SEROLOGICAL CHARACTERISTICS OF ABO BLOOD GROUP ANTIBODIES IN RELATION TO THE HEMOLYTIC DISEASE OF THE NEWBORN

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1 Journal of the Japan Society of Blood Transfusion 15 (3): , 1968, SEROLOGICAL CHARACTERISTICS OF ABO BLOOD GROUP ANTIBODIES IN RELATION TO THE HEMOLYTIC DISEASE OF THE NEWBORN by Hayato HASEKURA (Department of Legal Medicine, Tokyo Medical and Dental University, Director: Prof. Dr. T. Miki) INTRODUCTION Anti-A and anti-b isoantibodies in the sera of mothers with ABO incompatible infants show diversity in their serological characteristics through pregnancies or deliveries of incompatible infants which are considered to serve as immunological stimulus similar to the incompatible blood transfusions or immunization with blood group specific substances. Presence of such antibodies has been regarded as the evidence to show that the person was immunized with A or B antigen and further as the cause of severer hemolytic reactions in the next incompatible pregnancy or incompatible transfusion. For this reason, such antibodies have been called immune anti-a or anti-b, to be distinguished from so-called natural anti-a or anti-b. Main characteristics of immune anti-a or anti-b so far pointed out are: (1) increased titer, especially of the incomplete antibody, i.e., enhancement of the agglutinin titer by the use of colloid medium, proteolytic enzyme-treated cells, or anti-globulin technic (Coombs' test), (2) incomplete agglutinin activity which remains after the partial neutralization with blood group specific substances of animal or human origin, (3) stability to heating, especially of incomplete antibody activity, (4) ability to hemolyse A or B red cells, and (5) presence of mercaptoethanol-stable antibody activity. Proteins possessing known antibody activities are called immunoglobulins; there are at least three major classes among them referred to as r G, r A and Y M immunoglobulins. The ABO antibody activities are associated with all the three classes. Natural anti-a and anti-b mainly reside in y M, and after stimulation with ABO substances y G antibodies tend to be predominantly produced. But this is not necessarily the case. Natural or immune antibodies do not always signify y M or y G immunoglobulins, although the terms natural and immune are preferably used in discussing on the blood group incompatibility between mother and fetus. In the present report, various tests for anti-a and anti-b were compared on the sera with or without antigenic stimulation-normal males, normal mothers, moderately stimulated mothers with heavily jaundiced infants, and strongly stimulated mothers with fatally affected infants, and the heterogeneity of anti-a and anti-b antibodies was discussed in relation to the serological tests for the hemolytic diseases of the newborn due to ABO incompatibility.

