THE Rh BLOOD FACTOR; AN ANTIGENIC ANALYSIS* I. DAVIDSOHN AND B. TOHARSKY

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1 THE Rh BLOOD FACTOR; AN ANTIGENIC ANALYSIS* I. DAVIDSOHN AND B. TOHARSKY From the Department of Pathology, ount Sinai Hospital, Chicago, Illinois Landsteiner and Wiener discovered in 94 the existence of a new blood group factor in man following the injection of rabbits the blood of the acacus rhesus. The serum of the immunized rabbits clumped not only the blood of the rhesus monkeys but also the blood of about 85 per cent of human beings irrespective of their blood groups, thus revealing the presence in man of a new blood factor designated as Rh, because it was first found in the rhesus monkey. Present knowledge of the Rh factor can be summed up as follows: it is an antigenic substance in human blood cells similar in some ways to other previously discovered antigenic factors, the most important of which are those known as A, B,, and N. It is inherited as a endelian dominant as are the others; it occurs only in the red blood cells, resembling in this respect the factors and N, but differing from A and B, which occur in tissues and secretions of at least some persons; there are no normal agglutinins against the Rh factor in man, again resembling in this respect the factors f and N, while such agglutinins are present normally against A and B; on the other hand when blood containing the Rh factor (Rh) is introduced into a person out it agglutinins may develop against it, while they never develop if blood containing the or N factors is injected into persons out these factors in their blood. The Rh factor has isoimmunizing ability, while the and N factors lack it. That is why the and N factors are of no significance for blood transfusions. Soon after the discovery of the Rh factor Wiener and Peters studied the blood of patients who had hemolytic reactions after one or more previous uneventful transfusions of blood of the correct group. The serums of these patients contained atypical isoagglutinins which clumped blood the Rh factor. These serums behaved similarly as did the serums of the rabbits immunized monkey blood. The blood of the patients did not contain the Rh factor. Wiener and Peters explained the presence of the atypical agglutinins in the patients by assuming that Rhpositive blood was given to them during one or several of the transfusions which they had received previously. The fact that they did not possess the Rh factor in their own blood made it possible for the injected Rhpositive blood to act as an antigen and to stimulate production of antirh agglutinins. After these agglutinins developed Rhpositive blood was not suitable for these patients even if the donors belonged to the identical blood group. The administration of Rhpositive blood was followed by a hemolytic reaction. Subsequent reports confirmed these observations. It was found that patients manifesting hemolytic reactions following one or several previous un * Presented at the st Annual Convention of the American Society of Clinical Pathologists, Philadelphia, June,94. t Only five instances of normal anti isoagglutinins have been reported. 44 Downloaded from on 8 January 8

2 Rh BLOOD FACTOR 45 eventful transfusions of blood of the correct group, were as a rule Rhnegative and that some of them had antirh agglutinins in their blood, that the donors were Rhpositive and that Rhnegative blood could be subsequently administered out untoward reactions. The same mechanism was found to be responsible for reactions after first transfusions given to pregnant women. Here the development of the new agglutinin (antirh) was explained by the presence of the Rh factor in the blood of the fetus to whom it was transmitted by the father and by its passage through the placenta to the mother, who was thus immunized during pregnancy. 4 Levine suggested that fetal erythroblastosis may be due to a similar mechanism. Here the cycle of events goes further than the production of antirh agglutinins in the mother. The newformed agglutinins in the blood of the mother which resulted from the passage of the Rhpositive blood from the fetus, pass through the placenta in the other direction, from the mother to the Inheritance as a endelian dominant.. Occurrence in red blood cells Occurrence in tissues other than blood. Heteroimmunizing ability Isoimmunizing ability Normal isoagglutinins Immune isoagglutinins TABLE BLOOD FACTORS A t B t * * Only 5 cases reported. t Blood transfusion accidents. t After repeated transfusions of Rh blood into Rh recipients and in pregnancy. fetus, react blood of the fetus and damage it. Erythroblastosis is the result of this damage. The correctness of this hypothesis is based on the assumption of at least four conditions: () the father must have the Rh factor in his blood in order to transmit it to the fetus, () the fetus must have the inherited Rh factor in his blood, () the mother must lack the Rh factor in order to develop antirh agglutinins in response to the passage of the factor from the fetus through the placenta, (4) there must be a free exchange of antigenic substances and of antibodies through the pacenta from the fetus to the mother and vice versa. The fulfillment of the fourth of these conditions can be taken for granted. The passage of protein substances from the fetus to the mother and of antibodies from the mother to the fetus has been satisfactorily demonstrated. However, it may be that under certain, possibly abnormal, conditions the passage through the placenta is freer than normally. It may also be that there are quantitative differences in the freedom of passage in the two directions. Levine tested the first three conditions by a study of the blood of 5 mothers N Rh t Downloaded from on 8 January 8

