Use of a Solid Phase Red Blood Cell Adherence Method for Pretransfusion Platelet Compatibility Testing JANE M. RACHEL, MT(ASCP) M.A., TERRI C. SUMMERS, B.S., LYLE T. SINOR, PH.D., AND FRED V. PLAPP, M.D., PH.D. A solid phase red blood cell adherence method has been used for platelet antibody detection and crossmatching for refractory platelet recipients. Patient sera were first screened for HLA or platelet-specific antibodies, then crossmatched with potential apheresis platelet donors. The overall correlation of platelet crossmatch results with transfusion outcome was 97% in patients with no evidence of nonimmune platelet destruction. The solid phase red blood cell adherence method provided a feasible and effective alternative to HLA matching as a means of donor selection for refractory platelet recipients. The speed and simplicity of this method may allow most hospital laboratories to perform platelet antibody screening before routine platelet transfusions. (Key words: Solid phase; Platelets; Compatibility testing; Crossmatch; Platelet transfusions) Am J Clin Pathol 1988;90:63-68 IN RECENT YEARS, platelet usage has increased ten times more than red blood cells (RBCs). Transfusions of either whole blood or RBCs increased 58%, from 6.3 to 9.9 million units, between 1971 and 1980. During the same period, platelet usage increased 598%, from 0.4 to 2.8 million units. 12 The growing demand for platelets has brought about an increased awareness of several problems associated with platelet transfusion therapy. Foremost among these is the detection and management of platelet alloimmunization. Most platelet transfusions are administered as a pool of six to ten individual concentrates, selected at random. As HLA or platelet-specific antibodies develop, posttransfusion platelet increments diminish, because of immune destruction. Recipients who repeatedly derive little or no benefit from random-donor platelets are termed refractory, and alloimmunization is suspected. Because the antibodies are often directed against HLA antigens, refractoriness can sometimes be overcome by providing platelets from HLA-matched donors. A major disadvantage to this approach is the cost of establishing Received September 29, 1987; received revised manuscript and accepted for publication November 30, 1987. Supported by a grant from the Research Foundation of the American Association of Blood Banks. Dr. Sinor's current address is Immucor, Inc., Norcross, Georgia. Dr. Plapp's current address is Department of Pathology, St. Luke's Hospital, Kansas City, Missouri. Address reprint requests to Ms. Rachel: Community Blood Center of Greater Kansas City, 4040 Main Street, Kansas City, Missouri 64111. 63 Community Blood Center of Greater Kansas City, Kansas City, Missouri and maintaining the necessary panel of several thousand HLA-typed apheresis donors. 6 " In addition, extensive experience has determined that 18-35% of HLAmatched platelet transfusions are not beneficial.115 The reason for these failures is unclear. One possible explanation is the presence of antibodies directed against platelet-specific antigens. In fact, such antibodies have been detected in the sera of 22-25% of alloimmunized patients.7'9 Thus, the assumption that histocompatible platelets are serologically compatible may not be valid. Pretransfusion platelet crossmatching is a more direct means of identifying compatible donors for alloimmunized recipients. Consequently, several techniques that were initially developed for platelet antibody detection have been adapted to platelet crossmatching.1-5-81316"19 The major impediment to widespread use of these methods is that several hours are required to complete small numbers of tests. This is a significant limitation, because finding compatible donors for broadly alloimmunized patients may require extensive crossmatching. Recently, a solid phase RBC adherence (SPRCA) assay was developed specifically for platelet crossmatching. Platelet preparation was reduced to a single 10minute centrifugation step so that, if necessary, samples from large numbers of donors could be crossmatched in a single day. The SPRCA method was initially evaluated by retrospectively crossmatching platelet samples from apheresis donors with sera from refractory recipients. Eighty transfusions given to 20 patients were studied. Analysis of the data indicated that none of 20 crossmatch-incompatible transfusions had produced significant posttransfusion increments. Of the crossmatchcompatible transfusions, 63% (38 of 60) had been successful. 