2 88. Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 MATERIALS (a) Sera of normal males: About 10 ml of blood was taken from 20 healthy males aged 23-41, who had no history of blood transfusion, antitoxin injection, blood diseases, or other known causes of immunization against A or B antigen. The sera were separated and inactivated by incubation at 56C for 30 minutes. Red cells were submitted to blood grouping. Group A: 7 cases, group B: 6 cases, group O: 7 cases. (b) Sera of normal mothers: 86 mothers whose normally born infants showed no sign of hemolytic disease, such as heavy jaundice or anemia, were bled 10 ml each, several days before or after delivery. Group A: 36 cases, group B: 19 cases, group O: 31 cases. (c) Sera of mothers with heavily jaundiced infants: Five mothers, whose ABO incompatible newbornn infants suffered heavy icterus praecox (serum total bilirubin nearly 20mg/dl) but recovered without exchange transfusion, were bled after 2-40 days after delivery. Group A: 1 case, group O: 4 cases. (d) Sera of mothers with fatally affected infants: Eight mothers, whose ABO incompatible infants suffered fatal hemolytic disease, such as stillbirth, death from kernicterus, cerebral palsy, or heavy jaundice which was cured by exchange transfusion, were bled 3 days to 3 months after delivery or in the 3rd to 9th month of pregnancy. All cases were group O. In all the sera tested, no irregular antibody was detected with indirect anti-globulin test. METHODS (a) Titration: The sera were submitted to the two-fold serial dilution with saline 0.1 ml each in test tubes and were mixed with the same volume of 2% red cell suspension in saline. The cells were obtained daily from the designated group A (N.K.) and group B (H.H.) laboratory staff members. The mixtures were incubated at 37C for 30 minutes and were read for agglutinations after a slow centrifuge at 1,000 r.p.m. for 1 minute. Macroscopic end-point expressed in the number of tubes was recorded as the titer; titer of the sera showing negative reaction in the original solution was recorded as 0. Anti-globulin test was performed on tubes which showed negative or microscopic reaction in saline. (b) Neutralization test: Sera were mixed with the same volume of A, Se (N.K.) saliva or B. Se (H.H.) saliva, kept at room temperature for 30 minutes, and submitted to the titration as above. Both saliva were boiledd and centrifuged soon after the harvest to remove mucin, cell residues, and enzyme activity; the supernatants had the agglutination inhibition titer of 1:4,000 against anti-a or anti-b with the adjusted titer of 1:8. (c) Heating test: Sera were diluted with twice the volume of saline to prevent aggregation and was heated in the water-bath of 70C for 10 minutes. Titration was performed as above. (d) Mercaptoethanol-treatment test: According to Mollison32), sera were directly mixed with the same volume of 0.2 M2-mercaptoethanol (ph7.4), incubated at 37C for 2 hours, and dialysed with buffered saline overnight. Titration method was the same as in (a). (e) Hemolysis: The hemolytic tests were made in barbital buffer (ph7.2) to which Mg-ions were added. 0.1 ml of inactivated and serially diluted sera were mixed with the same volume of 1% suspension of group A or B cells and the same volume of undiluted AB, se, fresh human serum as the source of compliment, incubated at 37C for 1 hour, and read for hemolysis (-toiii) macroscopically. Complete hemolysis was taken as the end point. (f) Calculation of titers: Although the sera were diluted twice with saliva or mercaptoethanol in neutralization test or mercaptoethanol-treatment test, those sera were regarded as the original solution and titrated. Macroscopic weak agglutination (+), microscopic agglutination (+), or negative (-) reaction in the original solutionn was expressed as titer 2,1,0, respectively. For heating test, the sera were diluted 1:3, and +, +, or-reaction in the original solution was regarded as titer 2.5, 1.5, or 0, respectively. When hemolysis of undiluted sera was II, +, or -, titers were recorded as 0, -1, or -2, respectively. (g) Statistical analyses: In the calculation of the mean titers, the central values were used; e.g., the seraa with the titer 3 actually possess the titers between 3 and 4, so the central value of 3.5 was multiplied by the number of cases. The significance of the differences in the mean titers was calculated by the Student's t-test. F-tests were performed to see the differences in the variances. compared by the y2-test. 1. Antibody titers of untreated sera. Percentages of the sera sensitive or stable to each treatment were RESULTS (a) Saline method. Saline titers of untreated sera are shown in Table I (See page 98) and Figure 1

3 The means and the standard deviations of titers in this and the following tests are also shown in the tables. Titers of normal sera distributed from 5 to 11 (1:16-1,024), and the mode was 8 (1: 128). Titers of normal mothers' sera also ranged from 5 to 11 (1:16-1,024), and the mode was 8 (1: 128). Sera from mothers with heavily jaundiced infants ranged in titers 8-11 (1:128-1,024). Sera from mothers with fatally affected infants ranged from 10 to 14 (1:512-8, 192). (b) Anti-globulin method. The anti-globulin titers of untreated sera are also shown in Table I (See page 98)and Figure 1. Titers of sera from normal males ranged in 5-11 (1: 16-1,024), the mode being SALINE TITER ANTI-GLOBULIN TITER Fig.1. Distribution of anti-a, B agglutinin activity in untreated sera. Lower graph: normal cases. (Blank: normal males. Dappled: normal mothers.) Upper graph: hemolytic cases. (Shaded: mothers with heavily jaundiced infants. Dark: mothers with fatally affected infants.) 8 (1: 128). Titers of normal mothers' sera ranged in 6-13 (1: 32-4, 096), with the mode of 10 (1: 512). Titers of sera from mothers with heavily jaundice infants ranged in (1: 512-2, 046); titers of sera from fatally affected infants ranged in tubes (1: 1,024-16,384). (c) Comparison of the mean titers: There was a weakly significant difference between the mean titers of the normal mothers and the mothers with heavily jaundiced infants (d.f.=120, saline method: t=2.56, 0.05>p>0.01, anti-globulin method: t=2.54, 0.05>p>0.01); the difference of the mean