3 4 I. DAVIDSOHN AND B. TOHAESKY of infants erythroblastosis of whom only per cent were Rhpositive and 9 per cent Rhnegative. This is startlingly different from the finding of 8 per cent Rhpositive and 4 per cent Rhnegative individuals in a series of,5 controls. On the other hand all 89 husbands of Rhnegative mothers of infants erythroblastosis were Rhpositive and of infants erythroblastosis 99 per cent were Rhpositive. The absence of the Rh factor in one infant erythroblastosis and the presence of the Rh factor in 8 per cent of mothers of infants erythroblastosis indicates that there are probably other factors involved in the pathogenesis of erythroblastosis besides the Rh factor, but these findings do not invalidate the concept of isoimmunization as a cause of the disease. TECHNIC OF THE TEST FOR THE Rh FACTOR Human immune serums Serums of women, most frequently mothers of babies fetal erythroblastosis, are the best and at present the most reliable reagent. Due to the presence of isoagglutinins these serums can be used for testing cells of the same blood group as the individual from whom they were obtained and for cells of group O, unless they come from a person of group AB, in which case they can be used for cells of all groups. However, isoagglutinins can now be easily inhibited Witebsky's A and B purified blood group substances. 8 Animal immune serums Landsteiner and Wiener found that immune serums produced in rabbits could be purified only great difficulty. Attempts to remove the agglutinins for Rh cells were in most instances followed by adsorption also of the agglutinins for Rh cells. Immune serums produced in guinea pigs could not be purified at all, but occasional ones had significantly higher titers for Rhpositive cells and could be used in proper dilutions. The technic recommended by Levine 4 was employed. For the test, one to two drops of the serum in a test tube of mm. inside diameter is added to one drop of a one per cent suspension of cells. The mixture is shaken and placed for one hour in a water bath or incubator at C. and then centrifuged for one minute at 5 revolutions. The result is read the naked eye and if agglutination is absent it is checked by placing a drop of the mixture on a slide and viewing the microscope. Crossmatching is done in the same way using the serum of the recipient and the cells of the donor. The purpose of this presentation is to report observations which indicate: () that there are antigenic differences in the human Rh factor of different individuals and () that the Rh factors in man and in the acacus rhesus are not identical.* It is known that human anti Rh serums vary in their ability to react blood specimens of different persons. ' 8 Table is a record of reactions of three serums (Sl, S, S4) 9 blood specimens. were agglutinated by all three serums and 44 were not agglutinated by any of the serums, an agreement in 4 or 84 per cent. Six specimens were clumped by only one serum, three * The existence of qualitative differences in the Rh agglutinogens in human Rhpositive blood was first established by Wiener (8); similar differences in the Rh property in the bloods of man and rhesus monkey were reported by Landsteiner and Wiener (). Downloaded from on 8 January 8

Rh BLOOD FACTOR 4 by S and three by S4. Twentysix specimens were clumped by two serums. The fact that some of the bloods were clumped by the weaker serums and not at all by the strongest serum No.

