Vox Sang. 35: 58-64 (1978)
Association between HLA and Red Cell Antigens VI. Family Studies R . Nordhagen 1 Department of Immunology, Tissue Typing Unit and National Blood Group Reference Laboratory, National Institute of Public Health, Oslo
Abstract. The reactivity of the HLA antigens A28, B7 and B8 on red blood cells (RBC) has been studied, and the inheritance of the strength of reactivity examined in 13 families where the HLA haplotypes could be determined. Examination of individuals possessing more than one of the three antigens showed that the antigens seemed to be expressed independently of each other on RBC. The family studies showed that the HLA reactivity of RBC does not invariably follow the HLA haplotype determining the particular antigen. It was also shown that heterozygosity/homozygosity did not seem to have much influence on the strength of reactivity. Our conclusion is that several factors are likely to be responsible for the great variation in HLA reactivity of RBC.
Introduction HLA antigens are represented on RBC, but their expression varies considerably [4101. The correlation with the Bg antigens has been demonstrated by Morton et al. [4]. A few family studies concerning Bgl HLA antigens on RBC have been reported previously [2, 5, 141. Race and Sanger [12] state that: ‘by ordinary methods of testing, Bg+ children often have apparently Bg- parents’. Seaman The author thanks Margareta Been, Ellen Langeland and Tove Andaas Isene for their skilled technical assistance.
1
et al. [14] also reported that the ‘original’ Bg families did not show straightforward inheritance, but they believed this was due to the relative insensitiveness of the reactions. In their own studies, using the AutoAnalyzer (AA) technique, they found evidence that the Bg antigens are inherited as Mendelian dominant characters, and they did not find any Bg- x Bg- matings with Bg+ children. Since it is evident that these antigens are HLA antigens, it would be of special interest to study the inheritance of RBC HLA reactivity together with HLA typing of the lymphocytes. Morton et al. [4] studied some families using HLA haemagglutinins
Association between HLA and Red Cell Antigens
and the AA technique, where white blood cell typing was done simultaneously. They concluded that the HLA reactivity of RBC was not necessarily inherited in a straightforward way, following the HLA haplotype. However, only three families with HLA-B 17 and one with HLA-A28 were reported. A study of one family was reported by van der Hart et al. [2]. In their case, HLA-A2,B7 and WlO(w40) were demonstrated on the RBC from a healthy blood donor, but not on the RBC from any of the parents. This study was performed with a manual technique only. In our preliminary studies, we had noted that the HLA reactivity of RBC did not always seem to be inherited together with the HLA antigen. We intended to further investigate this phenomenon, by examining some families which were informative with regard to the HLA haplotypes. We have demonstrated that the AA method is the most sensitive and exact method for the examination of the HLA reactivity of RBC; the results of manual analyses are often difficult to interpret [lo]. In family studies, a combination of HLA lymphocyte typing and AA testing for the HLA reactivity of RBC, would give us the most exact information. We have pointed out the variation in HLA reactivity of RBC, both qualitatively and quantitatively [6-lo]. We, as well as other authors, have also shown that the HLA reactivity of RBC from a donor may vary somewhat from time to time [lo]. This makes it rather difficult to draw conclusions with regard to the inheritance of the strength of the HLA reactivity. However, the occasional variations are usually relatively small, and do not occur very often. We have, for instance, never
59
seen a donor changing from a negative reactor to a very strong reactor, or vice versa. We have further shown that some antigens are more strongly represented on RBC than others [6-lo]. The strongest HLA antigens on RBC seem to be HLA-A28, B7 and B8, the former two being about equal in strength, B8 considerably weaker. Since we had several good haemagglutinins against these three antigens, we have limited our family studies to their examination on RBC. We were also interested in the ‘pattern of reactivity’ in an individual. That is, if a donor, for instance, has both HLA-A28 and B7, will the HLA reactivity of RBC be of similar or different strength for the two antigens, since they seem to be comparable in strength in population studies? The frequency of reactors among HLA-A28- and B7-positive donors, respectively, is about the same, with the same range of reactivity. The purpose of this study was to investigate the HLA reactivity of RBC of some donors with two or more of the antigens A28, B7 and B8, and to examine families with informative HLA haplotypes with one or more of the same antigens.
