ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 15, No. 5 Copyright 1985, Institute for Clinical Science, Inc. HLA Alloimmunization with Leukocyte Concentrates from HLA-matched and HLA-non-m atched Donors in Patients with H unter s Syndrom e* RAM M. KAKAIYA, M.D.,t ROBERT G REEN STEIN, M.D.,i PATRICIA PISCIOTTO, M.D.,$ SHEILA K. SLOCUM, + DEBBYE ROSEN, R.N.,f RITCHARD G. CABLE, M.D.,f EDWARD E. MORSE, M.D.,$ and AN DREA GAINEY, M.S 4 famerican Red Cross Blood Services Connecticut Region and tdepartment of Pediatrics and Division of Blood Bank and Hematology, Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington, CT 06032 ABSTRACT The incidence and characteristics of HLA alloimmunization following transfusions of leukocyte concentrates as a source of enzyme replacem ent were determ ined in male patients with H unter s syndrome. Five patients were given leukocyte concentrates from HLA m atched donors (Group I) and another five patients received leukocyte concentrates from non-hla m atched donors (Group II). No other blood products were transfused in either group. Im m une response pattern of HLA alloimmunization m easured as the proportion of screening cells manifesting cytotoxicity with patient s sera obtained during th e follow-up period was similar in both groups. HLA alloimmunization is seen with transfused leukocyte concentrates from either HLA-matched or non-hla-matched donors in patients with H unter s syndrome. There appears to be a trend for an earlier onset of HLA alloim m unization w ith few er transfusions w hen leukocyte concentrates from non-hla-m atched donors are transfused as compared to leukocyte concentrates from H LA-m atched donors. O nce HLA alloim m unization occurs, im m une response p a tte rn s appear sim ilar w ith e ith e r leukocyte product. Introduction nom enon has b e e n w ell stu d ie d for A lloim m unization follow ing blood in duction of alloantibodies to re d cell transfusion occurs frequently. This phe- antigens.4 The tim e course of alloimmu- nization to white cell antigens and the * Address reprint requests to: Ram M. Kakaiya, specificities of the antibodies produced M.D., A ssociate D irector, Am erican Red Cross r. l.* i. r 1. i Blood Services, Connecticut Region, 209 Farmington Avenue, Farmington, CT 06032. m ultiple transfusions has not been investigated in as m uch detail as is the 435 0091-7370/85/0900-0435 $00.90 Institute for Clinical Science, Inc.
436 KAKAIYA, GREENSTEIN, PISCIOTTO, SLOCUM, ROSEN, CABLE, MORSE, AND GAINEY case for alloimmunization to red cell antigens. T he use of d iffe re n t transfusion protocols d esig n ed to m odulate the alloim m unization process is increasing.1 Thus, understanding the nature of HLA alloim m unization has therapeutic re le vance to a variety of clinical situations, such as kidney and bone m arrow transplantation, as well as in cell supportive th erap y for hem atologic m alignancies and enzym e re p la c e m e n t th era p y in genetic m etabolic diseases. O ur findings have b e e n previously re p o rte d on th e o ccurrence of HLA alloim m unization in patients receiving transfusions of HLA-m atched leukocyte co n cen trates for enzym e rep lacem en t therapy in patients w ith H u n te r s syndrom e.3 The im m une response was characterized by th e p ro d u ctio n of a w ide variety of HLA alloantibodies including monospecific, duospecific, and m ultispecific HLA an tib o d ies, and antibodies with apparent HLA specificities to the crossreactive antigens of the recipients. In addition to the group of five patients in our previous study, HLA alloim m u nization has been observed in five other patients with H unter s syndrome. These latter patients received leukocyte concentrates collected from random, non- HLA-matched donors. This provided us the opportunity to com pare HLA alloimmunization following transfusions of non- H L A -m atched leukocyte concentrates with our previously reported findings following transfusions of HLA-matched leukocyte concentrates. Serological findings on HLA alloimmunization in these two groups of patients are presented in this report. M aterials and M ethods Lym phocyte HLA typing for th e antigens of HLA-A and B loci was perform ed by the standard N IH microlymphocytotoxicity assay.5 H LA typing was p e r form ed using 58 a n tise ra defining 18 HLA-A locus specificities and 108 antisera defining 37 HLA-B locus specificities. The standard NIH microlymphocytotoxicity te st was also used for HLA alloantibody screening and identification with a panel of 60 screening cells phen o ty p ed for m ost HLA-A and B locus antigens, including the split antigens. A serum sample from each patient was obtained before and periodically th e re after d u rin g transfusion th era p y and stored frozen at 65 C for less than one m onth before testing. E ach serum sam ple was scre e n e d for lym phocytotoxic antibody using a panel of lym phocytes obtained from HLA phenotyped healthy norm al donors. U sually 60 p an el cells w ere u sed for each screening. T he results of those sera m anifesting lym phocytotoxic reactions w ere quantitatively expressed as the percent lymphocytotoxicity calculated by dividing the num ber of panel cells manifesting cytotoxicity by the total num ber of panel cells. The evidence for HLA alloim m unization was defined as the appearance of lymphocytotoxicity observed with a serum sample against at least 15 percent of the panel cells. The tim e at which HLA alloim m unization o ccu rred was calculated in two ways because the periodic screening of the serum for antibody was som ew hat irregular. First, the earliest possible tim e at which alloim m unization could have occurred was based on the absence of antibodies in the latest serum sample. Second, the observed onset of alloim m unization was calculated based on a serum sam ple which showed th e p resence of HLA antibody. An inform ed consent was obtain ed from parents of each study patient before transfusions of leukocyte concentrates were initiated. Leukocyte donors were selected based on a schema described by Duquesnoy et al2 for the first group of p a tie n ts receiving leukocyte co n cen trates from H LA -m atched donors. The