4 90 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 titers between the normal mothers and the mothers with fatally affected infants was highly significant (d.f.=124, saline method:t=9.62, p<0.01, anti-globulin method: t=11.75, p<0.01). The mean titers of the mothers with heavily jaundiced infants and that of the mothers with fatally affected infants differed with low significance (d.f.=12, saline method: t=2.24, 0.05>p>0.01, anti-globulin method: t=2.29, 0.05>p>0.01). 2. Titers of neutralized sera Distributions of agglutinin titers of neutralized sera in saline and in anti-globulin methods are shown in Table II (See page 98-99) and Figure 2. (a) Saline method. Titers of normal male sera ranged in 0-5 (negative-1: 16) and the mode was 0 (negative). Titers of normal mothers' sera had the range of 0-3 (negative-1: 4) and the mode of 0 (negative). Titers of sera from mothers with heavily jaundiced infants ranged from 3 to 6 (1: 4-32), and those from mothers with fatally affected infants had a wide range of 0-9 (negative-1: 256). (b) Anti-globulin method. Titers of normal male sera ranged from 0 to 6 (negative-1: 32), and the mode was 0 (negative). Those of normal mothers had the range of 0-9 (negative-1: 256) with the mode of 0 (negative). SALINE TITER ANTI-GLOBULIN TITER Fig.2. Distribution of anti-a, B agglutinin activity in neutralized sera. Lower graph: normal cases. (Blank: normal males. Dappled: normal mothers.) Upper graph: hemolytic cases. (Shaded: mothers with heavily jaundiced infants. Dark: mothers with fatally affected infants.) Among the normal samples, the significant difference was found between the titer of groups A and B and the titer of group O, i.e., in normal males, the sera with titers below 2 (1: 2) included more groups A and B, and the sera with higher titers (1:2 or above) included more group O individuals (d.f.=3, x2=199.2, p<0.01), and also in normal mothers, the sera with lower titers (1: 2 or below) included more groups A and B, and the sera with higher titers (1: 4 or above) included more group O (d.f.=3,