10 These results suggested that the SPRCA platelet crossmatch method could be effective if used before apheresis to select platelet donors for alloimmunized patients. We describe the results of a study designed to evaluate the effectiveness and feasibility of using the SPRCA method prospectively for pretransfusion platelet antibody detection and crossmatching. RACHEL ET AL. 64 POSITIVE NEGATIVE platelet monolayers JL serum anti-platelet antibody ^\ antl-lgG-coated Indicator RBCs FIG. 1. Schematic representation of positive (incompatible) and negative (compatible) reactions using the SPRCA method for platelet antibody detection and crossmatching. Methods The SPRCA method, as described previously,10 was used for platelet antibody detection and crossmatching. Briefly, test platelets were prepared by collecting 7 rnL of whole blood into ethylenediaminotetraacetate (EDTA) and centrifuging for 10 minutes at 200 X g to obtain platelet-rich plasma (PRP). No further isolation or washing ofplatelets was required, nor was it necessary to determine platelet counts of PRP obtained from apparently healthy donors. PRP was prepared and used within 48 hours from time of collection. Solid phase platelet monolayers were formed by adding two drops of PRP to polystyrene U-bottom microplate wells that had been pretreated to specifically bind platelets (Capture-P®, Immucor Inc., Norcross, GA 30091). Microplates were centrifuged for 5 minutes at 35 X g to form solid phase platelet monolayers. Unbound platelets were removed by gently washing the wells six to eight times with 0.85% (w/v) NaCl. One drop of test serum and three drops of a low ionic strength solution (Capture-P) were added to the appropriate wells and mixed. Microplates were incubated for 30 minutes at 37 °C, which allowed serum antibody to bind to platelet monolayers. After incubation, unbound serum components were removed by washing the wells five times with 0.85% (w/v) NaCl. Platelet-bound antibodies were detected by the addition of one drop of a 0.2-0.4% (v/v) suspension of anti-IgGcoated indicator RBCs (Capture-P). Microplates were then centrifuged at 700 X g for 1 minute, which allowed indicator RBCs to react with antibodies attached to the solid phase platelet monolayers. Positive reactions were thus characterized by the adherence of indicator RBCs over the surface of the wells, whereas negative reactions produced discrete pellets of indicator RBCs. These reactions were read visually and are shown schematically in Figure 1. Results of 96 test wells (one microplate) were A.J.C.P.-July 1988 available within one hour, including the time required to prepare platelet monolayers. The SPRCA method detected antibodies directed against platelet-specific, HLA, or ABO antigens. Patients included in the study were those for whom apheresis platelet transfusions were ordered by the attending physician. Most had proven refractory to random donor platelets, although apheresis platelets were occasionally requested for patients who were expected to require long-term platelet support. Diagnoses included leukemia, aplastic anemia, and various malignancies. Patients' blood samples were initially screened against platelet samples obtained from 12 group O whole blood donors, selected at random with no knowledge of their HLA or platelet antigens. The antibody screen results were not considered crossmatches but were used only to determine the presence and extent of alloimmunization and to estimate the number of potential donors needed for crossmatching in order to provide an adequate supply of compatible platelets. For example, patients whose sera reacted with 9 of the 12 platelet samples in the antibody screen were expected to be compatible with approximately 25% of donors. Recipients' sera were then crossmatched with platelet samples obtained from ABO-compatible potential donors. Whenever possible, crossmatch-compatible donors were selected before apheresis. If no potential donors were compatible, incompatible single donor platelets were provided at the attending physician's request. To assess transfusion outcome, pretransfusion and one-hour posttransfusion platelet counts were requested from hospitals where transfusions were administered. Information concerning the recipient's clinical status with regard to fever, active bleeding, sepsis, splenomegaly, or disseminated intravascular coagulation (DIC) was also requested. The corrected count increment (CCI) at one hour posttransfusion was calculated as follows2: CCI _ (post - pretransfusion platelet count) X BSA (m 2 ) number of platelets transfused (X10") Transfusions were considered successful when the onehour CCI was greater than 10 X 109/L (10 X 10 3 /ML)- 2 Crossmatch results were correlated with transfusion outcome as follows4: true positive (TP)—incompatible crossmatch and unsuccessful transfusion; false positive (FP)—incompatible crossmatch and successful transfusion; true negative (TN)—compatible crossmatch and successful transfusion; false negative (FN)—compatible crossmatch and unsuccessful transfusion. The sensitivity, specificity, predictive value, and efficiency of the SPRCA platelet crossmatch method were determined by use of standard formulas. 