chimpanzee red cells. In addition, antisera were available from many assigned to chimpanzee blood.2b

Transcription

1 INDIVIDUAL DIFFERENCES IN CHIMPANZEE BLOOD, DEMONSTRABLE WITH ABSORBED HUMAN ANTI-RHo SERA* BY ALEXANDER S. WIENER, J. MOOR-JANKOWSKI, EVE B. GORDON, AND CLYDE H. KRATOCHVTL SEROLOGICAL LABORATORY OF T11E OFFICE OF THE CHIEF MEDICAL EXAMINER OF NEW YORK CITY, DEPARTMENT OF FORENSIC MEDICINE, NEW YORK UNIVERSITY SCHOOL OF MEDICINE, AND 6571ST AEROMEDICAL RESEARCH LABORATORY, HOLLOMAN AIR FORCE BASE, NEW MEXICO Communicated by Michael Heidelberger, June 27, 1966 Tests with a battery of human Rh-Hr antisera have demonstrated that chimpanzee red cells have blood factors Rho and hr' but lack factors rh', rh", and hr", and therefore all belong to a blood type that may be designated as Rho.1 2a, 2b However, the Rho factor of chimpanzee red cells is not identical with that of human red cells, as demonstrated by the negative or weak reactions of chimpanzee red cells with saline-agglutinating Rho antisera. This led to their original erroneous classification as Rh negative.' In direct tests of ficin-treated chimpanzee red cells with anti-rho sera individual differences are not apparent, and only minor differences in titer are observed21' 4,' comparable to those with different human Rh-positive red cells. However, when human anti-rho sera are absorbed to negative reactivity with chimpanzee red cells, they still react in high titer with human Rh-positive red cells.4 Thus, supposedly monovalent human anti-rho sera can be fractionated by absorption with chimpanzee red cells into components of at least two distinct specificities, one reactive for chimpanzee red cells as well as human Rh-positive red cells, and the other for human Rh-positive red cells alone. To emphasize the difference between human and chimpanzee Rh agglutinogens, therefore, the symbol RhCh has been assigned to chimpanzee blood.2b Our results so far indicated that red cells from all chimpanzees reacted alike with Rh-Hr antisera. However, in tests on red cells from chimpanzees with eluates prepared from stromata of human Rh-positive red cells that had been coated with human Rho antibodies, Masouredis et al. observed that while most chimpanzees reacted as "Rh-positive," some reacted as "Rh-negative." We now report experiments carried out in order to reconcile the seeming difference between Masouredis' results and those obtained by us. Materials and Methods.-The blood specimens for testing were freshly obtained from chimpanzees maintained by the 6571st Aeromedical Laboratory at the Holloman Air Force Base, New Mexico. The testing methods used have been described previously.2a 5 The anti-rho sera were obtained from male volunteer Rh-negative donors hyperimmunized by repeated intravenous injections of human Rh-positive blood.7 Before use, the sera were absorbed with human group A, Rh-negative, and group B, Rh-negative, red cells, to remove isoagglutinins. The high titer of the antisera made possible their use at a starting dilution at which nonspecific heteroagglutinins no longer agglutinated chimpanzee red cells. In addition, antisera were available from many chimpanzees containing isoagglutinins produced by isoimmunization or by crossimmunization with human red cells.8' 9 The principles underlying the tests and the nomenclature used have been discussed in detail in a separate monograph.'0 458

2 Vol. 56, 1966 MICROBIOLOGY: WIENER ET AL,. 459 Results.-Red cells from one of the chimpanzees (Beverly #161) typed as Rh negative by Masouredis,6 using eluates, were ficin-treated and used to absorb a human anti-rho serum. The serum was titrated before and after absorption against the ficin-treated red cells of 1.5 chimpanzees, as well as control human Rh-positive and Rh-negative red cells. Representative results are shown ill Table 1. Before absorption, the anti-rho serum showed no significant differences ill reactivity for red cells from different chimpanzees. After absorption with chimpanzee Beverly's red cells, the serum no longer clumped the absorbing cells but still strongly agglutinated red cells from most other chimpanzees. Moreover, titrations disclosed differences among the chimpanzees with positively reacting cells, suggesting the existence of subtypes. The experiments were then extended to two additional human anti-rho sera. As shown in Table 2, similar results were obtained with all three antisera. Thus, while human anti-rho sera react with all chimpanzee red cells, they can be fractionated by absorption with red cells from particular chimpanzees so as to yield reagents capable of disclosing individual differences among chimpanzee red cells. Red cells from other chimpanzees besides Beverly have also been used successfully to produce absorbed reagents of the same specificity, namely, chimpanzees with red cells not reacting with the Rho antisera absorbed with Beverly cells, e.g., Rosie #232 of Table 2. The possibility suggested itself that the blood group specificity of the absorbed human anti-rho sera for chimpanzees' red cells might be duplicated by some of the most recently produced chimpanzee isoimmune sera under investigation in our laboratory.9 A comparison (cf. Table 2) revealed, not unexpectedly, that chimpanzee Beverly, which had been isoimmunized for 18 months, had produced antibodies which clumped the red cells of all chimpanzees except those not agglutinated by the absorbed human anti-rho serum. However, as shown in Table 2, Beverly's serum gave little reaction with human red cells, whether Rh positive or Rh negative. Comparison of Beverly's isoimmune serum with the absorbed human anti-rho serum was then extended to 15 other chimpanzees. Red cells from only one chimpanzee failed to agglutinate with the absorbed human anti-rho and only this chimpanzee blood was Beverly negative. Thus, of a total of 30 random chimpanzees there were two animals (not counting Beverly) which were negative with both reagents, while the remaining 28 were positive with both reagents. This leaves little doubt that chimpanzee Beverly's isoantiserum and the absorbed human anti- Rho serum were of similar specificity for chimpanzee red cells, despite the differences in their origin and in their reactions with human red cells. The parallel reactions of chimpanzee Beverly's isoimmune serum, and of human anti-rho serum absorbed with Beverly's red cells, in tests with chimpanzee red cells, indicate that the blood factor (or factors) of chimpanzee red cells detected by Beverly's isoimmune serum must be related to the human Rh-Hr system. We have already expressed our conviction that the C-E-F-H blood group system of chimpanzees"1, 12 is the counterpart in chimpanzees of the human Rh-Hr blood group system, since anti-cc, anti-ec, anti-fc, and anti-ho all react with greatest avidity and to the highest titers with red cells treated with ficin, a characteristic shared also by all human Rh-Hr antibodies. Therefore, one would expect that the

