Marchioro, TL

Starzl, TE

Terasaki, PI

English

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His tocornpa tibil ity Testing Report of a Conference and Workshop sponsored by the Division of Medical Sciences National Academy of Sciences National Research Council 7-12 June, 1964 Publication 1229 National Academy of Sciences-National Research Council Washington, D. C. 1965 Paul I .  Terasaki, Thomas L. Marchioro, and Thomas E. Star11 Departments of Surgo? University of Califonaia School of Medicine, Los Angeles University of Colorado School of AIedicine Denver Velerans Hospital , The  rationale behind attemDts to select ~at ients .  However. if the matching of anti- = (~amdurke; ct aL, 1962: ~ k e n t  a i d  hleda- Ymportant role in long-term survhal; a dis- - - - - - - - - , - - - - , et al., 1964a). How this might be .accom- trend wa; obtain&. lished is the immediate concern of this con- it: rerence. In this preliminary study we have : .' Methods L investigated the possibility of using allogenic antisera derived from women with multiple t k  i I a If Denver was flown to Los AngelesT Viable As the initial step in testing the efficacy of lymphocytes were isolated within 12 hr  from the lymphocyte cytotoxic reaction as a typing the time of bleeding. The method of isola- orncedure for homotrans~lantation. long- tion consisted of differential adherence of r - -  term kidney homograft sirvivors and theyr granulocytes on a polystyrene surface fol- donors were tested for compatibility. If for- lowed by centrifugation in capillary tubes to tuitous matching of transplantation antigens eliminate most of the erythrocytes (Terasaki had hren ~ r i n c i ~ a l l v  res~onsible for the el  al., in press; Terasaki and McClelland. patibilit; should be evident f r o g  the typing different human antisera. The sera were ob- ~ t i i d i ~ ~  Preri~e corrclatinn of survival time tained mainlv from women with multiple - ---- -1 r - -0 . since the method of immunosuppressibn was tained from mblt$le transfusions (#2, 6, regulated by the clinical course of individual and 15), and two from persons immunized by ---- - . .  ' , , long survival of renal transplants in these submitted for publication). Lymphocyte anti- selected ~at ients ,  a reasoiable demee of com- zens were identified by cytotoxicity of 50 I " .--.- ". - - ----- ---. -- . . with t v o i n ~  methods cannot be ex~ected, abortions and premancies; a few were bb- m Aided bv United States Public Health Service mants Ahf-02375, AX-04444. A- m 6283, A-63h. HE-07735, Ahf-Oi'ii2, AI-01452. and b ~ - 2 7 ,  and a USPHS research career program award AM-7513 from the National Institute of Arthritis and Meta- bolic Diseases. three or four consecutive skin homografts (#22 and 24): Antisera #15, 27, and 14 were from habitual aborters .** The microdroplet oil chamber method of testing has been described (Terasaki and Mc- Clelland, submitted for publication). Essen- tially, mixtures of 2,000 lymphocytes, 0.003 ml of absorbed rabbit complement, and anti- body dilutions by thirds from 0.003 to 0.00003 ml were incubated for 4 hr at room tempera- ture. The microreactions were accompl~shed in an oil chamber and assayed by counting the percentage of viable cells under phase- contrast microscopy at 620 x or 820 x mag- nification. Erythrocytes were typed with standard com- mercial antisera, using the Coulter Counter to measure agglutination (Terasaki et al., in press). Results Red-cell antigens. Many of the long-term surviving kidney homograft patients have survived despite one to three "minor" erythro- cyte-group ~ncompatibilities with their re- spective donors (Fig. 1). Most erythrocyte groups seemed innocuous, for incompatibili- ties were noted with E, Fy', M. C, D, s, and c. Among the limited numbers of survivors tested, no incom atibilities were found for A, B, N, or e. ~ [ e  fact that eight out of I1 of the 6-months-or-longer group possessed one incompatibility or none may suggest. however, that multiple incompatibilities may be deleterious. On the other hand, since two of the longest survivors had two or three incompatibilities, mismatched erythrocyte antigens may not be a critical factor to sur- vival of the graft. In patient #4, in whom there was a C and D mismatch, 10 months after transplantation antibodies were de- tected against donor erythrocytes. If it is assumed that no transfusions of Rh+ blood were given, these antibodies may have been elicited by antigens in the donor kidney. The fact that the kidney is functioning 10 months after transplantation demonstrates that the presence of such antibodies is not clinically deleterious to the kidney graft. Similar tests on papainized erythrocytes of the donor with the plasma of recipients #1, 2, 12, 14, 13, and 10 were negative. In  these Kindly contributed by Dr. Roy Walford; #22 and 24 are designated Bu and Mo. respectively, in Walford, Galla her, and Sjaarda (1964) . ** Generously contributed by %r. Phillip Levine. H I S T O C O M P A T I B I L I T Y  TESTING ni- Im- Pa- ter :he :he nd by be ak- 16 re- 3rs. A.as >at- ~ l e s  . of .ed. led 'ere Lon ties the 7D, -eas six lual ible ndi- .ave this nti- her- 7D cip- lave t of sed. eld- cer- han are the In- ems 2D. DOrS igen 2D for and ting (of old- :on" -anS- P A U L  I. TEIASAKI, T H O M A S  L. M A R C H ~ O R O I  A N D  T H O M A S  ,E. S T A R Z L  Figure 1.