if blood for other tests is needed at the same time would be an unnecessary demand on the phlebotomist; to discard the first few milliliters of blood before collecting the sample for coagulation tests if only coagulation tests were ordered would be a waste. Our own data obtained in 1997 From the Hemostasis Laboratory and Department of Pathology, Northwestern Memorial Hospital and Northiuestern University School of Medicine, Chicago, Illinois. Manuscript received July 2, 1997; revision accepted August 22, Address reprint requests to Dr C-H Ts'ao: Department of Pathology, Northwestern University School of Medicine, 303 E Chicago Ave, Chicago, IL (unpublished) support the findings and conclusions of these investigators. Another issue in line with resource utilization is the question of "add-on" tests. Add-on tests are a common experience for all disciplines of clinical laboratory service, including coagulation. Often, a request for an add-on test is made hours after the blood has been received in the laboratory and processed. These requests are made because a specific piece of information is needed at that time or because obtaining another specimen would be difficult. Our experience with add-on PT/APTT, for the most part, fell into one of two categories: a wrong test was marked on the requisition and the error was discovered after the result had been released, or an APTT was first ordered for patients receiving heparin, and a few hours later, a PT was requested on the same sample. Almost never was APTT requested as an add-on test to samples from patients receiving oral anticoagulation for whom PT was initially ordered. Based on our experience, we conducted experiments to determine when add-on PT or APTT can be honored. Our criterion was that add-on results should be similar to the results yielded when the tests are performed on a fresh samples. If there is a difference, the disparity 758

2 NEOFOTISTOS ET AL 759 Coagulation Add-On Tests should not lead to an erroneous interpretation of patient's hemostatic status or alteration of the medical management. We wish to share our findings with those who also face the add-on challenges. reagents, respectively. According to the manufacturer, reagents are stable for 5 days at 4 C to 8 C after reconstitution. All of our reagents were used within 8 hours after reconstitution, and they were stored at 4 C. MATERIALS AND METHODS Interpretation of Results Subjects Twenty healthy subjects donated blood for the study. Blood was drawn by experienced medical technologists, using the 5-mL blue-topped evacuated tube (Vacutainer system, Becton Dickinson, Franklin Lakes, NJ). A 5-mL blue-topped glass Vacutainer (siliconized, lot 7D864A) contains 0.5 ml buffered sodium citrate (a mol/L concentration) and evacuates 4.5 ml of blood into it. Blood was centrifuged at 2500 rpm for 20 minutes at room temperature. PT and APTT were performed immediately after centrifugation and repeated at 2, 4, and 8 hours on plasma that had been left at room temperature on spun-down blood cells. Thirty patients receiving warfarin therapy and another 30 patients receiving heparin therapy were also included in the study. Coagulation tests were ordered by their physicians. Blood was drawn by phlebotomists using the same Vacutainer system. The time the blood was received in the laboratory and centrifuged was considered time zero. The PT' for patients receiving warfarin and the APTT for patients receiving heparin were performed and reported at this time. Surplus plasma was allowed to remain on spun-down cells in the original capped tube at room temperature for 2, 4, and 8 hours when the PT or the APTT was repeated. The PT was performed in duplicate on surplus plasma of another 25 patients receiving heparin for whom an APTT, with or without a PT, was ordered. An additional PT was performed at the time the APTT was done when only the APTT was ordered. At 2, 4, and 8 hours, PT testing was performed on plasma that had been left on spun-down cells at room temperature. Instrumentation and Reagents The PT and APTT were performed on a coagulation instrument (MLA-1400, Hemoliance, Raritan, NJ), with a rabbit brain thromboplastin (Ortho Brain Thromboplastin, Hemoliance; International Sensitivity Index, 1.91) and a partial thromboplastin (Thrombosil, Hemoliance) as the PT and APTT For healthy subjects, an unacceptable add-on result was considered one in which the PT or APTT was more than 1.0 second or more than 2.5 seconds different, respectively, from the original results, or the addon PT or APTT results exceeded the upper or lower limits of the normal range (PT, seconds; APTT, seconds). These "difference criteria" were chosen because, within the reference ranges, a 1- second difference in PT and a 2.5-second difference in APTT would have no clinical importance. For samples from patients receiving warfarin, a difference of more than 1.5 seconds was considered unacceptable. For samples from patients receiving heparin, unacceptable differences between initial and add-on results depended on the initial APTT values. A difference of more than 1.5 seconds was unacceptable if the initial APTT was less than 40 seconds. A 5% or more than 10% difference was unacceptable if the initial APTT was 41 to 60 seconds or 61 to 100 seconds, respectively. These difference criteria were established in our laboratory for acceptance of duplicate checks, with the understanding that a 5% or 10% difference for the respective APTT ranges would not lead to a change in medical management. Healthy Subjects RESULTS The initial mean PT and APTT results for 20 healthy subjects were 11.5 seconds (range, seconds) and 27.8 seconds (range, seconds), respectively. In the majority of cases, the PT results on stored plasma at 2, 4, and 8 hours remained unchanged; some showed differences of 0.1 to 0.5 seconds (Fig 1, A). Overall, the PT tended to be shorter on stored than on the original plasma. This trend was also obvious for the PT for patients receiving warfarin. For the APTT, a trend of increased values appeared with time. However, this trend was not observed in the APTT for patients receiving heparin. At any rate, the greatest prolongation of APTT in a healthy subject at 8 hours was 1.2 seconds over the initial result (Fig 1, B). Vol. 1C No. 6

