After Office ours Ru,(D) IMMUNOGLOBULIN (RHOGAM): How IT CAME INTO BEING Robert Mittendorf, MD, and Michelle A. Williams, SM The story of Rh,(D) immunoglobulin (RhoGAM) begins 1909 with Theobald Smith, Fabyan Professor of Compara- tive Pathology at Harvard, and ends in the 1960s with completion of the clinical trials. Important in the develop- ment of RhoGAM were the search for a safe diphtheria vaccine, an interest in butterfly mimicry, the discovery ofthe Rhesus factor, and the contribution of Sing Sing prison inmates—seemingly unrelated occurrences. But, when all ‘were combined, the riddle of erythroblastosis fetalis was solved. (Obstet Gynecol 77:301, 1991) The Search for a Diphtheria Vaccine Early in this century, immunologists were hoping to produce a safe diphtheria vaccine, as prophylaxis with immune serum had been unsatisfactory. To shed light, on this problem, Theobald Smith conducted experi- ‘ments on pregnant guinea pigs with neutralized mix- tures of diphtheria toxin and antitoxin.? Although he "rom the Hareard School of Public Heat, St. Margarets Hospital jor Women, and Tufts Univesity School of Mine, Boston, Massachie sels VOL. 77, NO. 2, FEBRUARY 1991 never produced a vaccine, he did discover one of the principles of immunology: In the presence of excess passively acquired antibody, the corresponding anti- gen usually fails to immunize. Unfortunately, because of the state of knowledge of Rh disease of the newborn at that time, Smith was unaware of the enormity of his finding. Half a century would elapse before the prin- ciple of passively acquired immunity would be applied in the prevention of erythroblastosis fetalis. Much later, in 1950, Molle Barr published the results of her clinical research on human infants.” She had discovered a method to quantify the amount of diph- theria antitoxin that would interfere with active immu- nization. Firs, the titer of passively acquired immunity to diphtheria was measured in the cord blood at birth; then infants were actively immunized with toxoid as carly as 6 weeks of age. Barr found that if levels of maternally conferred antitoxin were greater than 0.1 UimL, subsequent immunization was inadequate. ‘Thus, the quantity of passively acquired antibody that could prevent active immunization was determined. Rh and Butterflies Earlier, in 1940, Landsteiner and Wiener had found a factor in human blood that was agglutinated by Rhesus sera (the discovery of Rh).” A year later, Levine et al* described isoimmunization in pregnancy (formation of maternal antibody to fetal antigen). They also pointed out that there were fewer ABO-incompatible matings in the families of children afflicted with erythroblasto- sis.° Because ABO-incompatible fetal red cells are coated by anti-A or anti-B antibody, then removed from the maternal circulation, the mother is usually not sensitized to the Rh factor. In 1952, Cyril Clarke, a physician who had an interest in mimicry and hybridization in Papilio butter- flies, met Philip Sheppard, a student of genetic poly- morphism. Their common interests soon led to a study of human blood group inheritance, which proved to be similar to the heredity of wing patterns in some but- terflies. Later, the same team studied the families of exythroblastotic children in Liverpool. Of the 14 ABO- incompatible matings in which the sensitizing fetus’ blood group could be determined with certainty, Clarke and Sheppard found that the fetal ABO group was always compatible with the mother.® Thus, Levine's earlier observation that ABO incompatibility may protect against erythroblastosis was confirmed. In 1960, Ronald Finn, who had joined the Clarke- Sheppard research team, suggested giving postpartum. mothers anti-D to aid in the elimination of Rh- incompatible fetal cells before immunization could occur. Finn assumed that most sensitization results 0029.784491183.50 301from fetomaternal bleeding during labor and delivery. Wiener had demonstrated previously that blood loss from infant to mother does occur, but he did not quantify the amount of the bleeding. Kleihauer and Betke Kicihauer et al® showed that fetal erythrocytes seen on. the peripheral smear could be differentiated from the adult type by using a citric acid phosphate butfer. Using the Kleihauer-Betke test, Clarke” established that fetomaternal bleeding was the most important factor in sensitizing an Rh(D)-negative mother. Ina series of 256 postpartum women unselected for ABO blood group and parity, but containing 206 Rh- negative mothers, fetal bleeding was assessed by the new test. Of the 85 Rh(D)-negative mothers who had Rh-positive infants, ten had fetal cells in their blood and three formed anti-D. None of the 75 mothers without evidence of fetal cells in the maternal circula- tion were sensitized. The author concluded that “transplacental hemorrhage at delivery was the most important factor in sensitizing Rh-negative women, and consequently we felt that it was entirely logical to begin experiments in male volunteers to see if we could prevent such sensitization.” But the fist clinical experiment with men failed.