5 x2=43.2, p<0.01). Calculation of the separate means showed that in normal males the mean of groups A and B was 1.73 (1: 1.66) and of group O was 3.00 (1: 4.00); in normal mothers mean titers were 1.88 (1: 1.84) for groups A and B and 4.65 (1: 12.5) for group O. Titers of sera from mothers with heavily jaundiced infants ranged from 4 to 7 (1: 8-64), and those from mothers with fatally affected infants ranged in 5-14 (1:16-8,096). (c) Comparison of the mean titers. Between the mean titers of the normal mothers and the mothers with heavily jaundiced infants, there was a highly significant difference by saline method but not by antiglobulin method (d.f.=120, saline method: t=15.4, p<0.01, anti-globulin method: t=0.91, p>0.05). Between the mean titers of the group O normal mothers and the mothers with heavily jaundiced infants, there was a highly significant difference by saline method (d.f.=65, t=13.1, p<0.01), but a low significant difference by anti-globulin method (d.f.= 65, (t=2.42, 0.05>p>0.01). The mean titers of the normal mothers and the mothers with fatally affected infants differed with high significance (d.f.=124, saline method: t=22.4, p<0.01, anti-globulin method: t=8.98, p<0.01). The titers of the mothers with heavily jaundiced infants and that of the mothers with fatally affected infants were compared; by the saline method the variances differed with low significance (d.f.=8/4, F=8.00, 0.05>p>0.01) but the mean titers showed little difference (d.f.=12, t=0.41, p>0.05); by the anti-globulin method, the difference of the mean titers was significant (d.f.=12, t=2.43, 0.05> p>0.01). 3. Heating test Distributions of agglutinin titers of heated sera in saline and in anti-globulin methods are shown in Table III (See page ) and Figure 3 (See page 92). (a) Saline method. Sera from normal males had titers with the range of (negative-1: 6) and the mode of 0 (negative). In normal mothers, the range was (negative-1: 48) and the mode U (negative). Significant difference was observed between blood groups; more sera with completely heatinactivated antibodies belonged to groups A and B, and the sera that retained titers 1.5 or more belonged to more group O than to groups A and B (n=3, x2=8. 43, 0.05>p>0.01). The mean of groups A and B was 1.29 (1: 1.22), and that of group O was 1.90 (1: 1.85). Mothers with heavily jaundiced infants had a titer-range of (1: 1.5-3) and those with fatally affected infants had a range of (negative-1: 12). (b) Anti-globulin method. Sera from normal males had the range of (negative-1: 24) with the mode of 0 (negative), and sera from normal mothers ranged from 0 to 6.5 (negative-1: 48), with the mode of 0 (negative). Significant difference was found between groups A and B and group O: the sera whose titers were decreased to 1. 5 or below included more groups A and B and sera which retained titers of 2.5 or over included more group O (d.f.=3, x2=22.0, p<0.01). The mean of groups A and B was 2.16 (1: 2.24) and of group O was 3.45 (1: 5.46). Sera from mothers with heavily jaundiced infants ranged in (1: 1.5-6) and those from mothers with fatally affected infants had the range of (1: 3-192). (c) Comparison of the mean titers. The mean titers of the normal mothers and the mothers with heavily jaundiced infants were not distinguished by the heating test (d.f.=120, saline method: t=1.43, p>0.05, anti-globulin method: t=0.06, p>0.05), and this was also the case in the mean titers

6 92 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 of group O normal mothers and the mothers with heavily jaundiced infants (d.f.=65, saline method: t=0.87, p>0.05, anti-globulin method: (t=0.93, p>0.05). As the variances of the saline titers of the normal mothers and the mothers with fatally affected infants differed significantly (d.f.=8/116, F=2.68, p<0.01), t-test was not done; the mean anti-globulin titers differed with high statistical significance (d.f.=124, t=5.75, p<0.01). Comparison between the titers of the mothers with heavily jaundiced infants and that of the mothers with fatally affected infants was performed; variances of the saline titer differed with high significance (d.f.=8/4, F=14.81, p<0.01) and the mean titers were nearly the same high; the difference was weakly significant between the variances of the anti-globulin titers (d.f.=8/4, F=7.39, 0.05>p>0.01) and tentative t-test gave a weakly significant difference of the mean titers (d.f.=12, t=2.34, 0.05>p>0.01). SALINE TITER ANTI-GLOBULIN TITER Fig.3. Distribution of anti-a, B agglutinin activity in heated sera. Lower graph: normal cases (Blank: normal males. Dappled: normal mothers.) Upper graph: hemolytic cases. (Shaded: mothers with heavily jaundiced infants. Dark: mothers with fatally affected infants.) 4. Mercaptoethanol-treatment test Distributions of agglutinin titers of mercaptoethanol-treated sera are shown in Table IV (See page ) and Figure 4 (See page 93). (a) Saline method. Titers of normal male sera distributed in the range of 0-4 (negative-1: 8) with the mode of 0. The mean of groups A and B only was 0.73 (1: 0.83) and that of group O was 2.86 (1: 3.62). This difference was significant (d.f.=25, t=4.1, p<0.01). Sera from normal mothers had the titer-range of 0-5 (negative-1: 16). The mean of groups A and B was 0.81 (1: 0.88), and the mean of group O was 2.16 (1: 2.24); there was a significant difference between them (d.f.=115, t=5.38, p<0.01).