Materials and Methods The family material comprised 14 matings with 40 children. Two generations were tested in each family, except in one, where three generations were examined. The haemagglutinating antisera against HLAA28, B7 and B8 have been used in our previous studies, and their specificities have been documented [6, 81. A few ‘new’ sera which showed exactly corresponding specificities were added. They had been tested for a long time in parallel with the ‘old‘ sera on the AA. Only family members being HLA-A28-, B7- or B8-positive were
60
Nordhagen
tested on the AA for HLA reactivity of RBC. All these donors and their family members could not be tested on the AA against all the antisera, mostly due to incompatible ABO groups. Thus, we had no sera available to examine donors of blood group B or AB. However, we have used several sera of each specificity in the investigation of every family. All members of a family were always tested on the A A simultaneously. The blood for AA testing was collected on the same day or on the day before the examination on AA. There was no change in AA equipment or method from our previous reports [&lo]. The HLA typing was performed in our laboratory, using 54 antisera typing for 20 antigens. Thc method was the same as in our previous experiments [6]. All propositi were HLA-typed at least twice, as were some of the family members in families showing unexpected findings.
Results and Discussion
When the HLA-A28 and B7 reactivity RBcfrom the Same donor with both antigens was compared, we found that sometimes HLA-A28 showed the strongest reactivity, sometimes B7. Table1 shows some of the results. In donors 1 and 2, the HLAA28 reactivity is evidently stronger than the B7 reactivity, demonstrated with all the sera. Donor 1 is a completely non-reactive B7, but a quite strong A28 reactor. The experjments were repeated several times with the same results. Donors 3 and 4 showed a slightly stronger reactivity of B7 than of A28, even though they both are relatively strong A28 reactors. Donor 4 is blood group Of
Table I. Comparison of HLA reactivity of RBC from some donors with at least two of the antigens A28, B7 and B8
Donor and HLA type
Antigens and serum No. Anti-HLA-A28 (A2) 5
90 113 120 181 350 394
Donor 1 All,28; B7,17
28 24 33
Donor 2 All,28;B7,wl5
15 16 20
Donor 3 A3,28; B7
33 26 34
Donor 4 A2,28; B7, 8
20
Donor 5 A l , 28 ; B8,12
21 26
Donor 6 A2.9; B7,8
Anti-HLA-B7
22
29 20
25
28
20
7
62 71 101 107 186 216 236 265
0
0
0
0
4
2
2
3
38
29 22 36
0
0 4
36
32
31
16
15
37 30
35
20
20
The reaction strength of the RBC on AA is given in A OD x 100. Donor 1 is 11.2 in family No. 12 (table 11); donor 4 is 1.2 in family No. 13 (table 11.)