4 Vol. 90 • No. 1 PLATELET COMPATIBILITY TESTING Results Platelet antibody screen and prospective crossmatch results are summarized in Table 1. A total of 2,878 crossmatches were performed for 87 patients, of which only 479 (17%) were compatible. These data were further evaluated by separating patients into one of three categories based on their initial antibody screen results. The first category included 35 patients whose sera were nonreactive when initially screened for platelet antibodies. Most potential donors (96%) were compatible with these patients. The few incompatible crossmatches (4%) may have resulted from antibodies directed against low-frequency antigens present on donor platelets but not on the 12 randomly selected platelet samples used for antibody screening. Although these patients were not considered alloimmunized, they were transfused with apheresis platelets, at the attending physicians' request. Category II consisted of 30 patients whose sera had positive results in the antibody screen against some platelet samples but not against all 12. These results indicated that patients were alloimmunized but not so broadly that compatible donors could not be found. These patients' sera were compatible with 31% of potential donors. In most instances, crossmatch-compatible platelets were provided for transfusion. The 22 patients in category III were those whose sera reacted with all 12 platelet samples in the antibody screen, indicating broad alloimmunization. A total of 2,144 crossmatches were performed for these patients, of which only 87 (4%) were compatible. The actual number of compatible donors per patient ranged from none to six (data not shown). Patients with three to six compatible donors were usually transfused with crossmatch-compatible platelets. Transfusions given to patients with less than three compatible donors were sometimes obtained from incompatible donors, at the attending physician's request. Recipients' samples were retested frequently in order to detect developing antibodies. As expected, patients often became more broadly alloimmunized during the course of therapy, so that previously compatible donors were subsequently incompatible. Another observation, which has previously been reported, 314 was the sudden and complete disappearance of antibody in patients known to be broadly alloimmunized. Loss of antibody could not be reliably attributed to incompatible platelet transfusions or changes in treatment. Antibodies usually reappeared within several days, so that crossmatches done during the period of antibody absence were no longer valid. During a one-year period of study, 621 apheresis platelet concentrates were transfused. Unfortunately, only 67 of these transfusions met our criteria for evalua- 65 Table 1. Summary of Platelet Antibody Screen Results and Correlation with Crossmatch Results Category I II III Antibody screen Neg Posl-11* Pos 12f Number of patients 35 30 22 Number of crossmatches Compatible 241(96%) 151(31%) 87(4%) Incompatible 10(4%) 332(69%) 2,057(96%) Total 251 483 2,144 Totals 87 479 2,399 2,878 * Patients whose sera reacted with 1-11 of 12 random platelet samples in the antibody screen. | Patients whose sera reacted with all 12 random platelet samples in the antibody screen. tion of transfusion outcome and correlation with crossmatch results. A large number of transfusions involved recipients who received platelets from an individual donor more than once. For the purposes of this study, transfusions involving unique donor-recipient pairs were evaluated only once, because repeat donations usually resulted from previously successful transfusions. Thus, inclusion of replicate donor-recipient pairs would have biased the study in favor of a successful outcome. In many instances, transfusions could not be evaluated because platelet counts either were not ordered by the attending physician or were not drawn