7 Titer of sera from mothers with heavily jaundiced infants ranged in 4-6 (1: 8-32) and those from mothers with fatally affected infants ranged from 5 to 10 (1: ). (b) Anti-globulin methods. Titers of normal male sera ranged from 0 to 9 (negative-1: 256). The mean of groups A and B only was 2.50 (1: 2.53), and of group O was 6.57 (1: 14.7); this difference was significant (d.f.=25, t=3.98, p<0.01). Sera from normal mothers had the wide titer-range of 0-10, the double mode of 3 (1: 4) and 6-7 (1: 32-64). The separate means of groups A and B (3.59, 1: 6.02) and group O (6.60, 1: 48.4) differed significantly (d.f.=115,t= 6.62, p<0.01). Titers of sera from mothers with heavily jaundiced infants ranged in 6-11 (1: 32-1,024), and sera from fatally affected infants ranged in 9-14 (1: 256-8, 192). (c) Comparison of the mean titers. The differences of the mean titers of the normal mothers and the mothers with heavily jaundiced infants were highly significant (d.f.=120, saline method: t=5.69, p<0.01, anti-globulin method: t=3.39, p<0.01) as well as the difference of the mean saline titer of the normal mothers and the mothers with fatally affected infants (d.f.=124, saline method: t= 10.90, p<0.01); the difference of the variances of anti-globulin titers was weakly significant (d.f.=116/8, F=3.42, 0.05>p>0.01) and the mean titers far differed (d.f.=124, t=7.02, p<0.01). Between the mean titers of the group O normal mothers and the mothers with heavily jaundiced infants, there was a highly significant difference (d.f.=65, saline method: t=4.37, p<0.01, anti-globulin method: t=2.91, SALINE TITER ANTI-GLOBULIN TITER Fig. 4. Distribution of anti-a, B agglutinin activity in mercaptoethanol-treated sera. Lower graph: normal cases. (Blank: normal males. Dappled: normal mothers.) Upper graph: hemolytic cases. (Shaded: mothers with heavily jaundiced infants. Dark: mothers with fatally affected infants.) p<0.01); highly significant difference was also observed between the mean titers of the group o normal mothers and mothers with fatally affected infants (d.f.=69, saline method: t=9.47,

8 94 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 p<0.01, anti-globulin method: t=7.89, p<0.01). There were significant differences between the mean titers of the mothers with heavily jaundiced infants and those of the mothers with fatally affected infants (d.f. =12, saline method: t=2.24, 0.05> p>0.01, anti-globulin method: t=2.35, 0.05>p>0.01). 5. Hemolysis (a) Titer. As seen in Table V (See page102) and Figure 5, hemolysin titers of normal males ranged from -2 (negative) to 3 (1: 4). For normal mothers, the range stood from-2 (negative) to 4 (1: 8). The range in the mothers with heavily jaundiced infants was-1 (weakly positive) to 3 (1: 4). Among the mothers with fatally affected infants, the range was -2 (negative) to 6 (1: 32). (b) Comparison of the mean titers. The mean hemolysin titers of the normal mothers and the HEMOLYSIN TITER Fig.5. Distribution of anti-a, B hemolysin activity in untreated sera. Lower graph: normal cases. (Blank: normal males. Dappled: normal mothers.) Upper graph: hemolytic cases. (Shaded mothers with heavily jaundiced infants. Dark: mothers with fatally affected infants.) mothers with heavily jaundiced infants differed with high statistical significance (d.f.=120, t=4.35, p< 0.01); the variances of the hemolysin titers of the normal mothers and the mothers with fatally affected infants had a significant difference (d.f.=8/116, F= 4.27, p<0.01) and the difference of the mean titers was large. The mean titers of the mothers with heavily jaundiced infants and the mothers with fatally affected infants were not separated statistically (d.f.=12, t=1.55, p>0.05). DISCUSSION The change which is noticed first after immune stimulus such as blood group incompatible pregnancy