3 46() MICROBIOLOGY: WIENER ET AL. PROC. N. A. S. TABLE 1 DEMONSTRATION OF INDIVII)UAL DIFFERENCES IN CHIMPANZEE RED CELLS WITH ABSORBED HUMAN ANTi-RhO SERUM Blood of. - Reaetions with Unabsorbed Anti-Rho Serum s-- chimpanzees IJndilute'd 1:2 1:4 1:8 1:16 1:32 1:64 1. Manuel#134 +± ± tr Rosemary # ± ± - 3. Rosie # ± - 4. Bob# ± ± ±fi 5. Meredith# ± Mel # ± ± ++± Seetee #250 ++±4 ++± ++± ± tr. 8. Beverly# tr. Blood of ---Reactions with Anti-Rho Serum Absorbed with Red Cells of Beverly - chimpanzees Undiluted 1:2 1:4 1:8 1:16 1:32 1. Manuel # Rosemary#182 ++± tr. 3. Rosie # tr. 4. Bob # _ tr. 5. Meredith # ± Mel#236 ++± Seetee #250 ++±4 ++ +± i tr. 8. Beverly #161 tr The strength of the reactions is indicated by the number of plus signs: is the strongest reaction possible, namely, a single clump containing almost all the red cells; tr. = trace. blood factor detected by Beverly's serum should be related to the C-E-F-H blood group system. Comparison of the reactions of chimpanzee Beverly's serum with the C-E-F-H blood types showed that the blood factor detected by Beverly's serum was different from Cc, Ec, Fc, and Hc, but could be the "allele" of blood factor CC, i.e., blood factor cc. Tests on a series of 158 chimpanzees for blood factor Cc have shown 54 per cent Cc positive and 46 per cent Cc negative. Therefore, "gene" c = V/Co neg. = V0.456 = 67.5 per cent, so that "gene" C = 33.5 per cent. (Cc-negative individuals will be genotype cc, where c is the gene determining agglutinogens having cc-negative specificity. If x is the frequency of gene c, then the frequency of genotype cc must be x2, so that x = V/Co neg.) Therefore, the expected frequency of cc-negative blood = (0.335)2 or 10.6 per cent. On a series of 105 chimpanzees, Beverly's serum gave 15 negative reactions, or 14.5 per cent, not far from the expected frequency. Moreover, all 15 chimpanzees with red cells not agglutinated by Beverly's serum proved to be Cc-positive. Discussion.-The fractionation of human anti-rho serum by absorption with chimpanzee red cells into reagents of various specificities is comparable to the fractionation of M-N antisera by absorption with red cells of gibbons.13 There are sound reasons14 to believe that all so-called "monovalent" blood grouping antisera contain a "spectrum" of antibodies rather than antibodymolecules of single, uniform structure. It has been known for a long time that human anti-a and anti-b sera can be fractionated by absorption with red cells from infrahuman species. We have accomplished a similar absorptive fractionation of lectins prepared from extracts of seeds. What relationship the specificity of human anti-rho absorbed with chimpanzee red cells bears to the blood factors RhA, RhB, RhC, RhD, rhg, etc., which are associated with the blood factor Rho of human Rh-positive blood,15 remains to be determined. The findings described here are reminiscent of the original observations on the Rh factor. The Rh factor of human blood was discoveredl6 by testing human red