° Each Rh(D)-negative subject was injected with Rh(D)-positive, ABO-compatible adult blood. The sub- jects received anti-D serum intravenously, but the controls got the Rh(D}-positive, ABO-compatible red cells only. The investigators administered complete (caline immunoglobulin M [IgM)) antibody because naturally occurring anti-A and anti-B are of that type. Unexpectedly, most cases formed antibodies. How- ever, a subsequent successful experiment used incom- plete (albumin IgG) anti-D antibody instead of the complete antibody. Stern et al! had shown previously that when Rh(D)-positive red cells coated in vitro with incomplete anti-D were given to Rh(D)-negative men, no antibody was induced. Thus, in the follow-up experiment," plasma containing incomplete anti-D ‘was administered to the subjects. Only three of 21 men receiving this prophylaxis developed immune antibod- ies after three or four exposures to Rh-positive blood, ‘whereas most of the controls became sensitized. Sing Sing The next advancement was made by Vincent Freda, an obstetrician, and his associates at Columbia Univer- In experiments conducted at Sing Sing prison,”? prophylactic anti-D gamma globulin, instead of whole serum, was given to inmate volunteers. In the first 302 Mittendorf and Williams RioGAM experiment, Rh-negative, group O male prisoners were challenged once a month for 5 months with Rh-positive whole blood. The subjects were given gamma globulin before each injection of the possibly sensitizing blood, whereas the controls received no preventive. None of the cases, compared with four of the five controls, became sensitized. However, would gamma globulin given after the injection of Rh-positive red cells prevent sensitization? To answer this question, the number of volunteers, ‘was increased from ten to 20 and the sensitizing blood was given first, followed by the antibody. However, Sing Sing warden Wilfred Denno was concerned that a short, predictable injection schedule involving so many inmates might encourage a prison break. In a compromise with the warden, the research team chose to extend the interval between the administrations of antigen and antibody from 24 to 72 hours (the basis for the maximum permissible interval between delivery and prophylaxis in current obstetric practice). Thus, the stage was set for testing recently delivered women.? In 1964, the British and American research groups began separate, independent clinical trials of anti-Rh gamma globulin. In New York,® Rh(D)-negative primi- paras who had just delivered Rh-positive, ABO- compatible infants were admitted for study only if the peripheral smear contained five or more fetal cells by the Kleihauer-Betke technique. Alternate subjects re- ceived a gamma globulin preventive. None of the 78 cases, but 19 of 78 controls, produced immune anti-D antibody within 6 months of the trial. In the smaller but more selective British trial, the findings were similar? Hence, more than 50 years after Theobald Smith’s search for a diphtheria vaccine, RoGAM came into being. Since then, this pharmaceutical has become one of the most important primary preventives in obstet- rics. Because of the contributions of so many basic science and clinical researchers, erythroblastosis fetalis has been largely eliminated in the developed world. References 1. Smith T. Active immunity produced by so-called balanced oF ‘eutzal mitures of diphtheria toxin and antitoxin. J Exp Med 1999;11241-56 2. Barr M, Glenny AT, Randall KJ. Diphtheria immunization in| young babies. A study of some factors involved. Lancet 1950: ea ‘3 Zimmerman DR. Rh: The inimate history ofa disease and its ‘conquest. New York: Macmillan, 1973:17-9, 22, 256-64 4 Levine P, Katzin EM, Buraham L, Isolmmunization in preg fancy. JAMA 1941116857. 5. Levine P. Serological factors as possible causes in spontaneous abortions. J Hered 19833471-80, Obstetrics & Gynecology6, Finn R, Clatke CA, Donohoe WTA, etal. Experimental studies on the prevention of Rl haemolytic disease, Br Med | 19611:486-50. 7, Wiener AS, Diagnosis and treatment of anemia ofthe newborn caused by occult placental hemorthage. Am J Obstet Gynecol 194835671722. 8, Klehaver E, Braun H, Betke K, Demonstration von fetaem Haemoglobin in den Eryivocyten eines Blutaustchs. Klin ‘Wochensehe 1957:35:637-8. 9, Clatke CA. Prevention of Rh fuemolytic disease. Vox Sang 1966;11:681-55. 10, Stem K, Goodman HS, Berger M. Experimental soimmunization to hemeantgens in man. | Immuno 196187:189-96 11, Chaka CA, Donohoe WTA, McConnell RB, eta. Further xper- ‘mental studies on the prevention of Rh haemolytic disease. Be Med J 1965;1979-84. 12, Freda Vj, Gorman JG, Pollack W. Successful prevention of ‘experimental Rh sensitization in man with an anti-Rh gamma? lobulin antibody preparation: A preliminary report. Transfusion T9ott26-22. VOL. 72, NO. 2, FEBRUARY 1991 ‘Address reprint requests Robert Mittendorf, MD Department of Epidemiology Harvard School of Public Health 677 Huntington Avenue Boston, MA 02115 Received July 27,1990. Received in reise form Seplembor 24, 1990. ‘Accepted October 1, 1980, Copyright © 1991 by The American College of Obstetricians and Gynecologists. Mittendorf and Williams RhoGAM_ 303