9 or incompatible blood transfusion is the increase of agglutinin titer. to 1: 1,000,000 in saline3). Titers also differ widely according to titration methods. It rises higher than 1: 512, even Test in hole-glasses is suitable for observations of avidity or aggregate sizes, but has a disadvantage in that it requires hasty reading of agglutinations when many sera have to be simultaneously titrated because the reagents dry up in a short time. From this reason, tube method was adopted in this study. Wiener's4) titration method by one-drop serial dilution, which Arai5) followed, has tendency to be inaccurate as drops stick to tube walls, so the author titrated with 0.1ml each of diluted sera and cell suspension. For detection of incomplete antibody, Wiener4) used acasia medium, Boorman et al. 6), and Arai5) used group AB human serum; the author performed indirect anti-globulin test in all the tests in this study. 0.1ml of 2% cell suspension was also most suitable for anti-globulin tests. Arai's data5) of saline and serum titers well accorded with this study with saline and anti-globulin methods in categories of normal mothers and mothers with hemolytic infants, though the former showed the averages one tube higher than this study. By agglutination titers, hemolytic cases could not be distinguished from the normal range, except a few which showed extraordinary high titers. Enhancement of saline agglutination titer by anti-globulin test was more distinct in normal mothers than in normal males and in group O than in groups A and B; hemolytic cases (average 1.4 tubes) and group O normal mothers (average 1.4 tubes) were equally enhanced. Mothers' sera differs according to their blood groups in the tendency to cause hemolytic disease of the infants. Rosenfield7) observed that among his 39 cases, whose ABO hemolytic infants' bloods gave positive direct Coombs' tests, 38 mothers were group O. If other groups were similarly liable to cause the disease, only 28 from 39 should be group O. Takahashi8) found that many group O human sera possess incomplete antibodies: titers of 64.1% of anti-a and 67.8% of anti- B were enhanced by indirect Coombs' test 2 tubes or higher. Nakamoto9) investigated the blood groups of 109 mothers and their jaundiced infants and reported that the frequencies of incompatible mother-child combination ran as 0-A>O-B>A-B>B-A. Some reports say that anti-a had immune characteristics more often than anti-b10,11) but the present author interprets this as a reflection of higher frequencies of group A than group B in general population. Rosenfield7) said that both group A and group B children were similarly hemolysed according to the frequencies of groups A and B. Also in the present study no difference was noticed between anti-a and anti-b. Rosenfield and Ohno12) observed that many group O sera contained anti-a or anti-b which can pass the placenta ; most of the newborn babies from group O mothers possessed iso-agglutinins in the sera, while only 35% of the babies from groups A and B mothers possessed the dur iso-agglutinins. They also showed the presence of cross-reacting antibody in the sera of group O mothers, which could be absorbed with either group A or B cells. This has been considered as antibody against C factor, the common antigen of A and B18), and as the main cause of the hemolytic disease of the newborn14). Zuelzer et al. 15) reported that a group O mother delivered fraternal twin; one was group B and suffered hemolytic disease and the other was group A and was not affected. This may show that the cross-reacting antibody does not necessarily play the essential part in the erythroblastosis neonatorum. The present author also found that anti-a and anti-b in the mercaptoethanol-treated serum of group O mothers with hemolytic infants were not cross-absorbed with group A and B saliva34). Positive reaction in serum medium after losing agglutinating activity in saline by neutralization with

