ANTI-HUMAN HEMAGGLUTININS AND ANTI-RH ANTIBODY GUINEA PIG AND HUMAN SERUMS*

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1 ANTIHUMAN HEMAGGLUTININS AND ANTIRH ANTIBODY GUINEA PIG AND HUMAN SERUMS* E. E. ECKER, EILEEN W. ERLANSON MACFARLANE AND T. C. LAIPPLY From the Institute of Pathology, Western Reserve University, and the University Hospitals, Cleveland, Ohio IN I. ANTIHUMAN HEMAGGLUTININS Landsteiner and Wiener 1 have shown that testserums for the Rh factor may be obtained from guinea pigs injected with the blood of Rhesus monkeys, from the blood of patients who have had hemolytic transfusion reactions from homologous bloods, or from the blood of mothers of infants with erythroblastosis foetalis. In a subsequent paper Wiener 2 stated that "while the majority of guinea pigs will be found to show differences between the sorts of blood, Rhpositive and Rhnegative, only one or two sera out of every ten will prove suitable for practical diagnosis". He further indicated that "in occasional sera containing appreciable amounts of antia and antib agglutinins it may be necessary to absorb with small amounts of group A and group B blood". Because of a lack of knowledge concerning the native hemagglutinins of guinea pig serums it was decided to test the serums of a number of these animals for antihuman hemagglutinins. Technic Washed cells from citrated or clotted human blood of known groups were used in two per cent suspension. The blood from the guinea pigs was obtained by cardiac puncture. The tests were carried out on open slides in a moist chamber at room temperature. Equal volumes of cell suspension and serum were employed. The mixtures were well stirred and examined microscopically at intervals of 1, 3 and 6 minutes. The sex and coat color of each guinea pig were recorded. The individual serums from one hundred animals were tested with over a thousand human blood suspensions. Results The serums of 66 of 1 animals tested agglutinated A cells to varying degrees; 25 of these also agglutinated B cells; and 34 gave negative results with all blood groups (see table 1). When freshly drawn, the serums which later proved negative, gave some weak nonspecific agglutinations with several bloods, even with O cells. Heating at 55 C. for 3 minutes did not destroy this effect, but it disappeared after the serums stood overnight at from 3 to 4 C. In testing for the stable heteroagglutinins, therefore, the serums were employed on the day following the bleeding. For comparison agglutinations were also carried out by the centrifugal method at low speed (58 r.p.m.). The reactions obtained were the same as on the open slides with the same bloods and testsera. The titers of the antia agglutinins were nearly all low, averaging about from 1:4 to 1:8. However, two Downloaded from by guest on December 31, 2 * Aided by a grant from the Commonwealth Fund. 8

2 ANTIRH ANTIBODY 9 colored and two white animals possessed serums with higher titers; and two, including one white guinea pig, had serums with titers as high as 1:. In only three of the serums with both antia and antib agglutinins, was the antib stronger than the antia agglutinin, and in two of these (both obtained from colored animals) the antib titer reached 1:1. These six serums with titers of heterohemagglutinins of higher than 1:8 were the only ones that might interfere in the 1:1 dilutions used to test for the Rh factor, unless first absorbed with A and B cells. Absorption tests were carried out on the normal serums and showed that the homologous hemagglutinins may be removed. However, heating at 55 C. for 3 minutes did not alter the hemagglutinative capacities of the serums. Ten serums (seven from colored and three from white animals) showed marked lytic effects: six on A cells only; one on A and B cells; one on certain A and on certain cells; one on certain B and certain O cells; and one on cells of all four groups. These lysins were likewise not inhibited by heating at 55 C. AntiA lysins were present in one of the otherwise negative serums obtained from a white guinea pig. Colored White Totals TABLE 1 SHOWING ANTIHUMAN HEMAGGLUTININS OP 1 NORMAL GUINEA PIG SEBUMS TYPE OF COAT TOTAL ANTIA ONLY ANTIA AND ANTIB NO HETERO HEMAGGLUTINS PER CENT NEGATIVES There was no difference in the distribution of heteroagglutinins between the male and female guinea pigs, but a greater proportion of the serums obtained from white than of colored pigs were negative (table 1). About 25 per cent among guinea pigs with colored coats, were negative, while about 51 per cent of the white animals had no heteroagglutinins. The variation in titer was as great in the two kinds of guinea pigs, but it appears as though the heteroagglutinative character is less developed in the white guinea pigs. It is not known whether native heteroagglutinin titers vary in the lifetime of the animal or remain constant. In order to simplify the procedure it is therefore recommended that the blood of guinea pigs be examined prior to immunization with Rhesus monkey blood and to select only those showing no heteroagglutinins. White guinea pigs were therefore chosen and those found to have negative serums were injected with Rhesus blood. A summary of these results reveals that about twothirds of normal guinea pigs have serums containing antihuman hemagglutinins, chiefly antia. About twentyfive per cent also had antib hemagglutinins. Ten in one hundred lysed human cells, usually those of group A, but occasionally cells of other Downloaded from by guest on December 31, 2