Association between HLA and Red Cell Antigens
A, and only a few antisera could be used in the testing of her RBC. As expected from previous population studies, B8 is usually weaker than A28 and B7 on the RBC, as shown in table1 for donors 5 and 6. However, many of our strong A28 or B7 reactors are strong B8 reactors as well. This is seen in some of the family studies in table 11. Donor 4 is hitherto the only one we have observed who showed a reactivity of B8 stronger than A28, and nearly as strong as her B7 reactivity. Since she is blood group A, serum No. 82 was the only haemagglutinating antiHLA-B8 that could be used, but this is not our strongest anti-HLA-B8 serum, and it usually shows much weaker reactions than
Antigens and serum No. Anti-HLA-B8 82
125
127
252
21
0
0
0
0
0
61
the haemagglutinating anti-HLA-A28 sera (the results of the studies of the family of donor 4 are shown in tableII, family 13, donor 4,1.2). The results from the examination on AA of RBC from donors possessing at least two of the antigens HLA-A28,B7 and B8 have shown that on the RBC from one individual, the strength of the three different antigens does not always show the same ‘order of reactivity’. Individual reactivity seems to exist for each antigen. We have tried to evaluate the inheritance of this trait in 13 families, with ‘informative’ HLA haplotypes. The results are shown in table 11. The family members tested on the AA are divided into non-reactors (N), weak (W),medium (M) and strong (S) reactors. This definition is relative, and given after running the RBC on AA against known, good (often several) antisera. For the apparently equally strong antigens A28 and B7, S = A OD x 100 > 20, M = 10-20, W < 10. Since B8 is a weaker antigen, a strong B8 reactor (S) is defined as A OD x 100 > 10, M = 5-10, and W < 5 . In table 11, the reactivity is given in A OD x 100 for one of the sera used in each family, but several sera of each specificity were usually employed in all families. All HLA haplotypes could not be exactly determined since homozygosityheterozygosity is uncertain in some individuals. In the three-generation family 10, child 11.3 is the mother of the third generation, married to 11.4. In family 12, there has apparently been a recombination. However, there are only two children, and we cannot tell in whom the recombination has taken place. The inheritance of strength of reactivity has been investigated for other RBC antigens before. It may be easy to see that the
Nordhaaen
62
Table II. Studies of HLA reactivity of RBC in 13 families Family HLA haplotypes
A28
Family I 1.1 1,8/2,12 1.2 2,7/-,12 11.1 2,12/2,7 11.2 2,12/-,12 11.3 1,8/-,12 11.4 2,12/2,7 Family 2 1.1 1,8/3,7 1.2 2,8/9,w35 11.1 3,7/9,w35 11.2 3,7/2,8 Family 3 1.1 10,w40/9,w15 S 1.2 2,7/28,5 M 11.1 lO,w40/28,5 11.2 9,w15/2,7 11.3 9,w15/28,5 S S 11.4 lO,w40/28,5 Family 4 1.1 3,27/-,8 1.2 3,27/28,12 S/M S 11.1 3,27/28,12 11.2 -,8/28,12 M Family 5 1.1 1,8/2,w40 1.2 9,-(w40?)/2,w40 11.1 2,~40/9,-(~40?) 11.2 1,8/2,w40 11.3 2,~40/9,-(~40?) Family 6 1.1 1,8/2,7 1.2 2,7/3,-(7?) 11.1 2,7/2,7 11.2 1,8/2,7 Family 7 1.1 1,8/11,-(8?) 1.2 28,7/1 l?,w35 (deduced) 11.1 1,8/11,w35? 11.2 1,8/28,7 M 11.3 1,8/11,w35? 11.4 1,8/11,w35? 11.5 1,8/28,7 M
B7
B8
M 16 M/W 11
M/W 11 M
13
M/S 18 S 25
N O S 14
s
11
27 17 30 27
20 32 13 S 25
S 27
s NT M
w
35 20 10
N O
19
w
10
NT N O
17
w
10
N O
Family HLA haplotypes
A28
Family 8 1.1 1,-(7?)/3,7 1.2 2,wl5/2,5 11.1 3,7/2,w15 11.2 3,7/2,5 Family 9 1.1 3?,7/3?,w40 1.2 3,7/l,w15 11.1 3?,7/3,7 11.2 3?,40/3,7 Family 10 1.1 -(2?),12/2,wl5 1.2 3,7/10,7 11.1 2,w15/3,7 11.2 2,w15/10,7 11.3 2,w15/10,7 11.4 2,w15/3,7 (husband of 11.3) 111.1 3,7/10,7 111.2 3,7/10,7 Family I I 1.1 1,7/-,12 1.2 3,7/1,w37 11.1 1,7/3,7 11.2 1,7/3,7 11.3 -,12/1,~37 11.4 1,7/3,7 11.5 -,12/3,7 Family 12 1.1 28,7?/3,17? N 0 1.2 2,13/11,7 11.1 28,7/11,7 M 18 11.2 28,17/11,7 M 22 Family 13 1.1 1,8/3,1.2 2,7/28,8 S/M 20 N O 11.1 1,8/28.8 11.2 3,-/2,7
B7
B8
s
35
w s
10 35
S 27 M 13 W 8 M/S 16
S
M NT
27 17
w w
10 10
S S
25 27
M N S S
20 O 30 32
s S
34 29
N M W N
0 18 10 0
S
30
w
12
N O S 27 w 5
The reaction strength is given in A OD x 100 for one of the sera used in each family. S = Strong reactor; M = medium reactor; W = weak reactor; N = non-reactor; NT= not tested on AA.