3 760 COAGULATION AND TRANSFUSION MEDICINE Original Article Patients Receiving Warfarin The initial PT for this group of patients ranged from 14.4 to 31.8 seconds, with a mean of 19.2 seconds. The PT values of individual patients tested at 0, 2, 4, and 8 hours are shown in Figure 2. The differences in the results between the initial PT and the PT at later times ranged from -1.2 to +0.8 seconds. Unexpectedly, there were more patients with shorter PTs at later hours than at time zero. Except for two cases with a PT 1.2 seconds shorter at 8 hours than the initial values, other patients had PT differences less than 1.0 second from initial testing at any time. A higher initial PT was generally but not consistently associated with greater differences on subsequent tests.. Patients Receiving Heparin The initial APTT of patients receiving heparin ranged from 41.0 to 99.7 seconds (5 patients, <50 seconds; 17 patients, seconds; 8 patients, >81 seconds). The mean APTT for this group of patients was 71.4 seconds. None of the repeated testing at 2, 4, and 8 hours had the same clotting time as the initial value. The APTT for individual patients at the various times are shown in Figure 3. The difference between the initial and repeated clotting times was as little as 0.1 second or as much as 7.6 seconds (9.1% in one case with an initial APTT of 83.0 seconds). These differences were almost equally scattered on the plus and minus sides of the initial clotting time. Generally, FIG 1. Differences in (A) prothrombin time (PT) and (B) activated partial thromboplastin time (APTT) results from the initial values (represented by horizontal lines) for normal plasma retested at 2,4, and 8 hours. Initial values were obtained at time zero after obtaining the blood sample and preparing plasma. Plasma was left on spun-down blood cells in the original evacuated tube, capped, at room temperature. FIG 2. Prothrombin time (PT) results for patients receiving warfarin therapy assayed at 0, 2, 4, and 8 hours. Sample handling was the same that as described in the legend for Figure 1. In the majority of patients, the PT tended to be 0.1 to 1.2 seconds shorter when assayed at later times compared with the initial values. AJCP June 1998

4 NEOFOTISTOS ET AL 761 Coagulation 'd-on Tests specimens with higher initial clotting times that were retested later showed greater discrepancies from the initial results than specimens with shorter initial clotting times that were retested sooner. However, exceptions were evident in several cases. For example, the biggest difference for a sample with an initial APTT of 99.7 seconds was +4.3 seconds. The difference was +7.6 seconds for a sample with an initial APTT of 83.0 seconds. In no case was the difference beyond what we would accept for duplicates. PT on Prolonged APTT Specimens In the group of 25 patients with prolonged APTT due to heparin administration, 17 also had PT values greater than 13.5 seconds, indicating simultaneous administration of oral anticoagulant therapy. The APTT for this group of patients ranged from 36.4 seconds to more than 100 seconds (1 patient), with a mean value of 59.9 seconds (the APTT > 100 seconds was excluded from the calculation of the mean). The initial PT ranged from 12.2 to 24.5 seconds (mean, 15.8 seconds). The PT values for 0 to 8 hours are shown in Figure 4. In 1 case at 2 hours, the PT was 1.5 seconds higher than the initial result; at 4 and 8 hours, the PTs were -0.4 and -0.1 seconds less, respectively, than the initial value. In another case, the PT was 1.9 seconds greater at 4 hours, but it was only 0.8 seconds higher at 8 hours FIG 3. Activated partial thromboplastin time (APTT) results for patients receiving heparin therapy tested at 0, 2, 4, and 8 hours. Greater differences from initial values, although none we considered clinically significant, were noted at 4 and 8 hours than at 2 hours. FIG 4. Prothrombin time (PT) results for patients receiving heparin assayed at 0, 2, 4, and 8 hours. These patients had prolonged activated partial thromboplastin times as a result of heparin therapy. Some of them were receiving concomitant oral anticoagulants. Vol. No. 6