3 17 B. E. ECKER, E. W. E. MACFARLANE AND T. C. LAIPPLY groups. Only six serums in one hundred possessed heterohemagglutinins with a titer greater than 1:8. More white guinea pigs than those with colored coats were found to lack antihuman hemagglutinins, although for statistical reasons still larger numbers should be examined. For the production of antirh test serums white guinea pigs were therefore chosen with serums free from antihuman hemagglutinins. II. AN ANALYSIS OP THE ANTIRhANTIBODY IN GUINEA PIG AND HUMAN SERUMS The finding that more white guinea pigs (than those with colored coats) lack antihuman hemagglutinins led to the selection of these animals free from antihuman hemagglutinins for the production of antirh testing serums. Technic The method of immunization was essentially the same as reported by Landsteiner and Wiener 2, except that some animals were also injected by the subcutaneous route. The inactivated sera of three injected animals, heated at 55 C. for 3 minutes, strongly agglutinated human red cells of all groups. They were, therefore, analyzed separately for their titers of antirh agglutinins. Three bloods of known content of Rh factor were available, namely: Group O Rhnegative, group A Rhpositive, and group AB'Rhnegative. The serum of one immunized animal (No. 57) showed an agglutinin titer of 1: with both group and group AB Rhnegative cells. A moiety of this serum was then absorbed with A and B cells, and after absorption the antirh titer was found to be 1:8 with the same cells (table 2). Two group cell suspensions, in which the Rh factors were unknown, were then used against both the absorbed and the unabsorbed serum No. 57. One serum gave titers of 1:32 and 1:8 respectively and the other titers of 1:64 and 1:32 respectively. It was, therefore, thought that the first was Rhnegative and the second Rhpositive. Unknown cells of group A and group B types agglutinated at 1:64 and 1:128 with the unabsorbed serum and 1:32 with both in the case of the absorbed serum. Both types of cells were probably Rhpositive cells. With the absorbed serum No. 57 group cells (No. 3) gave no reaction with dilutions weaker than 1:8, and this blood was later concluded to be Rhnegative by definition (see table 2). Another group (No. 4) (see table 1) was selected at random and was tested as before with different dilutions of the test serum No. 57. The unabsorbed serum gave strong agglutination in all dilutions up to 1:32, a marked positive reaction at 1:64 and a reaction at 1:128. With the absorbed serum a negative reaction was obtained at a dilution of 1:64 and a weak positive at 1:32. This blood then was considered to be Rhpositive by definition. A group A and a group B blood (Nos. 5 and 6) (table 1) were tested as before. Both these cell suspensions gave an antirh titer of 1:128 in the unabsorbed serum (weak positive) and a titer of 1:32 in the absorbed serum (weak positive) so that they were also considered as Rhesus positive by definition. From these preliminary tests it appeared that all human red cells were "Rhesus positive" with this particular guinea pig serum when used undiluted, absorbed and unabsorbed. A dilution of 1:1 in the case of the absorbed serum Downloaded from by guest on December 31, 2