4 Rh BLOOD FACTOR 4 by S and three by S4. Twentysix specimens were clumped by two serums. The fact that some of the bloods were clumped by the weaker serums and not at all by the strongest serum No., indicates that the differences in the agglutinating properties of the serums are not merely quantitative. The relatively low number of Rhf in this series is due to the fact that included in it is a number of selected cases of mothers of babies erythroblastosis and it therefore does not represent a true random series. In a random series of determinations serum No. failed to agglutinate only four specimens which were clumped by another serum. It reacted about 85 per cent of the specimens. Serum No. reacted per cent and serum No. 4 8 per cent. Although the 9 blood specimens which are included in the table do not represent a random series, they were used because each specimen was tested all three serums at the same time. TABLE COPARISON OP THREE HUAN ANTiRh SERUS NUBER OP BLOOD SPECIENS S S S Total Total Rh, 8% Rh, % Number of specimens agglutinated by one or more of the serums, 5 (.5%). Number of specimens agglutinated by each serum: 5 agglutinated 48 (9.4% specificity5.% sensitivity) 5 agglutinated 8 (84.% specificity.% sensitivity) S4 agglutinated 4 (9.4% specificity.4% sensitivity) Sensitivity of serum: ability to react the Rh factor in general ( specimens of blood selected at random). Specificity of serum: ability to react the Rh factor in specimens of blood known to contain the Rh factor. What is the cause for the difference in the reactions? It could be due to differences in the agglutinating ability of the serums or in the agglutinability of the red cells or to both. Table records an adsorption experiment which throws some light on this question. Three antirh serums were used (S, S, S). The ORh cells were divided in three groups: ORh () were those clumped by all three serums, ORh () were clumped by two serums (S, S) and ORhf () were clumped by one serum (S). Technic of adsorption: One cc. of serum and cc. of packed cells were mixed for the adsorption. After standing one hour at C. and being shaken every fifteen minutes during that time, the mixtures were centrifuged and the supernatant fluids were used. S was each of the three varieties of cells until the agglutinins for the homologous cells were removed and was then tested for agglutinins for the other two varieties of cells. S was ORh () and ORhf () only. Downloaded from on 8 January 8

5 48 I. DAVIDSOHN AND B. TOHARSKY ANALYSIS OF RESULTS Cells ORh () which were clumped by all three serums removed also the agglutinins for the other cells (ORh () and ORh ()). Cells ORh () and ORh () removed theagglutininsforthemselvesandonlyoccasionallyfororht ()andorh () respectively and never for ORh (). This suggests antigenic differences in the Rh factor. It is possible that the Rh factor consists of several fractions e.g. Rh ; Rh, Rh s, etc. Some persons may have all these fractions, others only some of them. TABLE ANTIGENIC ANALYSIS OF HUAN Rh FACTOR ANTIRh SERU CELLS S S S S Rh S Rh S Rh S Rh S Rh ORh ORh ORh 8* * The numbers indicate the highest dilution of the serum which still clumped the cells. The symbol indicates absence of agglutination in the lowest dilution (:.5). t Adsorption may or may not occur in these instances. It varies cells from different individuals. TABLE 4 COPARISON OF Rh FACTOR IN HUAN AND IN ACACUS RHESUS BLOODS t ot HUAN ANTIRh SERU (ISOIUNIZATION) GUINEA PIG ANTIRH SERU (IUNIZED WITH. RHESUS CELLS) CELLS NORAL HUAN SERU Adsorbed cells ORh No. No. No. (9) () GUINEA PIG SERU Un Un Adsorbed cells: ORh No. No. No. (9) () ORh ORh ORh (9) (lll) * rhesus cells. * The numbers indicate the highest dilution of the serum which still clumped the cells. The symbol indicates absence of agglutination in the lowest dilution (::5). Is the Rh factor in man identical the factor in the acacus Rhesus? Table 4 records an attempt to answer this question. Adsorption of a human antirh serum human red cells removed the antirh agglutinins for human cells completely or partly, while the agglutinins for monkey cells were completely removed. The remnants of agglutinins for the cells of the monkey after adsorption are probably identical those present in the normal serum. The differences in adsorption of the agglutinins for human Downloaded from on 8 January 8

