0 1984 S. Karger AG, Basel 0042-9007/84/0476-0421 $?.75/0 Vox Sang. 47: 421-426 (1984)

Immunoglobulin Class Heterogeneity of Platelet Alloantibodies

Karen Miller, Thomas S. Kickler, Paul M. Ness, Hayden Braine Department of Pathology, Division of Immunohematology, and Oncology Center, The Johns Hopkins Medical Institutions, Baltimore, Md. (USA)

Abstract. The immunoglobulin classes of platelet alloantibodies were studied using monospecific antisera in a radiolabeled technique. 14 of 28 consecutively studied multiply transfused oncology patients demonstrated platelet antibodies. These 14 patients were also refractory to random donor platelets. The immunoglobulin classes of these platelet anti- bodies were found to be: IgG alone 4, IgM alone 4, IgG and IgM 5, IgM plus IgA 1. The results in this study suggest that using monospecific antisera other than anti-IgG alone, one can detect additional antibodies involved in the immune destruction of platelets.

Introduction

There have been few studies utilizing monospecific anti-immunoglobulin re- agents in the determination of the immuno- globulin class of platelet alloantibodies [ll, 121. It has been established that in most cases of autoimmune thrombocytopenic purpura, it is an autoantibody of the immunoglobu- lin-G class which is responsible for the de- struction of platelets [4-6,101. However, few systematic studies have been performed in determining the immunoglobulin of platelet alloantibodies.

There have been several methods devel- oped to detect platelet antibodies principally demonstrating antibodies of the IgG class [l, 2,6,12]. Increasingly, laboratories are utiliz- ing these assays to perform platelet cross-

matches to aid in the selection of the most compatible platelet transfusions, thereby in- suring a successful outcome. Most studies employ only an anti-IgG technique and the occurrence of false-negative crossmatches has been noted [I, 71. One possible explana- tion that may account for these nonpredic- tive crossmatches is that antibodies of the IgM and/or IgA classes may be mediating the immune destruction of transfused plate- lets. For this reason, we studied 28 multiply transfused patients for the presence of plate- let alloantibodies of different immunoglobu- lin classes using a radiolabeled antiglobulin technique. Platelet antibodies of all three classes were detected. The data presented in this report suggest that IgM and IgA antibod- ies may play a role in mediating refractori- ness to platelet transfusions. The predictive

422 Miller/Kickler/Ness/Braine

Table I. Determination of the optimal temperature of reactivity of IgM platelet antibodies

Incubation temperatures', "C RAGT index

4/4 4/22

22/22 37/37

3. I 2.2 2.2 I .7

I The first temperature is the sensitization incuba- tion; the second temperature is the radiolabeled antig- lobulin incubation.

value of platelet crossmatching may be in- creased by using antiglobulin reagents recog- nizing all immunoglobulin classes.

Materials and Methods

Patients. In the examination of platelet alloantibod- ies, 28 consecutive multiply transfused oncology pa- tients were studied (table I). Patients were considered refractory to platelet transfusions when on at least two occasions they had less than 5% of expected increment to 6 or more units of random donor platelets.

Lymphocytotoxic Antibody Testing. The standard NIH microlymphocytotoxicity technique [9] was used to screen patients' sera for HLA antibodies. Sera were screened against a 20-cell lymphocyte panel [7].

Antisera. Affinity purified goat anti-human IgG, IgM, and IgA were obtained from Kierkegaard-Perry (Gaithersburg, Md.). All three were radiolabeled with "'I [7]. On immunoelectrophoresis, the anti-IgG reacted specifically with human IgG, the anti-IgM reacted specifically with human IgM, and the anti-IgA reactedspecifically with human IgA. No antilight chain activity was detected.

Controls. Normal plasmas were obtained from blood donors and laboratory personnel who had no prior history of transfusion or pregnancy. The plasmas and platelets used as the negative controls were col- lected and prepared on the day of testing. The positive control used in the IgG assay was produced by sensitiz- ing PLAl-positive platelets with anti-PLAl, obtained from a plasma of a patient who had posttransfusion

purpura. In the IgM assay, the positive control was produced by incubating group A platelets with group 0 plasma containing isoagglutinins of a titer of I : 512. A plasma from a patient with autoimmune thrombocyto- penia which contained platelet antibodies reactive in the IgA assay was used as a positive control for the detection of IgA-reactive antibodies.