Association between HLA and Red Cell Antigens
trait is inherited, as for the D" antigen [ll, 131, but it may also be difficult. It has, for instance, been difficult to prove that the variation in PI reactivity is inherited, even though a great number of families have been investigated [ l , 31. In some of our families, the inheritance of an HLA haplotype seems to give about the same reactivity in parents and offspring (families 1, 3, 5), but there are many exceptions (families 2, 4, 6, 8, 9, 11-13). HLA antigens showing a strong reactivity of RBC in one of the parents, may give weak or even no reactivity in some of the children inheriting the same HLA haplotype. This is seen in families 6, 8, 9, 12, 13. On the other hand, strongly reactive children may have inherited the HLA haplotype from a considerably weaker-reacting parent (families 2, 4, 12). Thus, it may be concluded that the HLA haplotype does not per se determine the HLA reactivity of the RBC. The strength of the HLA reactivity of RBC may be influenced by both haplotypes, even if only one of them determines the presence of the HLA antigen investigated on RBC. There is no evidence from these family studies that any particular antigen determined by the other haplotype, or by the other locus on the same haplotype, should influence the reactivity, as a parallel to the 'position effect' in the Rh system. In five of our families, families 1, 3, 7, 10 and 11, we have apparently HLA-identical sibs, and in four of these the identical sibs show almost identical HLA reactivity of their RBC. In the fifth family, however (family 3), a relatively great difference was observed. This could mean that the strength of reactivity is not governed from the HLA locus only, but the difference found is not greater than the occasional variation seen
63
in some cases. This variation has occasionally been observed to exceed 20 ( A OD x 100) in rare cases (lo), but is usually less than 10. Unfortunately, we were not able to retest the members of family 3. If only one family had been examined, e.g. family 10, we would think that homozygosity is deciding reactivity, because all the homozygous individuals show a stronger reactivity than the heterozygous. However, many exceptions are seen in other families (families 6, 9, 11-13). In previous population studies, we have also noted that homozygosityheterozygosity does not seem to be important for the HLA reactivity of RBC [6, 8,91. If the reactivity were determined by a single dominant gene outside the HLA system, we would not expect any offspring tc. show greater reactivity than any of the parents, if both carry the antigen. We have no families in this study where both parents are non-reactors for the same antigen with reactor children (that is negative x negative matings with positive children), but family 11 is close to this pattern. Both parents carry HLA-B7 (medium and non-reactors, respectively). All the children are strong reactors, including 11.5, the heterozygous one. The members of this family were all examined with several antisera, all showing the same as the one listed in the table. Again, the difference might be explained by occasional variation. However, this is not seen very often. It is not evident after these family studies that the strength of HLA reactivity of RBC is inherited at all. We may see different variations of reactivity within the families, but it will require very extensive family studies to see if these variations are smaller than in population studies. We might, how-
64
Nordhagen
ever, note that a strong B8 reactivity, which is a rare trait, is found in two propositi (families 2 and 9, and the same strong B8 reactivity is also found in one of the parents in both families. Thus, if the strength of HLA reactivity of RBC is genetically determined, it is probably polygenic, and may possibly also be influenced by other, non genetic factors.