6 Rh BLOOD FACTOR 49 blood were of the kind that had to be expected from the previously described experiments. Adsorption monkey cells removed the antirh agglutinins for the monkey cells completely, but not for the human cells. The result suggests antigenic differences between the Rh factor in man and in the monkey. This was confirmed by adsorption experiments an antirhesus immune serum produced by immunization of a guinea pig macacus rhesus blood. Adsorption human blood removed the agglutinins for human cells, but not for monkey blood, while the blood of the monkey removed completely the agglutinins for human and monkey blood. In both instances the antigen removed completely the homologous antibody. TABLE 5 COPARISON OF HUAN ANTIRI SERUS WITH A GUINEA PIG IUNE ANTIRHESUS SERU NUBER OF BLOOD (4%) REACTIONS WITH HUAN ANTIRh SERUS 4 (%) 4 _ 4 (%) REACTIONS WITH RHESUS SERU 4 (%) Complete agreement, 5 cases (8%). Complete disagreement, 5 cases (8.%). Partial disagreement, cases (9.8%). Production of antirhesus immune serum in guinea pigs According to the method of Landsteiner and Wiener, guinea pigs were injected intraperitoneally twice ( cc. of whole pooled monkey blood, washed twice and the cells brought up to volume saline). The injections were made five days apart. A week after the last injection the guinea pigs were bled and the serum was titrated Rh and Rh cells. Of the twelve guinea pigs injected five showed slight differences in titers for Rhf and Rh cells. However only one guinea pig showed a sufficiently pronounced difference to permit its practical use. How do human antirh serums compare immune antirhesus serums produced in guinea pigs and rabbits? Comparison of reactions of three human antirh serums a guinea pig immune serum in a series of sixtyone human bloods (Table 5) showed complete agreement in 8 per cent of cases, complete disagreement in 8. per cent, and partial disagreement in 9.8 per cent. Human serum S () clumped 45 (4 per cent) of the specimens, S () clumped 4 ( per cent), S (4) clumped 4 ( per cent) and the guinea pig serum clumped 4 or per cent. In this group as well as in the one recorded in the next paragraph the reason for the low number of Rh positive specimens is the same as was previously stated. Twentyfive human blood specimens were tested three human antirh serums and two rabbit immune serums (Table ). Human serum () and (4) clumped 8 or Downloaded from on 8 January 8

7 44 I. DAVIDSOHN AND B. TOHARSKT per cent, S () clumped 5 or per cent, the rabbit serums clumped 9 or per cent and or 8 per cent of the blood specimens respectively. There was complete agreement in 8 cases ( per cent), complete disagreements in 4 ( per cent) and partial disagreements in ( per cent). Production of antirhesus immune serums in rabbits Eleven rabbits which had been previously immunized either acacus rhesus cells or human Rh cells and which had been rested for four months until the antihuman agglutinin titer had dropped were given daily intravenous injections of. cc.. Rhesus cells (washed three times) for a period of a week. Another series of injection was given after a rest of three weeks. The titers for Rh cells ranged from :5 to :. The titers for Rh cells ranged from :5 to :5. Various adsorption procedures were tried in order to remove tha agglutinins for the Rh cells and to leave only the agglutinins for the Rh cells. The adsorption Rh cells, inhibition Witebsky's blood group specific A and B purified substances, inhibition a mixture of A and B saliva, have thus far been ineffective. TABLE COPARISON OF HUAN ANTIRII SERUS WITH RABBIT IUNE ANTIRHESUS SERUS NUBER OF BLOOD REACTIONS WITH HUAN ANTIRh SERUS REACTIONS WITH RABBIT ANTIRHESUS SERUS (%) 5 (%) 8 (%) (8%) 9 (%) Complete agreement, 8 cases (%). Complete disagreement, 4 cases (%). Partial disagreement, cases (%). Inhibition of several serums pooled human AB serum was attempted. Two cc. of AB serum and.5 cc. of the rabbit serum were mixed and were allowed to stand at room temperature at least one hour before the titration. In one instance (rabbit ) there appeared a marked difference between the anti Rh titer and the anti Rh titer. The former was :448, the latter varied from : to : various blood specimens of Rh cells. The dilution of the rabbit serum the human serum was taken into account in figuring the dilutions of the rabbit serum. What is the significance of the disagreements between the human and the animal antirh serums? The instances where the animal serums clumped and the human serums did not, could be explained by the absence of an appropriate agglutinin in the three human serums that were employed. If we had more human serums available, there might be one or more found which would react the blood specimens that were clumped by the animal serum. On the other hand the instances in which the animal immune serums failed to detect the Rh factor in human blood suggests antigenic differences between the Rh factors in the blood of man and of the acacus rhesus. Downloaded from on 8 January 8