10 96 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 blood group substance was regarded by some researchers as the essential character of immune antibodies3, 16). Schiff17) immunized men of groups A and O with intramuscular injection of B and A saliva, neutralized the sera with serially diluted saliva, and titrated them in saline and acasia for agglutination; great heterogeneity of immune anti-a and anti-b was shown by this study. Ervin et al. 18) Discussed the necessity of searching for the adequate quantity of blood group substances to inhibit the agglutinating activity in saline and leave the anti-globulin activity. Gibbs19) made quantitative studies on neutralization of anti- A with group A specific substance of pork origin, and found that, compared with immune anti-a, natural anti-a showed less steep log-probit analysis line of agglutination inhibition, needed relatively greater quantity of blood group substance for agglutination-inhibition, and was easily dissociated from the bound antigen. Gibbons et al. 20) found that group B specific substance purified from human ovarian cyst fluid had a strong neutralizing activity against immune anti-b but had little effect on complete anti-b agglutinin activity. Also Winstanley et a1. 21) studied the absorbability of anti-a using group A pork red cell (AP) and found that absorption of immune anti-a with Ap cells removed hemolysin against human Al cells but did not reduce the titer of saline agglutinin, and that the antibody involved in the hemolytic disease of group A infants had the specificity of anti-ap. Tovey22) applied this for antenatal prediction of ABO hemolytic disease of group A infants by measuring the fall in anti-a titer after absorbing the sera of pregnant women with Ap cells. Bore135) described that potentially dangerous anti-a sera could be quite easily detected by their ability to lyse human A, Ap, and sometimes sheep R and cattle J cells, to agglutinate Ap cells, and to agglutinate human A cells using an anti-globulin test after partial neutralization with soluble specific blood group substances. The neutralization test examined by the present author which was aimed at the inhibition of agglutinating activity of natural antibodies by saliva, made a relatively good distinction of hemolytic cases from normal mothers. Separation of immune and natural antibodies by stability to heat was recommended by Boorman and Dodd23), who performed anti-globulin test after heating the sera at 75C for 20 minutes. As this intensive heating often inactivated strong incomplete antibodies detectable by neutralization tests, the author selected the condition of 70C 10 minutes according to Mollison24). Reepmaker et al. 25) observed that still many immune antibodies lose the activity by heating at 70C for 10 minutes. At the laboratory of the present author, this heating test had been mainly performed as ABO incompatibility test, but it was found to give relatively unreliable results from the present comparative study. Many investigators regard the presence of hemolysin as an important character of immune sera. Mollison24) reported that sera from mothers with hemolytic disease of the newborn, mixed with the same volume of 5% red cell suspension and absorbed fresh group O sera as the source of complement, usually gave complete hemolysis in the undiluted sera and incomplete hemolysis in the 1: 4 dilution. Reepmaker et al. 25) observed hemolytic disease in about 1/3 infants whose mother possessed hemolysin against their cells. Crawford et al. 26) reported 11 cases of ABO hemolytic disease whose mothers' sera inevitably hemolysed the adult cells of the same blood groups with the infants. In the present study, all the sera from mothers with hemolytic infants completely hemolysed the incompatible standard cells with titers of one or more tubes, except one case of negative reaction. Fudenberg et al.27) separated human sera by ultra-centrifugation and found that natural anti-a and anti-b agglutinins sedimented with macroglobulins with the sedimentation constant of 19S, and group