4 ANTIRH ANTIBODY 171 was needed to give a negative reaction with Rhnegative cells and this is the dilution of absorbed guinea pig test serum recommended by Landsteiner and Wiener. Some cells seem to be four times as sensitive to the antirh serum as others. The latter are the Rhnegative cells. The former are classified as positive and give a titer of 1:32 to the absorbed serum. Gallagher and Jones, 3 using a dilution of 1:8 absorbed guinea pig serum, concluded that 15 per cent were Rhesus negative. They do not show nor mention what reactions were obtained with greater concentrations of the serum. The serum from guinea pig No. 61, before absorption with A and B cells, gave, when mixed with known Rhpositive group A cells, a titer of 1:. After absorption this serum gave a negative reaction with the same cells even when used in full strength. Therefore, all the antirh factor was removed in the TABLE 2 ANTIRH TITER IN GUINEA PIG SERUMS FROM PIGS Nos. 57, 61 AND 63 BLOOD (2%)SERUM Gr. O (RhNeg) #57 unabs Gr. O (RhNeg) #57 abs Gr. AB (RhNeg) #57 unabs... Gr. AB (RhNeg) #57 abs Gr. O (Rh?) #57 unabs Gr. O (Rh?) #57 abs Gr. O (Rh?) #57 unabs Gr. O (Rh?) #57 abs Gr. B (Rh?) #57 unabs Gr. B (Rh?) #57 abs Gr. AB (Rh. Neg) #61 unabs... Gr. AB (Rh. Neg) #61 abs Gr. A (Rh. Pos) #61 unabs Gr. A (Rh. Pos) #61 abs Gr. A (Rh. Pos) #63 abs 1:4 1:8 1:12 I SERUM DILUTIONS 1: 1:32 course of absorption. However, this serum was not drawn until from 4 to 5 weeks after all injections of monkey cells were discontinued. Absorbed serum No. 57 with these same group A cells gave a titer of 1:32. The antirh in serum No. 61 was therefore too weak for use. Absorbed serum No. 63 at a dilution of 1:4 gave a negative reaction with group (known Rhnegative) and a positive test with a group A (known Rhpositive) blood. This serum was also considered too weak for routine use. 1:64 1: Downloaded from by guest on December 31, 2 Agglutinogen and AntiA Agglutinin According to Boyd 4 the Rhesus monkey red cells contain agglutinogen M and the serum of this monkey may contain the antia agglutinin. Injected guinea pigs may, therefore, develop the antim agglutinin as well as a species

5 172 E. E. ECKER, E. W. E. MACFARLANE AND T. C. LAIPPLY agglutinin (antiprimata). If after absorption with A and B cells some antim remains it would be expected that type M cells would test positive with the antirh serums. It was found, however, that Rhnegative bloods occurred in all groups (see table 3). Distribution of RhPositive and RhNegative Bloods One hundred and twentyfive bloods of known groups were tested with the pooled antirh serums from guinea pigs Nos. 57 and 63. The serums were absorbed with A and B cells and diluted 1:1. Bloods negative with this serum were tested with the undiluted pooled antirh serum (Nos. 57 and 63). Table 3 shows the results obtained with 125 human bloods. The percentage of group B bloods in the sample is noticeably high, 18.4 per cent. The pooled and diluted antirh guinea pig serum gave 19 negative reactions in the 125 bloods examined or 15.2 per cent. This agrees well with the findings of Landsteiner and Wiener, 5 Landsteiner, Wiener and Matson, 6 Levine, Vogel, TABLE 3 ANTIRH TESOS ON 125 HUMAN BLOODS OF KNOWN GBOUP AND TYPE (SERUMS #57 AND 63 POOLED) DILUTED 1:1 ) Nos. in Rh in Type Nos. in Rh in 48 6 M 36 3 A GKOUP B 23 1 N 23 4 AB 1 2 TOTALS Katzin and Burnham, 7 and Gallagher and Jones. 3 Negative bloods belonged to all groups and types (table 3) including 4 type N bloods. Only one group B blood has been found to be negative. Centrifuged with an equal volume of undiluted pooled serum (Nos. 57 and 63) all 19 gave strong agglutinations, 3 in 15 cases and 2 in 4 cases. With an equal volume of unabsorbed pooled serum diluted 1:1 thirteen were negative and a weak reaction was obtained in 6 cases (O, ABN, ON, AN, B, O) Downloaded from by guest on December 31, 2 Removal of AntiRh by Absorption A portion of the pooled absorbed serum (Nos. 57 and 63) was absorbed with a quarter of its volume of strongly Rhesuspositive human red cells (No. 12 BM). After absorbing for a half hour at room temperature blood No. 12 and two other positive bloods gave only weakly positive reactions. After overnight absorption at 34 C. no reactions were obtained with the absorbed serum and known Rhpositive cells, that is, cells that had been agglutinated by the same serum before absorption, including No. 12.