ALLOIMMUNIZATION TO LEUKOCYTES 437 degree of H LA -m atching betw een the donor and th e recipient for all transfusions a d m in iste re d to this group I patients has been described in detail in our previous report.3 The second group of patients w ere provided leukocyte conc e n trate s from non-h L A m atched donors. In fact, in th re e of G roup II p a tie n ts, th e HLA p h e n o ty p e of the recip ie n ts was not d e te rm in e d. Both groups of patients received leukocytes from ABO com patible blood donors. Leukocyte concentrates for transfusion to study p a tie n ts w ere p re p a re d as d e scrib ed by us3 previously and contain ed, on average, 6.1 ± 2.2 x 109 (±S D ) leukocytes. The lymphocyte and platelet content of this product was 5.9 ± 2.1 x 109 and 5.5 ± 5.0 x 1010 (mean ± SD), respectively. Statistical analysis was perform ed with a M ann-w hitney U test or a t-test. Results T he characteristics of patients receiving e ith e r HLA m atched (G roup I) or non-hla-m atched (Group II) leukocyte concentrates are shown in table 1. The range and distribution of ages in the two groups were similar. Since the duration of follow-up was less for the Group II as compared to Group I patients, they also received fewer transfusions. The average (±S D ) frequency of all the transfusions was 18.2 ± 25.7 days (N = 214 transfusions) and 21.6 ± 30.3 days (N = 117 transfusions) for G roup I and II patients, respectively. T herefore, transfusion frequency betw een the two groups was similar (p > 0.05). The transfusion frequency was also calculated for transfusions that preceed ed the observed onset of alloim munization and gave an average (±S D ) frequency of 13.4 ± 26.2 days (N = 75 transfusions) and 8.1 ± 12.4 days (N = 33 transfusions) for group I and II p a tie n ts, respectively. This difference was statistically not significant (p > 0.05). As described in the M ethods section, the onset of HLA alloimmunization was calculated in two ways. First, the earliest possible tim e at which alloimmunization could have o ccu rred was calculated TA BLE I C h aracteristics o f P atien ts with Hunter's Syndrome Receiving HLA-matched (Group I) or Non-HLA Matched Leukocytes (Group II) A ge ( y r s ) D u r a t i o n o f F o l l o w - u p (mo) E a r l i e s t P o s s i b l e A llo im m u n i z a t i o n (mo) O b s e r v e d O n s e t o f A llo im m u n i z a t i o n (m o) T r a n s f u s i o n s B e f o r e O b s e r v e d O n s e t o f A llo im m u n i- z a t i on T o t a l T r a n s f u s i o n s G ro u p I G ro u p I I 1 8 33 7 13 27 5 8 2 20 27 5 6 19 57 3 8 25 3 3 7 44 4 2 19 NK* 4 10 22 5 19 28 8 16 12 33 M ean ± SD 11,.4 ± 7. 8 2 6. 4 ± 5. 1 8. 4 ± 5. 8 15 ± 8 4 2. 8 ± 6 18 30 1 1. 5 6 43 7 6 8 NK 5. 0 ( d a y s ) 3 15 8 5 19 1 2. 0 5 22 9 10 7 4 6. 0 11 13 10 18 14 NK 4. 0 8 24 M ean ± SD 8..1 ± 6. 3 1 5. 6 ± 9. 4 2. 7 ± 2..3 6. 6 ± 3. 0 2 3. 4 ± 0. 4 2 1 0.0 7 5 0. 1 1 4 0. 0 4 8 0. 0 2 8 0. 0 4 'NK = N o t know n
438 KAKAIYA, GREENSTEIN, PISCIOTTO, SLOCUM, ROSEN, CABLE, MORSE, AND GAINEY based on the absence of antibodies in the latest seru m sam ple. Second, th e observed onset of alloim m unization was calculated based on a serum sample which showed the presence of HLA antibody. It should be n o ted th at th e alloim unization could have o ccu rred b e tw een th e tim e at w hich one seru m sample was negative and the following one which showed the presence of HLA antibodies. This tim e lag for group I patients, namely, patients #1, #2, #3, and # 5, was si?^ one, zero, and eight m onths, respectively. F or p a tie n t # 4, the first post-transfusion sample obtained at four m onths was positive and, th e re fore, this tim e lag could not b e calculated. The tim e lag for group II patients, namely, patients #6, #8, and #9, was 0.5, one, and two months, respectively. For patients # 7 and #10, the first followup sample obtained at five days and four months, respectively, was positive and, therefore, this tim e log could not be calculated. As shown in table I, for G roup II patients, th e observed onset of alloimm unization was som ew hat earlie r and with fewer transfusions as com pared to th e G roup I p a tie n ts, p = 0.048 and 0.028, respectively. All patients becam e alloim m unized. The im m une response pattern for each patient was m easured as the proportion of p anel cells m anifesting cytotoxicity with a given serum sample. As seen in figures 1 and 2, once th e im m une response was generated, it was generally well sustained in each patient. Only one patient, namely, patient #8, appeared to be a low responder as judged by a low percent reactivity with the patient s sera obtain ed at d ifferen t intervals. This patient also received the least num ber of transfusions and was follow ed for the shortest interval (seven months). Except for this p a tie n t, th e resp o n se p a tte rn ap p eared sim ilar am ong all o th e r p a tients. Months Months Months F i g u r e 1. Patients # 1 to 5 received leukocytes from HLA-matched donors. Percent cytotoxicity is the percentage of screening cells manifesting cytotoxicity with patient s serum.