References 1 Fisher, Sir Ronald: The variation in strength
2
3
4
5
6
of the human blood group P. Heredity 7: 8189 (1953). Hart, M. van der; Szaloky, A.; Berg-Loonen, E. M. van den; Engelfriet, C. P. et Loghem, J. J. van: PrCsence d’antighnes HL-A sur les hematies d’un donneur normal. Noun Revue fr. HCmat. 14: 555-563 (1974). Ilenningsen, K.: On the heredity of blood factor P. Acta path. microbiol. scand. 26: 769785 (1949). Morton, J. A.; Pickles, M. M., and Sutton, L.: The correlation of the Bga blood group with the HL-A7 leucocyte group: demonstration of antigenic sites on red cells and leucocytes. Vox Sang. 21: 536-547 (1969). Morton, J. A.; Pickles, M. M.; Sutton, L., and Skov, F.: Identification of further antigens on red cells and lymphocytes. VOXSang. 21: 141153 (1971). Nordhagen, R. and Orjastzter, H.: Association between HL-A and red cell antigens. An AutoAnalyzer study. Vox Sang. 26: 97-106 (1974).
7 Nordhagen, R.: Association between HL-A and red cell antigens. 11. Absorption and titration analyses. Vox Sang. 27: 124-133 (1974). 8 Nordhagen, R.: Association between HL-A and red cell antigens. 111. Studies of haemagglutinins in cytotoxic anti-HL-A7- and anti-HL-AS-related sera. Vox Sang. 29: 23-35 (1975). 9 Nordhagen, R.: Association between HLA and red cell antigens. IV. Further studies of haemagglutinins in cytotoxic HLA antisera. Vox Sang. 32: 82-89 (1977). 10 Nordhagen, R.: Association between HLA and red cell antigens. V. A further study of the nature and behaviour of the HLA antigens on red blood cells and their corresponding haemagglutinins. Vox Sang. 35: 49-57 (1978). 11 Race, R. R.; Sanger, R., and Lawler, S. D.: The Rh antigen Du. Ann. Eugen. 14: 171-184 (1948). 12 Race, R.R. and Sanger, R.: Blood groups in man; 6th ed., p. 499 (Blackwell, Oxford 1975). 13 Renton, P. H. and Stratton, F.: Rhesus type Du. Ann. Eugen. 15: 189-209 (1950). 14 Seaman, M. J.; Benson, R.; Jones, M. N.; Morton, J. A., and Pickles, M. M.: The reactions of the Bennett-Goodspeed group of antibodies with the AutoAnalyzer. Br. J. Haemat. 13: 464-473 (1967).
Received: July 18, 1977 Accepted: October 8, 1977
Dr. Rannveig Nordhagen, National Institute of Public Health, SIFF, Postuttak Oslo 1 (Norway)
Association between HLA and Red Cell Antigens VI. Family Studies R . Nordhagen 1 Department of Immunology, Tissue Typing Unit and National Blood Group Reference Laboratory, National Institute of Public Health, Oslo
Abstract. The reactivity of the HLA antigens A28, B7 and B8 on red blood cells (RBC) has been studied, and the inheritance of the strength of reactivity examined in 13 families where the HLA haplotypes could be determined. Examination of individuals possessing more than one of the three antigens showed that the antigens seemed to be expressed independently of each other on RBC. The family studies showed that the HLA reactivity of RBC does not invariably follow the HLA haplotype determining the particular antigen. It was also shown that heterozygosity/homozygosity did not seem to have much influence on the strength of reactivity. Our conclusion is that several factors are likely to be responsible for the great variation in HLA reactivity of RBC.
Introduction HLA antigens are represented on RBC, but their expression varies considerably [4101. The correlation with the Bg antigens has been demonstrated by Morton et al. [4]. A few family studies concerning Bgl HLA antigens on RBC have been reported previously [2, 5, 141. Race and Sanger [12] state that: ‘by ordinary methods of testing, Bg+ children often have apparently Bg- parents’. Seaman The author thanks Margareta Been, Ellen Langeland and Tove Andaas Isene for their skilled technical assistance.