Preparation of Platelets. For the indirect platelet assay, platelets were collected in EDTA from group 0 donors. The platelet-rich plasma was prepared by cen- trifugation (800g for 5 min) and a platelet button was then obtained after a second centrifugation (3,000 rpm for IOmin). The platelet button was washed4 timeswith Tyrode's buffer and the count adjusted to 100,000/mm3 with buffer. The patient's samples were screened for antibody against a pool of platelets from 5 donors pos- sessingHLAspecificities(Al,A2, A19, A3,88, B7, B40, B12). These HLA antigens were selected based on the frequency of antibody specificity encountered in our patient population.

lndirect Platelet Assay. The indirect platelet assay is a modification of the radiolabeled antiglobulin test [2, 71. For each experiment, negative as well as positive controls were carried out in parallel with the patient samples and all were run in duplicate. In the assays to detect IgG and IgA platelent antibodies, 10' platelets (obtained from normal group 0 donors) in Iml of Tyrode's buffer were incubated with Iml of patient plasma for 30 min at 37 "C. The sensitized platelets were then washed 3 times and resuspended to a I-ml volume. The lZ5I anti-lgG or '''1 anti-lgA was added and the mixture incubated again for 30 min at 37 'C. After wash- ing the platelets 4 times with buffer, the amount of platelet-associated radioactivity was determined in a gamma scintillation counter. Methods to detect IgM platelet antibodies were similar to those used in the procedure described above. This assay also consists ofa sensitization phase with the exception of it being carried out at 4°C for 30 min. Washing is followed by the addi- tion of lZ5I anti-IgM and another incubation at 4'C for 30min. The optimal incubation temperature for the IgM assay was determined by comparing the results of the positive and negative controls after the assay was performed at various temperatures (table I).

The test results of all assays are expressed as a ratio of test counts per minute to the average counts per minute of the negative controls, and is called the radio- labeled antiglobulin test index (RAGT-index) [7]. In a previous study using "'I anti-IgG, it had been deter- mined that the average RAGT index of80 untransfused

Heterogeneity of Platelet Alloantibodies 423

Table 11. Serologic results of 14 patients with platelet alloantibodies

Patient Diagnosis Transfusion History, units Lymphocytotoxic anti- Platelet antibody red cells platelets bodies, O/o of reactivity assays

with a 20-cell lymphocyte panel IgG IgM IgA

A. G. J. C. T. H. V. F. E. H. D. J. H. K. w. s. v. w. W. D. M. C. A. P. G. B. L. M.

AA ALL AML AML AML AML AML AML AML AML AA AA AML AML

43 20 56 16 29 10 31 22 44 41 16 32 57 4

1,252 65

3 60 88

189 84

222 96

354 126 400 616 170 24

+ 50 - 0 + 10 + 20 - 0 + 50 - 0 - 0 + 10 + 100 + 100 + 100 + 80 + 10

AA = Aplastic anemia; AML = acute myelogenous leukemia; ALL = acute lymphocytic leukemia.

male donors was I.O8f(SD) 0.3 [7]. Therefore, any index greater than three standard deviations above the normal (greater than 2.0) was considered positive. Sim- ilarly, the average RAGT index was found to be 0.93 with a standard deviation off0.3 using the Iz5I anti- IgM. Therefore, any RAGT index greater than 1.8 was considered to be positive for IgM platelet antibodies. Using 1251 anti-IgA, an average RAGT index of 1.20 with a standard deviation o f f 0.3 was found. Therefore, any index greater than 2.1 was considered to be positive for IgA platelet antibodies.

forming the indirect platelet assay at various temperatures using positive and negative controls (tableI). As seen in the results, when the assay was performed using both incubations (the sensitization and antiglob- ulin test) at 4"C, the RAGT index was sig- nificantly higher than those which used higher incubation temperatures. On the ba- sis of this, incubations at 4°C were used throughout the study for the IgM assays.

Indirect Platelet Assay Results The sera of 28 consecutive thrombocyto-

penic patients, all of whom had been mul- tiply transfused with both red cells and platelets, were studied. 14 of these patients demonstrated platelet-reactive antibodies in the indirect assays (table 11). These 14 pa-

Results

Optimal Temperature of Reactivity of A ntisera The optimal temperature of reactivity for

the Iz5I anti-IgM was determined by per-

424 Miller/Kickler/Ness/Braine

Table 111. Summary of the immunoglobulin classes of the alloantibodies detected

Antibody Number of O/O of total class patients

IgG only 4 IgG and IgM 5 IgM only 4 IgM and IgA 1

28 36 28

7

tients were refractory to random donor platelets. IgG antibodies were found in 9 of these 14 (64%) patients. In 5 of the 9 (36%), IgM was found to be associated with IgG. 4 patients out of the total of 14 demonstrated IgM antibodies alone. IgA was found in only 1 case, and it was associated with IgM. In addition, 10 ofthese 14 patients had lympho- cytotoxic antibodies. A summary of the im- munoglobulin classes can be seen in table 111. The remaining 14 patients had no de- monstrable platelet alloantibodies by the ra- diolabeled antiglobulin technique, nor by lymphocytotoxicity. These antibody-nega- tive patients were not refractory to platelet transfusions.