5 762 COAGULATION AND TRANSFUSION MEDICINE Original Article DISCUSSION Add-on test requests are a daily occurrence and most of them, in our experience, are made within 8 hours of the initial request. Because of varying stability of plasma components involved in the coagulation process, add-on coagulation tests warrant special consideration. Our experience has been that in most cases, the request for a PT was added to a request in which only an APTT had been ordered. In others, APTT was inadvertently marked on the requisition for a sample intended for PT testing, or vice versa. Occasionally, a PT, an APTT, or both are requested on a specimen drawn earlier for a fibrinogen or D-dimer assay. Clotting factors, especially the labile factor V, deteriorate on storage. Advancement in coagulation instrumentation allows cap-piercing and auto-sampling. Because of demands for cost containment by hospital administration and third-party payers and for shorter turnaround time by physicians, most PT and APTT testing is done on plasma on spun-down blood cells in the original Vacutainer. Transferring plasma to a separate tube after centrifugation of whole blood for PT and APTT testing has become a matter of the past. Plasma is usually left on spundown cells at room temperature after the ordered test is performed and before the blood is discarded. If an add-on coagulation test is requested, the test would be done on that plasma. In addition to storage-related deterioration of clotting factors, procoagulants and inhibitors of coagulation in the blood cells could be released on storage to alter coagulation test results. The present study was designed to determine how different the clotting times would be if the PT, APTT, or both were performed on plasma that had been left on spun-down cells for up to 8 hours compared with test results from fresh samples. If there are differences, would the differences foster a different interpretation of the patient's hemostatic status, leading to alteration of medical management? Three groups of subjects were included: healthy subjects, patients receiving oral anticoagulation therapy, and patients receiving heparin therapy with or without concomitant oral anticoagulation. Not surprisingly, coagulation test results obtained from stored plasma were somewhat different from initial values. This was more evident with samples from patients receiving heparin. However, the differences are not larger than what we would accept for duplicate checks on those samples. Furthermore, it is unlikely that those differences would lead to a different interpretation of therapeutic intensity to alter patient care. For the most part, differences in PT and APTT results of add-on tests were scattered on the plus and minus sides of the initial values. The differences probably reflect the divergent nature of PT and APTT testing, in which dozens of components are involved to produce a simple clotting time value. This should be particularly evident when the activity of clotting factors is inhibited or rendered nonfunctional by exogenous interventions. Our data did not show a consistent change of clotting components in the stored plasma to the point that was indicated by PT and APTT assay results. In other words, we believe that within an 8- hour period, deterioration of clotting factors in plasma and leakage of procoagulants and inhibitors from spun-down blood cells, if any, are not significant enough to alter PT and APTT results and medical decision making. Koepke et al 4 collected several tubes of blood from each of 10 healthy subjects. The samples were centrifuged and kept at room temperature up to 24 hours. Immediately after centrifugation, PT and APTT were performed on plasma of one tube and repeated on the other tubes at 2, 4, 6, and 24 hours. They found no change in PT and APTT at 6 hours and a 10% to 15% prolongation of APTT after 24 hours. Although we carried our study to only 8 hours, we had similar findings with patients receiving anticoagulant therapy with prolonged initial PT and APTT. Baglin and Luddington 5 obtained five tubes of citrated blood from patients receiving oral anticoagulant therapy and determined the international normalized ratio (INR) daily on each tube of blood with nine different thromboplastin preparations. Blood was left at room temperature unspun until the day of testing. They found essentially no difference in INR before day 3. The small increases in INR registered thereafter were clinically insignificant. The National Committee for Clinical Laboratory Standards recommends that coagulation testing be done within 2 hours of venipuncture if the blood is maintained at 22 C to 24 C. 6 In light of our findings and those of others, a reevaluation of the recommendation may be warranted. Testspecific guidelines probably should be devised. Our data indicate that add-on PT and APTT can be performed on plasma that has been resting on blood cells and left at room temperature for up to 8 hours. Whether our findings would also hold true for samples stored beyond 8 hours or for plasma of persons with a congenital factor deficiency or presence of inhibitors remains to be determined. AJCP June 1998

6 NEOFOTISTOS ET AL 763 Coagulation Add-On Tests REFERENCES 1. Yawn BP, Loge C, Dale J. Prothrombin time: one tube or two. Am J Clin Pathol. 1996;105: Gottfried EL, Adachi MM. Prothrombin time and activated partial thromboplastin time can be performed on the first tube. Am J Clin Pathol. 1997;107: Carroll WE, Jackson RD. Prothrombin time: one tube or two. Am j Clin Pathol. 1997;107:133. Letter. 4. Koepke JA, Rodgers JL, Ollivier MJ. Pre-instrumental variables in coagulation testing. Am ] Clin Pathol. 1975;64: Baglin T, Luddington R. Reliability of delayed INR determination: implications for decentralized anticoagulant care with off-site blood sampling. Br I Haematol. 1997;96: Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and Performance of Coagulation Assays. 2nd ed. Baltimore, Md: National Committee for Clinical Laboratory Standards; NCCLS Document H21-2A. Vol 11. No Vol. 109 No. 6