1
et al. [14] also reported that the ‘original’ Bg families did not show straightforward inheritance, but they believed this was due to the relative insensitiveness of the reactions. In their own studies, using the AutoAnalyzer (AA) technique, they found evidence that the Bg antigens are inherited as Mendelian dominant characters, and they did not find any Bg- x Bg- matings with Bg+ children. Since it is evident that these antigens are HLA antigens, it would be of special interest to study the inheritance of RBC HLA reactivity together with HLA typing of the lymphocytes. Morton et al. [4] studied some families using HLA haemagglutinins
Association between HLA and Red Cell Antigens
and the AA technique, where white blood cell typing was done simultaneously. They concluded that the HLA reactivity of RBC was not necessarily inherited in a straightforward way, following the HLA haplotype. However, only three families with HLA-B 17 and one with HLA-A28 were reported. A study of one family was reported by van der Hart et al. [2]. In their case, HLA-A2,B7 and WlO(w40) were demonstrated on the RBC from a healthy blood donor, but not on the RBC from any of the parents. This study was performed with a manual technique only. In our preliminary studies, we had noted that the HLA reactivity of RBC did not always seem to be inherited together with the HLA antigen. We intended to further investigate this phenomenon, by examining some families which were informative with regard to the HLA haplotypes. We have demonstrated that the AA method is the most sensitive and exact method for the examination of the HLA reactivity of RBC; the results of manual analyses are often difficult to interpret [lo]. In family studies, a combination of HLA lymphocyte typing and AA testing for the HLA reactivity of RBC, would give us the most exact information. We have pointed out the variation in HLA reactivity of RBC, both qualitatively and quantitatively [6-lo]. We, as well as other authors, have also shown that the HLA reactivity of RBC from a donor may vary somewhat from time to time [lo]. This makes it rather difficult to draw conclusions with regard to the inheritance of the strength of the HLA reactivity. However, the occasional variations are usually relatively small, and do not occur very often. We have, for instance, never
59
seen a donor changing from a negative reactor to a very strong reactor, or vice versa. We have further shown that some antigens are more strongly represented on RBC than others [6-lo]. The strongest HLA antigens on RBC seem to be HLA-A28, B7 and B8, the former two being about equal in strength, B8 considerably weaker. Since we had several good haemagglutinins against these three antigens, we have limited our family studies to their examination on RBC. We were also interested in the ‘pattern of reactivity’ in an individual. That is, if a donor, for instance, has both HLA-A28 and B7, will the HLA reactivity of RBC be of similar or different strength for the two antigens, since they seem to be comparable in strength in population studies? The frequency of reactors among HLA-A28- and B7-positive donors, respectively, is about the same, with the same range of reactivity. The purpose of this study was to investigate the HLA reactivity of RBC of some donors with two or more of the antigens A28, B7 and B8, and to examine families with informative HLA haplotypes with one or more of the same antigens.
Materials and Methods The family material comprised 14 matings with 40 children. Two generations were tested in each family, except in one, where three generations were examined. The haemagglutinating antisera against HLAA28, B7 and B8 have been used in our previous studies, and their specificities have been documented [6, 81. A few ‘new’ sera which showed exactly corresponding specificities were added. They had been tested for a long time in parallel with the ‘old‘ sera on the AA. Only family members being HLA-A28-, B7- or B8-positive were
60
Nordhagen
tested on the AA for HLA reactivity of RBC. All these donors and their family members could not be tested on the AA against all the antisera, mostly due to incompatible ABO groups. Thus, we had no sera available to examine donors of blood group B or AB. However, we have used several sera of each specificity in the investigation of every family. All members of a family were always tested on the A A simultaneously. The blood for AA testing was collected on the same day or on the day before the examination on AA. There was no change in AA equipment or method from our previous reports [&lo]. The HLA typing was performed in our laboratory, using 54 antisera typing for 20 antigens. Thc method was the same as in our previous experiments [6]. All propositi were HLA-typed at least twice, as were some of the family members in families showing unexpected findings.