ALLOIMMUNIZATION TO LEUKOCYTES 439 Patient #6 Patient #7 Patient *10 10 20 10 20 Months Months Months F i g u r e 2. Patients #6 to 10 received leukocytes from non-hla-matched donors. Percent cytotoxicity is the percentage of screening cells manifesting cytotoxicity with patient s serum. Discussion The findings dem onstrate the occurrence of HLA alloimmunization to transfusions of non-hla-m atched leukocyte concentrates in patients w ith H u n te r s syndrom e. This is c o n sisten t w ith our previous observations in w hich HLAalloimmunization was seen in a group of patients transfused with HLA-matched leukocyte concentrate.3 Donor-recipient HLA incompatibilities w ere not known for the group of patients who received non-h L A -m atched leukocyte co n cen trates. In contrast, the donor-recipient HLA in com patibilities w ere clearly d efin ed in p a tien ts receiv in g HLAm atched leukocyte concentrates. In fact, in these latter patients, as per our p revious re p o rt,3 12.7 p e rc e n t of HLA-A locus and 21.7 percent of HLA-B locus donor antigens w ere incom patible with th e re c ip ie n t s HLA antigens. E ven then, a trend towards an earlier onset of HLA alloimmunization with fewer transfusions was noticed with the non-hlam atched leukocyte concentrates as com p a red to th e H L A -m atched leukocyte concentrates. This trend was not due to a difference in th e age or in th e fre quency of transfusion betw een the two groups of p atien ts. E ventually, all patients developed HLA alloim m unization regardless of the type of leukocyte p ro d u ct. O nce alloim m unization was detected, the im m une response pattern that followed was similar in both groups and was ch aracterized by a co n sisten t high percent lym phocytotoxicity found with the patient s sera during follow-up period. It should be noted that the num ber of patients with H unter s syndrom e studied by us is small. Also, m ost of our patients were children. Thus, the implications of our findings to other situations in which
440 KAKAIYA, GREENSTEIN, PISCIOTTO, SLOCUM, ROSEN, CABLE, MORSE, AND GAINEY HLA alloim m unization occurs should co n sid er th e lim itations m en tio n ed in our study. T he d ata suggest th at th e im m une resp o n se p a tte rn is sim ilar, once it occurs, regardless of w h e th e r HLAm atched or non-hla-m atched leukocyte co n cen trates are transfused. T h ere appears to be a trend towards an earlier o n set of HLA alloim m unization w ith few er transfusions w ith non-h L A - m atched as compared to HLA-matched leukocyte concentrates. References 1. B r e n t, L., F a b r e, J. W., E l v e s, M. W., and S e l l s, R. A.: Proceedings of the Ninth International Congress of the Transplantation Society. The blood transfusion effect in man and in experimental animals. Transplant. Proc. i5 :915-1026, 1983. 2. D u q u e s n o y, R. J., F i l i p, D. J., R o d e y, G. E., R i m m, A. A., and A s t e r, R. H. : Successful transfusion of platelets mismatched for HLA antigens to alloim m un ized th rom b ocytopen ic patients. Am. J. Hematol. 2:219-226, 1977. 3. K a k a iy a, R. M., G r e e n s t e i n, R., P i s c i o t t o, P., S l o c u m, S., R o s e n, D., C a b l e, R. G., and M o r s e, E. E.: Characteristics and natural history o f alloim m unization fo llo w in g HLAmatched leukocyte transfusions. Ann. Clin. Lab. Sci. 14:276-284, 1984. 4. M o l l i s o n, P. L.: Blood Transfusion in Clinical M edicine, 7th ed. Oxford, Blackwell Scientific Publications, 1983. 5. S t a f f : Transplantation and Immunology Branch (1979-1980). NIH lymphocyte microcytotoxicity tech nique. N IA ID M anual o f T issue Typing Techniques; 1979-1980. Ray, J. G., ed. NIH Pub. No. 80-545, pp. 39-4 1, 1980.