within one hour after transfusion. Strict adherence to this time limit was considered necessary in order to minimize the effects of nonimmune platelet destruction on posttransfusion increments. In general, unsatisfactory platelet increments at one hour posttransfusion are thought to represent immune destruction, whereas platelet counts obtained after this time are more likely to be affected by clinical factors such as fever, sepsis, bleeding, splenomegaly, or DIC. 2 The correlation of crossmatch results with transfusion outcome for 67 transfusions involving 28 patients is expressed in Figure 2. The data include all transfusions for which crossmatch results were available and a one-hour CCI could be calculated, regardless of the patient's status with regard to fever, active bleeding, sepsis, splenomegaly, or DIC. Some transfusions occurred when one or more of these complications were known to be present. Although most studies evaluating platelet crossmatch methods exclude such patients,1'5,8'16"19 we were unable to obtain the clinical information necessary to allow such exclusions to be made fairly. We thus chose to express the total data, with the assumption that many false negative results may have resulted from nonimmune platelet destruction. Of the 67 transfusions evaluated, only 32 were given to patients whose clinical status at the time of transfu- A.J.C.P.- July 1988 RACHEL ET AL. 66 CCI TN FP 50 45 40 35 30 25 20 Evaluation of the data derived from both patient populations is shown in Table 4. Chi-square analysis is highly significant for both groups. The sensitivity, specificity, and predictive values most representative of actual transfusion practice are those of the total patient population. However, the ability of the SPRCA platelet crossmatch method to predict serologic compatibility is best represented by data from the selected patient population, which is presumably unaffected by nonimmune platelet destruction. Comparisons of this study with others should be based on these latter figures, because most platelet crossmatch methods have been evaluated by use of selected patient populations. Discussion • • 15 • 10 m • FN TP °„ o n r, U n O n n nO noun crossmatch incompatible crossmatch compatible mean CCI 4.1 SD 5.1 mean CCI 16.3 SD 9.6 FIG. 2. Correlation of posttransfusion CCI (X109/L) (103/ML) and crossmatch results for the total patient population. Data represent 67 apheresis platelet transfusions given to 28 patients. Replicate donorrecipient pairs are excluded. Many hospitals and blood centers are currently using both HLA matching and platelet crossmatching to select donors for alloimmunized recipients. The question that has not been answered is whether or not platelet crossmatching can be used instead of HLA matching. This study represents an extensive investigation into the feasibility of using platelet crossmatching as the sole crite- CCI FP TN 50 45 sion was reliably reported and did not include fever, active bleeding, sepsis, splenomegaly, or DIC. The correlation of crossmatch results with transfusion outcome for this selected patient population is expressed in Figure 3. As expected, the number of false negative reactions is greatly reduced, but two false positive results are also excluded. One of these transfusions involved a reportedly bleeding patient who benefitted from crossmatchincompatible platelets. The other false positive result occurred when incompatible platelets were given to a febrile patient. Tables 2 and 3 contain additional information concerning transfusions evaluated for the total and selected patient populations, respectively. Initial antibody status of the recipients, as described in Table 1, is correlated with the crossmatch results expressed in Figures 2 and 3. Both tables indicate that most of the transfusions analyzed were given to alloimmunized recipients, that is, those in categories II and III of Table 1. This distribution is important, because inclusion of a disproportionate number of transfusions involving recipients in category I may have biased the outcome in favor of success. 