8 Rh BLOOD FACTOR 44 SUARY Human antirh serums differ in their sensitivity, i.e., in their ability to react specimens of blood selected at random and in their Rh specificity, i.e. in their ability to react known Rh positive bloods. It is essential to use at least three different antirh human serums for tests for the Rh factor. At least one of them must be similar to serum of our series and react about 85 per cent of unselected blood specimens. Adsorption experiments confirm the existence of antigenic differences in the Rh factors of different Rhpositive individuals. Definite antigenic differences were also found to exist between the Rh factors in man and in the acacus rhesus. REFERENCES () LANDSTEINER, K., AND WIENER, A. S.: An agglutinable factor inhuman blood recognized by immune sera for rhesus blood. Proc. Soc. Exper. Biol. & ed., 4:, 94. () DAVIDSOHN, I. AND ScHIRER, E.: Normal anti isoagglutinins. A case report. Proceedings of Chicago Pathological Society, October, 94. () WlENEK, A. S., AND PETERS, H. R.: Hemolytic reactions following transfusions of blood of the homologous groups, three cases in which the same agglutinogen was responsible. Ann. Int. ed., :, 94. (4) LEVINE, P., AND STETSON, R. E.: An unusual case of intragroup agglutination. J. A.. A., :, 99. (5) LEVINE, P., KATZIN, E.., AND BURN HA, L.: Isoimmunization in pregnancy. J. A.. A., : 858, 94; LEVINE, PH., BTJRNHA, L., KATZIN, E.., AND VOGEL, P.: The re of isoimmunization in the pathogenesis of erythroblastosis fetalis Am. J. Obst, and Gynec, 4:95,94. () (a) WITEBSKY, E., KLENDSHOJ, N., AND SwANSON, P.: Reduction or elimination of the antia antibody in O blood by means of the addition of the "A" specific substance. J. Inf. Dis., : 889, 94. (b) WITEBSKY, E., AND KLENDSHOJ, N. C.: The isolation of the blood group specific B substance. J. Exper. ed., :, 94. () LANDSTEINER, K., AND WIENER, A. S.: Studies on an agglutinogen (Rh) in human blood reacting anti Rhesus sera and human isoantibodies. J. Exper. ed., 4: 9, 94. (8) WIENER, A. S.: Hemolytic reactions following transfusions of blood of the homologous group. II. Further observations on the re of property Rh, particularly in cases out demonstrable isoantibodies. Arch. Path., : 5, 94. Downloaded from on 8 January 8

DIAGRAMMATIC REPRESENTATION OF THE RH BLOOD TYPES*

DIAGRAMMATIC REPRESENTATION OF THE RH BLOOD TYPES* ALEXANDER S. WIENER, M.D. From the Serological Laboratory to the Office of the Chief Medical Examiner of New York City The clinical pathologist and the