Results and Discussion
When the HLA-A28 and B7 reactivity RBcfrom the Same donor with both antigens was compared, we found that sometimes HLA-A28 showed the strongest reactivity, sometimes B7. Table1 shows some of the results. In donors 1 and 2, the HLAA28 reactivity is evidently stronger than the B7 reactivity, demonstrated with all the sera. Donor 1 is a completely non-reactive B7, but a quite strong A28 reactor. The experjments were repeated several times with the same results. Donors 3 and 4 showed a slightly stronger reactivity of B7 than of A28, even though they both are relatively strong A28 reactors. Donor 4 is blood group Of
Table I. Comparison of HLA reactivity of RBC from some donors with at least two of the antigens A28, B7 and B8
Donor and HLA type
Antigens and serum No. Anti-HLA-A28 (A2) 5
90 113 120 181 350 394
Donor 1 All,28; B7,17
28 24 33
Donor 2 All,28;B7,wl5
15 16 20
Donor 3 A3,28; B7
33 26 34
Donor 4 A2,28; B7, 8
20
Donor 5 A l , 28 ; B8,12
21 26
Donor 6 A2.9; B7,8
Anti-HLA-B7
22
29 20
25
28
20
7
62 71 101 107 186 216 236 265
0
0
0
0
4
2
2
3
38
29 22 36
0
0 4
36
32
31
16
15
37 30
35
20
20
The reaction strength of the RBC on AA is given in A OD x 100. Donor 1 is 11.2 in family No. 12 (table 11); donor 4 is 1.2 in family No. 13 (table 11.)
Association between HLA and Red Cell Antigens
A, and only a few antisera could be used in the testing of her RBC. As expected from previous population studies, B8 is usually weaker than A28 and B7 on the RBC, as shown in table1 for donors 5 and 6. However, many of our strong A28 or B7 reactors are strong B8 reactors as well. This is seen in some of the family studies in table 11. Donor 4 is hitherto the only one we have observed who showed a reactivity of B8 stronger than A28, and nearly as strong as her B7 reactivity. Since she is blood group A, serum No. 82 was the only haemagglutinating antiHLA-B8 that could be used, but this is not our strongest anti-HLA-B8 serum, and it usually shows much weaker reactions than
Antigens and serum No. Anti-HLA-B8 82
125
127
252
21
0
0
0
0
0
61
the haemagglutinating anti-HLA-A28 sera (the results of the studies of the family of donor 4 are shown in tableII, family 13, donor 4,1.2). The results from the examination on AA of RBC from donors possessing at least two of the antigens HLA-A28,B7 and B8 have shown that on the RBC from one individual, the strength of the three different antigens does not always show the same ‘order of reactivity’. Individual reactivity seems to exist for each antigen. We have tried to evaluate the inheritance of this trait in 13 families, with ‘informative’ HLA haplotypes. The results are shown in table 11. The family members tested on the AA are divided into non-reactors (N), weak (W),medium (M) and strong (S) reactors. This definition is relative, and given after running the RBC on AA against known, good (often several) antisera. For the apparently equally strong antigens A28 and B7, S = A OD x 100 > 20, M = 10-20, W < 10. Since B8 is a weaker antigen, a strong B8 reactor (S) is defined as A OD x 100 > 10, M = 5-10, and W < 5 . In table 11, the reactivity is given in A OD x 100 for one