-Erythrocyte-group incompatibilities between kidney homognft donors and their recipients are shown in the dark ban. planted from a brother to sister, remained proper diagnosis and tlierapy of "rejection" anuric for 2 weeks, after which it was re- crises, are essential (Starzl et al., 1964b). A moved. Tests of the recipient's serum before lesser degree of matching presumably would grafting revealed cytotoxlc antibodies against have led to rapid rejection. The possibility the donor's lymphocytes. Serum taken on that the apparent correspondence was merely the day of transplantation just before graft- the result of similar but nonspecific reactions ing was cytotoxic to 70 percent of cells (using of the antisera was tested by comparing the 0.003 ml of serum, 0.005 ml of C', and 2,000 typing reactions of random hypothetical lymphocytes) and became slightly less toxic 2 donors with the donor actually used. In  and 6 days after grafting. These antibodies most instances, the actual donor was found could have been elicited by transfusions and to be one of the better donors with few in- may be suspected of being responsible for compatibilities, whereas many other donors the immediate rejection. possessed numerous antigens in excess of the recipient (Table 3). Of considerable sig- Discussion nificance is the fact that for some recipients a nonrelated individual was an even more suitable donor than the close relative used. A P A U L  I. TERASAKI, T H O M A S  I.. M A R C H I O R O .  A N D  T H O M A S  L. S T A R Z L  Figure 3.-Lymphocyte-cytotoxicity titration of a donor (wife) and unrelated recipient (#7). The resultr are plotted as in Fig. 2. Of particular interest is the fact that the donor possessed only one antigen that the recipient did not, whereas a graft in the opposite direction would have been incompatible for An incompatibility in which the donor re- saki, submitted for publication; Terasaki acts with a given antiserum and the recipient et al., in press). Van Rood has earlier en- does not is not open to the same polyvalent- countered a similar phenomenon with leuko- antiserum criticism. Cells of the donor may agglutinins which he termed agglutination- react on the basis of A, B, C, . . . Z antigens, negative-absorption-positive reaction van whereas the recipient can be taken to lack Rood and van Leeuwen. 1963), and similar all these antigens from a negative reaction. reactions have been found with erythrocytes This type of incompatibility, however, does (Race and Sanger, 1962). Absorption experi- suffer from another difficulty, in that a non- ments may therefore be essential to prove reactive cell may be capable at times of that an antigen is lacking, for the amount of absorbing out cytotoxicity (Becker and Tera- antigen present may be sufficient to absorb ur- . 2  I& '!Y m 'he le- in l is- !& I t s  In- 1st ;e, ld les he ti- - P A U L  I .  T L R A S A I I .  T H O M A S  L. M A R C H I O R O ,  A N D  T H O M A S  E.  STARZL culties of non-specific agglutination found with leukoagglutination tests (van Rood, van Leeuwen, and Bosch, 1962; Dausset, 1962). The present miaodroplet technique permits performance of numerous tests with extremely small quantities of rare human antisera. Moreover, since lymphocytes are H I S T O C O M P A T  ~ B I L I T Y  T E S T I N G  of "minor" erythrocyte g ~ o u p s  i n  trans- following transplantation were tested for plantation is given by Rogers (1963). From compatibility with their respective donors. the present initial trials, survival for more T h e  degree of incompatibility expressed in than 6 months was obtained in spite of terms of numbers o r  units of mismatched incompatibilities with E, Fy', M, C, D, s, and  antigens was relatively small when compared c (Fig. 1). Perhaps some significance could with that of hypothetical grafts from unre- be attached to the fact that seven out  of lated donors typed by the same methods. 