6 ANTIRH ANTIBODY 173 Erythroblastosis Foetalis. Serum Titers Case 1. The bloods of father, mother and fetus were all negative with the guinea pig antirh pooled test serum in a 1:1 dilution; at a 1:5 dilution all three were weakly positive (), and the undiluted pooled serum gave strong agglutination with all. The baby's cells were not affected by centrifugalization for 3 minutes with an equal volume of the mother's serum. Left overnight at 34 C. the baby's cells were lysed by the mother's serum, but small distorted clumps of cells still remained. The mother's serum had no effect on seven different group O cells after centrifugation. Case 2. The reaction of 2 drops of the mother's serum on 1 drop of cell suspension after centrifugation with seven different bloods is shown in table 4 and compared with the reaction of the 1:1 pooled antirh guinea pig serum. Two Rhnegative bloods with test serum were also negative to the human serum, but the five Rhpositive bloods all gave a weaker positive reaction with the human serum, although 2 drops instead of 1 were used; one of the Rhpositive bloods gave a doubtful () reaction with the human serum. Gallagher and TABLE 4 SERUM FROM ERYTHROBLASTOSIS FOETALIS MOTHER COMPARED WITH ANTIRH GUINEA PIG POOLED SERUM Group Type Guinea pig serum 1:1 Mother's serum II B M BLOOD NO N 125 B N Jones found, in comparing the reactions of two human "antirh" serums with guinea pig antirh serums, that one gave a corresponding number of negative reactions with the same series of bloods, while the other gave many more negatives than the guinea pig serums. Throughout the literature human "antirh" serums from the mothers in cases of erythroblastosis foetalis are reported to give more negative reactions than diluted guinea pig test serums. Wiener explains this as due to a series of allels Rhi, Rh2, etc. From the data here present this might be due to the lower titer of the "antirh" in the human serum. If, as has been suggested above, the "antirh" agglutinin is probably "anti Primata" agglutinin, it would appear that the sensitivity (or amount) of the responsible antigen varies on a broad gradient, particularly in the Whites or Europoids. Unless the cells of the fetus are very strongly Rhpositive and those of the mother very weakly so, the "antirh" produced in the mother's serum will not reach the titer of the true antirh in a guinea pig serum at 1:1dilution. In the second case of erythroblastosis foetalis the titer of the "antirh" in the mother's serum was only 1:4, while in the diluted test serum it was 1:, using the strongly positive cells # 12 (BM) in order to determine the titer. Downloaded from by guest on December 31, 2

7 174 E. E. ECKER, E. W. E. MACFARLANE AND T. C. LAIPPLY CONCLUSIONS Twothirds of normal guinea pigs have serum containing antihuman hemagglutinins, chiefly antia. More white guinea pigs than colored guinea pigs were found to lack antihuman hemagglutinins. For the production of antirh test serums it is recommended that white guinea pigs free from antihuman hemagglutinins be chosen. The antirh agglutinin in guinea pigs differs from other artificial agglutinins such as the antim and antin in that it reacts with all human cells unless diluted to a specific strength. The Rh antigen seems to be present in the red cells of all individuals but in some it is much less sensitive. Even the undiluted antirh guinea pig serum differentiated the two classes of red cells, one of which gave a titer with a strong serum two or more times as high as did the other class of less sensitive cells. The latter are the Rhnegative cells. For general use the guinea pig antirh serum should be diluted (1:1). (1) LANDSTEINER, K., AND WIENER, A. S.: An agglutinable factor in human blood recognized by immune sera for rhesus blood. Prov. Soc. Exper. Biol. & Med., 43: 223, 194. (2) WIENER, A. S.: Hemolytic transfusion reactions. III. Prevention with special reference to the Rh and crossmatch tests. Am. J. Clin. Path., 12: 32, (3) GALLAGHER, F. W., AND JONES, L. R.: Preparation and use of Rh testing serum, J. Immunol., 46: 9, (4) BOYD, W. C: Blood Groups, Tabulae Biologicae XVII. The Hague 1939, pp REFERENCES (5) LANDSTEINER, K., AND WIENER, A. S.: Studies on an agglutinogen (Rh) in human blood reacting with antirhesus sera and with human isoantibodies. J. Exper. Med., 74: 39, (6) LANDSTEINER, K., WIENER, A. S., AND MATSON, ALBIN, G.: Distribution of the Rh factor in American Indians. J. Exper. Med., 76: 73, (7) LEVINE, P., VOGEL, P., KATZIN, E. M., AND BURHNAM, L.: Pathogenesis of erythroblastosis fetalis: Statistical evidence. Science, 94: 371, LEVINE, P.: The pathogenesis of fetal erythroblastosis. N. Y. State Jour, of Med., 42: , Downloaded from by guest on December 31, 2