11 O sera often showed anti-a and anti-b activity also in 7S gamma-globulin; especially mothers with ABOhemolytic infants always possessed strong 7S antibodies. Kochwa et al.28) also demonstrated the importance of 7S antibodies as the cause of erythroblastosis fetalis by blood group incompatibility from the finding that anti-a and anti-b in cord blood sera were confined to 7S gamma-globulin. Rawson et a1. 29) separated isoagglutinins in normal human sera into 19S macroglobulin and 7S gamma-globulin, and found that 19S antibody was active in saline and 7S antibody had strong activity by anti-globulin method. Kekwick et a1.30) considered that so-called natural antibodies consisted of 19S antibodies and immune incomplete antibodies consisted of 7S antibodies. Kochwa28) eluted 7S gamma-globulin antibodies from ion-exchange cellulose and reported that when the mothers' 7S antibodies were neutralized with soluble blood group specific substance, the infants showed no ill effects and the 7S antibodies of mothers with hemolytic infants were not neutralized. Grubb and Swahn31 found that treatment of sera with 2-mercapto-ethanol or mercaptoethylamine destroyed the activity of 19S macroglobulin antibodies but did not affect the activity of 7S gammaglobulin antibody. Mollison32) detected incomplete anti-a and anti-b by a modification of this treatment, which the author adopted in the present study seeing at the possibility that this would be applied for ABO incompatibility test. Fong et al. 33) recommended, as practicable ABO incompatibility tests estimation of antibody activity against Ap cells and neutralization test with blood group specific substances. It is generally considered that the presence in the maternal serum of immune antibodies is not the only variable involved in the development of the hemolytic disease of the newborn, and that prediction of the disease is difficult from the serological test of the maternal serum only, however, it is also evident that the serological tests give the clinicians considerable informations of practical value. Among the tests compared in this study, mercaptoethanol-treatment test seemed to give the statistically most reliable results. Especially, one of the essential drawbacks of the other tests were inability to deny the involvement of ABO incompatibility in the infants' hemolytic disease even when titers were low, though they could predict the disorders if the antibodies had extraordinary high titers; while, in mercaptoethanol-treatment test, the weakest antibody from the mother with the fatally affected infant overlapped with the titers of normal mothers by only one from 117 cases (0.85%) in saline method and by three cases (2.56%) in anti-globulin method. So this test has a great advantage in that it can deny the hemolytic disease of the newborn due to ABO incompatibility if the titer falls in the normal range. is adopted at the department of the author as mercaptoethanol-stable Now this test antibody test for routine antenatal prediction of hemolytic disease of the newborn2). In order to express the titer more precisely, the titration scores3) are used for the routine test instead of the end point of macroscopic agglutination. SUMMARY Human sera were classified into four classes: normal males, normal mothers, mothers with heavily jaundiced infants, and mothers with fatally affected infants. The anti-a and anti-b agglutinin activities were examined in the untreated sera, the neutralized sera, the heated sera, and the mercaptoethanol-treated sera, with saline and anti-globulin methods. The hemolytic activity was also examined on the same

12 98 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 Table I. Antibody titers in untreated sera. S.d.: Table II. Antibody titers in neutralized sera. S.d.: Standard

13 Standard deviation, M.w.-i.: Mothers with-infants. deviation, M.w.-i.: Mothers with-infants, *See the text

14 100 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 Table III. Antibody titers in heated sera. S.d.: Standard Table IV. Antibody titers in mercaptoethanol-treated sera. S.d.:

15 deviation, Mw.-i.: Mothers with-infants, *See the text Standard deviation, Mw.-i.: Mothers with-infants, *See the text

16 102 Journal of the Japan Society of Blood Transfusion, Vol. 15, No. 3 materials. Results of the various tests did not necessarily coincided. The mean titers of the mothers with fatally affected infants were much higher than the mean titers of the normal mothers in any tests, but the statistical separation of the mean titers between the normal mothers and the mothers with heavily jaundiced infants as well as between the mothers with heavily jaundiced infants and the mothers with fatally affected infants was significantly possible only by mercapto-ethanol-treatment test and less significantly by titration of untreated sera; neutralization test gave the next good separation. Also from the point of view that the overlapping of the normal titers and the titers of the hemolytic Table V. Hemolysis S.d.: Standard deviation, M.w.-i.: Mothers with-infants, *See the text cases was the narrowest by mercaptoethanol-treatment test, it was suggested that this test will find a good application as a routine test for antenatal prediction of hemolytic disease of the newborn due to ABO incompatible pregnancy. ACKNOWLEDGMENTS The sera from normal mothers were kindly offered by Dr. M. Yanagida of Tsukiji Metropolitan Maternity Hospital. Information on the clinical findings of hemolytic infants were generously given by Dr. K. Ichinomiyaa of the Department of Obstetrics and Gynecology, Tokyo Medical and Dental University, and other obstetricians and pediatricians. Members of the Department of Legal Medicine gave helpful advices to the author. The author is deeply indebted to them all and expresses cordial appreciations.

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