10 long-term survivors (10 t o  18 months) Though many improvements in  the typing possessed two or three antigen incompatibil- system remain to  be made, it  is suggested rties. Moreover, i n  one patient, anti-CD that with further experience, this method of antibodies were found together with good histocompatibility typing may be.practicable renal function 10 months after grafting. T h e  in uncovering major transplantation "types." conclusion which might be indicated is that matching of "minor" erythrocyte antigens may increase the chance of success, though Acknowledgments complete matching may not be obligatory. We wish to  acknowledge the aid given us by Continental Airlines in  making possible the rapid shipment of blood from Denver to Los Angeles o n  numerous occasions for A microdroplet lymphocyte-cytotoxicity re- this study. We also thank Miss Theresa action was tested as a means of predicting Kaufmann and Mr. Jonathan P o  for their histocompatibility matching by titrations of excellent technical assistance and  Dr. Jose h 50 d i L r e n t  allogenic human andsera. As Van de  Water and  Dr. Herbert L c i r  c r  the trial system, 11 kidney homograft recipi- serum samples from women with multiple ents who have survived 6 t o  18 months abortions. References Bach, F., and K. Hinchhorn. Lymphocyte interaction: a potential histocompatibility test in vitro. Science 143: 813-814, 1964. Bain, B., M. R. Vas. and L. Lowenstein. The development of large immature mononudear cells in mixed leukocyte cultures. Blood 23: 108-116, 1964. Becker, L.. and P. 1. Terasaki. An absorption by recipient test for homograft compatibility. (sub- mitted for publication) Brent. L., and P. B. Medawar. Tissue transplantation: a new approach to the "typing" problem. Brit. Med. J .  2 (i.e. 2: 269-272) : 269-272, 1963. Brent, L., P. B. Medawar, and M. Ruszkiewia. Serological methods in the study of transplantation antigens. Brit. J. Exp. Pathol. 42: 464-477, 1961. Bridges, J. M., S. D. Nelson, and M. McGeown. Evaluation of lymphocyte transfer test in normal and uraemic subjects. Lancet 1: 581-584, 1964. Dausxt, J. The leukoagglutinins. Transfusion 2: 209-215, 1962. Dempster. W. J. Reassessment of the anurias after kidney transplantation. Brit. Med. J. 1 (i.e. 1: 1697-1701) : 1697-1701.1963. Goldman, R., W. E. Goodwin, D. C. Martin, J. J. Kaufman, R. J. Class&, and D. Nourak. Ex- perience with sixteen cases of renal homotransplantation. (submitted for publication) Corer, P. A. Some recent work on tumor immunity. Advanas in Cancer Res. 4: 149-186, 1956. Hamburger. J., J. Vaysse, J. Crosnier, J. Auvert, C. M. Lalanne, and J. Hopper. Renal homo- transplantation in man after radiation of the recipient. Experience with six patients since 1959. Am. J. Med. 32: 854-871. 1962. . hfarchioro. T. L., R. S. Brittain, C. R. Hermann, J. Holmes, W. R. Waddell, and T. E. Stanl. Use of living donors for renal homotransplantation. Arch. Surg. 88: 711-720, 1964. Mcllhann, C. F. Weak histocompatibility genes: The effect of dose and pretreatment of immuniz- ing cells. J. Immunol. 88: 500-504, 1962. Medawar, P. B. The use of antigenic tissue extracts to weaken the immunological reaction against skin homografts in mia .  Transplantation 1: 21-38, 1963. Moller, E., and G. Moller. Quantitative studies of the sensitivity of normal and neoplastic mouse cells to the cytotoxic action of isoantibodies. J. Exp. Med. 115: 527-553, 1962. Peer. L. A.. I. S. Walia, and R. Pullen. Observations on partial tolerance to skin homografts in man. Transplant. Bull. 26: 115-118, 1960. Pirofsky. B.. M. S. Cordova, and T. L. lmel. The  nonimmunologic reaction of globulin molecules with the T h r o c y t e  surface. Vox Sanguinis 7: 334-%7. 1962. Race, R. R., an R. Sanger. Blood groups in man. 4th Ed. Blackwell: Oxford, 1962. 456 p. Rogers, B. 0. Genetics of transplantation in humans. Diseases Nervous System 24: 7 4 3 ,  1963. P A U L  I .  T E R A S A K I ,  T H O M A S  L .  h I A R C H l O R 0 .  A N D  T H O M A S  E. STARZL tic Surg. 5: 238-242. 1951. Discussion rather long-term survivor that had many in- compatibilities with the donor lymphocytes DR. CEPPELLINI: What is the repre by your typing method. ducibility of your test? H I i T O C O M P A T I B I L 1 T Y  TEST I N C  TABLE 1 Lymphocyte Cytotoxicity in Relation to Time of Skin Grafting and Leukocyte Injection. Skin Grafts at Approximately 36-day Intervals; Leukocyte Injection 210 Days After Last Graft. Titer of Recipient Serum vs. Donor Lymphocytes Serum Specimen 1:2 1:8 192 1:128 1312 1:2048 - - - - - -  I4 days after 1st set 14 days Pfter 2nd ~t 15 days after 3rd set 80 50 14 12 days after 4th set 86 70 14 7 days dkerlcukocyte injection 95 61 58 67 51 

Sero-typing of human lymphocyte antigens: Preliminary trials on long-term kidney homograft survivors

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Sero-typing of human lymphocyte antigens: Preliminary trials on long-term kidney homograft survivors

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