40 35 30 25 20 15 10 crossmatch incompatible crossmatch compatible mean CCI 3.8 SD 3.1 mean CCI 19.2 SD 8.0 FIG. 3. Correlation of posttransfusion CCI (X109/L) (l03/fL) and crossmatch results for a selected patient population. Data represent 32 apheresis platelet transfusions given to 17 patients. Replicate donorrecipient pairs are excluded. PLATELET COMPATIBILITY TESTING Vol. 90 • No. 1 Table 2. Correlation of Recipient Antibody Status with Crossmatch Results for Transfusions Shown in Figure 2 67 Table 3. Correlation of Recipient Antibody Status with Crossmatch Results for Transfusions Shown in Figure 3 Category* Category* Compatible transfusions Incompatible transfusions Totals 17 0 17 II III 19 3 22 8 20 28 * As described in Table 1. ridn of donor selection for alloimmunized platelet recipients. The results indicate that use of the SPRCA method for both platelet antibody screening and crossmatching before apheresis is an effective approach. Antibody screen results provide useful information concerning the presence and extent of alloimmunization and the likelihood of finding compatible donors. The sensitivity, specificity, and predictive values obtained for the SPRCA platelet crossmatch method indicate that the ability of this method to select compatible platelet donors is better than that generally reported for HLA matching. 115 Refractoriness in the first group of patients listed in Table 1 may have resulted from either nonimmune platelet destruction or undetectable levels of alloimmunization. Most prospective donors (96%) were compatible. The high degree of correlation between antibody screen and crossmatch results indicates that a panel of 12 randomly selected platelet samples provides adequate pretransfusion platelet antibody detection. Prospective platelet crossmatching appeared to be most beneficial for patients who were alloimmunized but not broadly. The 30 patients in category II were incompatible with slightly more than two-thirds of potential donors. Pretransfusion testing thus provided an adequate supply of compatible platelet donors, while preventing many apheresis procedures that would have resulted in ineffective transfusions. Data expressed in Table 1 also suggest that crossmatching for patients who are broadly alloimmunized may not be productive. Only 4% of all donors were compatible with these patients' sera. However, the speed and simplicity of the SPRCA method allowed 100 crossmatches to be completed within eight hours by a single technologist. It thus seems reasonable to initially crossmatch 100-200 potential donors. Identification of even a few compatible donors may be worthwhile if their use is restricted to periods of extreme need. If no compatible donors are found, additional crossmatching cannot be justified. In most instances, potential donors were friends and family members of the intended recipient. Some pa- II III Compatible transfusions Incompatible transfusions 6 10 Total 16 * As described in Table 1. tients who were broadly alloimmunized recruited hundreds of potential donors in an attempt to obtain compatible platelets for transfusion. This was most often accomplished by organizing mass phlebotomies at the recipient's place of business or during meetings of interested church or social groups. Platelet samples were crossmatched with serum from the intended recipient within 48 hours, to eliminate the need for further isolation of test platelets from autologous plasma. The logistics of this system were complex at best. The most practical solution to this problem would be the establishment of a large group of committed apheresis donors, whose platelets could be stored and crossmatched against all patients' sera. In our study, 25% of patients for whom apheresis platelets were requested were broadly alloimmunized when initially screened for platelet antibodies (category III). These 22 patients required 2,144 crossmatches, of which only 87 (4%) were compatible. Subsequent plate- Table 4. Evaluation and Statistical Analysis of the Correlation Between Crossmatch Results and Transfusion Outcome for Both Total and Selected Patient Populations Total Patient Population Crossmatch correlation Number of: True positive results False positive results True negative results False negative results Total Chi-square analysis X2 P Crossmatch evaluation Sensitivity Specificity Predictive values Incompatible crossmatch Compatible crossmatch Efficiency 21 2 34 10 67 Selected Patient Population 11 0 20 1 32 28.57 <0.0005 27.93 <0.0005 68% 94% 92% 100% 91% 77% 82% 100% 95% 97% 68 RACHEL ET AL. let transfusions were often incompatible and ineffective. The disappointing outcome for this group of patients might have been improved if samples had been referred for platelet antibody screening at an earlier time, when they were presumably less broadly alloimmunized. Unfortunately, pretransfusion detection of platelet alloimmunization has been impeded by the lack of a method suitable for routine use in hospital transfusion service laboratories. Consequently, most