Discussion

Many sensitive techniques for the detec- tion of platelet immunoglobulins in patients with thrombocytopenia have been described in recent years. While many of these meth- ods were developed to detect autoantibodies, they have become useful in detecting platelet alloantibodies as well. It has been estab- lished that the radiolabeled antiglobulin test is not only very sensitive, but is also a fairly

simple and rapid method [l , 71. This study employed the radiolabeled antiglobulin method and adapted it to determine the immunoglobulin classes of platelet antibod- ies by using radiolabeled monospecific anti- sera. Although it is well recognized that patients receiving multiple transfusions may have platelet alloantibodies present in their sera, very few studies ofthe immunoglobulin classes of these antibodies have been report- ed. A limited number of studies show that the immunoglobulin class of these alloanti- bodies is IgG [3, 121. In addition to IgG, our study presents data in which platelet idloan- tibodies produced in response to multiple transfusions were found to be of the IgM and IgA classes as well. IgA was found in only 1 case, and it was found to be associated with IgM. IgG circulating antibodies were found in 9 of 14 (64%) of the patients. In 5 of the 9 (36%), IgM was found to be associated with IgG. 4 patients out of the total of 14 demon- strated IgM antibodies alone. It should be mentioned that in each of the 4 cases their transfusion therapy only recently had begun within the last month. This suggests that IgM platelet antibodies may develop before IgG as they do in red cell antibodies, but further sequential studies would be neces- sary to determine this.

The reactivity of the IgM antibodies de- tected was maximal at lower temperatures. It is possible that these antibodies are clini- cally insignificant just as red cell antibodies that are cold reactive do not lead to short- ened red cell survival. Further in vivo studies are needed to delineate the immune destruc- tion of platelets by different immunoglobu- lin classes ofantibody. In our 4 patients with only IgM antibody, their clinical importance was suggested by the patient’s refractory state to platelet transfusions.

Heterogeneity of Platelet Alloantibodies 42 5

It is well established that the serologically defined antigens of the HLA system play a major role in platelet alloimmunization. Much of the recent literature presents evi- dence that despite close HLA matches, transfusion failures still occur [l, 7, 101. It is for this reason that platelet crossmatching may be a useful adjunct to HLA-matching when selecting platelets for these refractory patients. Although platelet crossmatching has been found to be predictive of the trans- fusion outcome, false-negative crossmatches do occur [l, 6,121. Because most crossmatch- ing procedures employ only an anti-IgG technique, it is possible that some of these false-negative crossmatches are due to plate- let antibodies other than those of the IgG class. Evidence is presented in this study that antibodies of the IgM and IgA immunoglob- ulin classes do exist as well as IgG, which suggests that monospecific antisera may be useful in detecting these additional antibod- ies. These antibodies may be especially im- portant in those cases in which false-nega- tive crossmatches occur. Other explanations for the occurrence of false-negative cross- matches include insensitivity of the assay system, the presence of other, as yet uniden- tified, HLA antibodies or the nonimmune destruction of platelets. It is interesting to note that in 4 out of the 5 patients who dem- onstrated IgM and/or IgA antibodies with- out IgG, there were no HLA antibodies de- monstrable by the lymphocytotoxicity tech- nique. This suggests that the radiolabeled antiglobulin technique may be a more sen- sitive test or alternatively that these IgM platelet alloantibodies are platelet-specific antibodies.

In conclusion, the radiolabeled anti- globulin technique was found to be easily adapted for the determination of the immu-

noglobulin classes of platelet antibodies. By using monospecific antisera, antibodies of the IgM and IgA classes were detected as well as IgG in alloimmunized patients. These antibodies may play a part in the immune destruction of platelets, and their detection opens the possibilities of more accurate platelet crossmatching.

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Received: March 3, 1983 Accepted: October 10, 1983

Thomas S. Kickler, MD, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Md. 21205 (USA)