4 VOL. 56, 1966 MICROBIOLOGY: WIENER ET AL. 461 TABLE 2 COMPARISON OF THE REACTIONS OF HUMAN ANTI-Rh0 SERUM ABSORBED WITH RED CELLS OF CHIMPANZEE BEVERLY, AND THOSE OF THE ISOIMMUNE SERUM OF BEVERLY Reciprocal Titers of Antisera for Ficin-Treated Red Blood Cells -n - Anti-Rho #1-- -Anti-Rho #2-,- Anti-Rho f3- Absorbed Absorbed Absorbed Chimpanzee with with with Beverly's Red cells of Un- Beverly Un- Beverly Un- Beverly isoimmune chimpanzees absorbed red cells absorbed red cells absorbed red cells serum 1. Manuel B' Rosemary # Sam II # Rosie # Bob # Meredith # Mel #236 8( Glory # Shorty # Floyd # Clay # Spindy # Seetee # Hanzie #a Charlie # Beverly # Controls Human, type Rhjrh Human, type rh cells with antisera prepared in rabbits and guinea-pigs by immunization with red cells of rhesus monkeys, yet potent isoimmune anti-rh sera prepared in man by immunizing rhesus-negative individuals with rhesus-positive blood fail to react with the red cells of rhesus monkeys-a so-called "nonreciprocal reaction."'7 Similarly, human anti-rho sera after absorption with chimpanzee Beverly's red cells defined individual differences in the blood of chimpanzees, yet Beverly's own isoimmune serum which gave parallel reactions with red cells of chimpanzees reacted only feebly with human red cells whether Rh-positive or Rh-negative. The observations also reconcile the seeming contradiction between the results obtained by Masouredis with eluates and our own previous findings with unabsorbed, potent Rho antisera. The belief that eluates should give the same results as the original antiserum is based on the assumption that testing sera are truly "monovalent" and that there is a one-to-one correspondence between antigens and antibodies. Instead, as we and others have pointed out, most if not all antisera contain a spectrum of antibodies that can be separated by absorption. Absorption removes those antibodies having the highest avidity for the absorbing red cells, leaving behind the fraction with least avidity and therefore the fraction most readily eluted from the red cells. This explains the similarity of the results with Masouredis' eluates and our current absorbed antisera. Summary.-A "new" blood factor of chimpanzee red cells has been described, which is detected by two sorts of antisera, namely, by human anti-rho serum absorbed with red cells of particular chimpanzees and by isoimmune chimpanzee serum. The blood factor detected by these two reagents appears to be reciprocally related to blood factor cc of the C-E-F-H system, and therefore has been assigned the symbol ct. The authors wish to acknowledge the technical assistance of Miss Pat Ryan, Mrs. Dina Santana Bracco, Miss Sybil Gordon, Miss Toby Steckel, Mr. Bruce McPherson, and Mr. Hugh Binley.

5 462 MICROBIOLOGY: WIENER ET AL. PROC. N. A. S. * This study was aided in whole by U.S. Public Health Service grants GM and GM , and U.S. Air Force contract AF29(600) The serological studies were assisted by the use of the facilities of the Hofstra University, Hempstead, L.I., N.Y. I To avoid ambiguity, symbols for blood factors and their corresponding antibodies are printed in boldface, symbols for genes and genotypes are printed in italics, and symbols for agglutinogens, phenotypes, and blood group systems are printed in regular type. 2a Wiener, A. S., J. A. Gavan, and E. B. Gordon, Am. J. Phys. Anthropol., 11, 39 (1953). 2'Wiener, A. S., and E. B. Gordon, Am. J. Phys. Anthropol., 19, 52 (1961). 3Wiener, A. S., and M. Wade, Science, 102, 177 (1945). 4Wiener, A. S., J. Moor-Jankowski, and E. B. Gordon, Am. J. Human Genet., 16, 246 (1964). 5 Wiener, A. S., and E. B. Gordon, Am. J. Clin. Pathol., 23, 429 (1953). 6 Masouredis, S., et al., J. Immunol, in press. 7 Wiener, A. S., Proc. Soc. Exptl. Biol. Med., 70, 576 (1949). 8 Wiener, A. S., J. Moor-Jankowski, C. H. Kratochvil, and J. Fineg, Nature, 210, 498 (1966). 9 MoorJankowski, J., A. S. Wiener, C. H. Kratochvil, and C. Hanly, in preparation. 10 Wiener, A. S., and M. Shapiro, in Advances in Blood Grouping, II (New York: Grune & Stratton, 1965), pp. xiii-xxiv. 11 Wiener, A. S., J. Moor-Jankowski, A. J. Riopelle, and N. F. Shell, Transfusion, 5, 508 (1965). 12 Moor-Jankowski, J., A. S. Wiener, C. H. Kratochvil, and J. Fineg, Intern. Arch. Allergy Appl. Immunol., 29, 82 (1966). 13 Wiener, A. S., J. Moor-Jankowski, E. B. Gordon, and N. F. Shell, Transfusion, in press. 14 Wiener, A. S., Blood, 27, 110 (1966). 15 Wiener, A. S., in Advances in Blood Grouping (New York: Grune & Stratton, 1961), pp Landsteiner, K., and A. S. Wiener, Proc. Soc. Exptl. Biol. Med., 43, 223 (1940). 17 Wiener, A. S., and I. B. Wexler, Rh-Hr Syllabus (New York: Grune & Stratton, 1963), 2nd ed., p. 10.