platelet transfusions are administered with no prior antibody screening or compatibility tests. Platelet alloimmunization is usually detected clinically, rather than serologically, when a recipient repeatedly fails to benefit from random platelets. During the time required to demonstrate refractoriness, large numbers of platelet concentrates are wasted, and the recipient is exposed, without benefit, to platelets from a large number of donors. Our experience during this study indicates that the SPRCA method is comparable in terms of speed and simplicity to those currently in use for RBC compatibility testing and could thus be used in hospital laboratories before most platelet transfusions to detect platelet alloimmunization. Patients with negative antibody screens could still be transfused with pools of randomly selected platelet concentrates. Alloimmunized patients would be identified earlier and could then be supplied with platelets obtained by apheresis from crossmatchcompatible donors. The delay encountered would certainly be less than the time currently required for clinical documentation of refractoriness. We thus conclude that pretransfusion platelet compatibility testing may now be feasible. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Acknowledgment. The authors thank B. J. Schulenburg for review17. ing the manuscript. References 1. Brand A, van Leeuwen A, Eernisse JG, van Rood JJ: Platelet transfusion therapy. Optimal donor selection with a combination of lymphocytotoxicity and plateletfluorescencetests. Blood 1978;51:781-788. 2. Daly PA, SchifTer CA, Aisner J, Wiernik PH: Platelet transfusion 18. 19. A.J.C.P.-July 1988 therapy. One-hour posttransfusion increments are valuable in predicting the need for HLA-matched preparations. JAMA 1980,243:435-438. Dutcher JP, SchifTer CA, Aisrier J, Wiernik PH: Long-term follow-up of patients with leukemia receiving platelet transfusions: identification of a large group of patients who do not become alloimmunized. Blood 1981;58:1007-1011. Galen RS, Gambino SR: Beyond normality: the predictive value and efficiency of medical diagnoses. New York: John Wiley and Sons, 1975:10-14. Kickler TS, Braine HG, Ness PM, Koester A, Bias W: A radiolabeled antiglobulin test for crossmatching platelet transfusions. Blood 1983;61:238-242. Lohrmann HP, Bull MI, Decter JA, Yankee RA, Graw RG: Platelet transfusions from HL-A compatible unrelated donors to alloimmunized patients. Ann Intern Med 1974;80:9-14. Murphy MF, Waters AH: Immunological aspects of platelet transfusions. Br J Haematol 1985;60:409-414. Myers TJ, Kim BK, Steiner M, Baldini MG: Selection of donor platelets for alloimmunized patients using a platelet-associated IgG assay. Blood 1981;58:444-450. Pegels JG, Bruynes ECE, Engelfreit CP, von dem Borne AEGKr: Serological studies in patients on platelet- and granulocytesubstitution therapy. Br J Haematol 1982;52:59-68. Rachel JM, Sinor LT, Tawfik OW, et al: A solid-phase red cell adherence test for platelet cross-matching. Med Lab Sci 1985;42:194-195. SchifTer CA, Keller C, Dutcher JP, Aisner J, Hogge D, Wiernik PH: Potential HLA-matched platelet donor availability for alloimmunized patients. Transfusion 1983;23:286-289. Surgenor DM, Schnitzer SS: The nation's blood resource: a summary report. NIH Publication #85-2028. National Institutes of Health, March 1985. Tamerius JD, Curd JG, Tani P, McMillan R: An enzyme-linked immunosorbent assay for platelet compatibility testing. Blood 1983;62:744-749. Tejada F, Bias WB, Santos GW, Zieve PD: Immunologic response of patients with acute leukemia to platelet transfusions. Blood 1973;42:405-412. Tosato G, Applebaum FR, Deisseroth AB: HLA-matched platelet transfusion therapy of severe aplastic anemia. Blood 1978;52:846-854. van5lder Velden KJ, Sintnicolaas K, Lowenberg B: The value of a Cr platelet lysis assay as crossmatch test in patients with leukaemia on platelet transfusion therapy. Br J Haematol 1986;62:635-640. Ware R, Reisner EG, Rosse WF: The use of radiolabeled and fluorescein-labeled antiglobulins in assays to predict platelet transfusion outcome. Blood 1984;63:1245-1248. Wu KK, Hpak JC, Koepke J A, Thompson JS: Selection of compatible platelet donors: a prospective evaluation of three crossmatching techniques. Transfusion 1977;17:638-643. Yam P, Petz LD, Scott EP, Santos S: Platelet crossmatch tests using radiolabeled staphylococcal protein A or peroxidase anti-peroxidase in alloimmunized patients. Br J Haematol 1984;57:337-347.
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