of the sera used in each family, but several sera of each specificity were usually employed in all families. All HLA haplotypes could not be exactly determined since homozygosityheterozygosity is uncertain in some individuals. In the three-generation family 10, child 11.3 is the mother of the third generation, married to 11.4. In family 12, there has apparently been a recombination. However, there are only two children, and we cannot tell in whom the recombination has taken place. The inheritance of strength of reactivity has been investigated for other RBC antigens before. It may be easy to see that the
Nordhaaen
62
Table II. Studies of HLA reactivity of RBC in 13 families Family HLA haplotypes
A28
Family I 1.1 1,8/2,12 1.2 2,7/-,12 11.1 2,12/2,7 11.2 2,12/-,12 11.3 1,8/-,12 11.4 2,12/2,7 Family 2 1.1 1,8/3,7 1.2 2,8/9,w35 11.1 3,7/9,w35 11.2 3,7/2,8 Family 3 1.1 10,w40/9,w15 S 1.2 2,7/28,5 M 11.1 lO,w40/28,5 11.2 9,w15/2,7 11.3 9,w15/28,5 S S 11.4 lO,w40/28,5 Family 4 1.1 3,27/-,8 1.2 3,27/28,12 S/M S 11.1 3,27/28,12 11.2 -,8/28,12 M Family 5 1.1 1,8/2,w40 1.2 9,-(w40?)/2,w40 11.1 2,~40/9,-(~40?) 11.2 1,8/2,w40 11.3 2,~40/9,-(~40?) Family 6 1.1 1,8/2,7 1.2 2,7/3,-(7?) 11.1 2,7/2,7 11.2 1,8/2,7 Family 7 1.1 1,8/11,-(8?) 1.2 28,7/1 l?,w35 (deduced) 11.1 1,8/11,w35? 11.2 1,8/28,7 M 11.3 1,8/11,w35? 11.4 1,8/11,w35? 11.5 1,8/28,7 M
B7
B8
M 16 M/W 11
M/W 11 M
13
M/S 18 S 25
N O S 14
s
11
27 17 30 27
20 32 13 S 25
S 27
s NT M
w
35 20 10
N O
19
w
10
NT N O
17
w
10
N O
Family HLA haplotypes
A28
Family 8 1.1 1,-(7?)/3,7 1.2 2,wl5/2,5 11.1 3,7/2,w15 11.2 3,7/2,5 Family 9 1.1 3?,7/3?,w40 1.2 3,7/l,w15 11.1 3?,7/3,7 11.2 3?,40/3,7 Family 10 1.1 -(2?),12/2,wl5 1.2 3,7/10,7 11.1 2,w15/3,7 11.2 2,w15/10,7 11.3 2,w15/10,7 11.4 2,w15/3,7 (husband of 11.3) 111.1 3,7/10,7 111.2 3,7/10,7 Family I I 1.1 1,7/-,12 1.2 3,7/1,w37 11.1 1,7/3,7 11.2 1,7/3,7 11.3 -,12/1,~37 11.4 1,7/3,7 11.5 -,12/3,7 Family 12 1.1 28,7?/3,17? N 0 1.2 2,13/11,7 11.1 28,7/11,7 M 18 11.2 28,17/11,7 M 22 Family 13 1.1 1,8/3,1.2 2,7/28,8 S/M 20 N O 11.1 1,8/28.8 11.2 3,-/2,7
B7
B8
s
35
w s
10 35
S 27 M 13 W 8 M/S 16
S
M NT
27 17
w w
10 10
S S
25 27
M N S S
20 O 30 32
s S
34 29
N M W N
0 18 10 0
S
30
w
12
N O S 27 w 5
The reaction strength is given in A OD x 100 for one of the sera used in each family. S = Strong reactor; M = medium reactor; W = weak reactor; N = non-reactor; NT= not tested on AA.
Association between HLA and Red Cell Antigens
trait is inherited, as for the D" antigen [ll, 131, but it may also be difficult. It has, for instance, been difficult to prove that the variation in PI reactivity is inherited, even though a great number of families have been investigated [ l , 31. In some of our families, the inheritance of an HLA haplotype seems to give about the same reactivity in parents and offspring (families 1, 3, 5), but there are many exceptions (families 2, 4, 6, 8, 9, 11-13). HLA antigens showing a strong reactivity of RBC in one of the parents, may give weak or even no reactivity in some of the children inheriting the same HLA haplotype. This is seen in families 6, 8, 9, 12, 13. On the other hand, strongly reactive children may have inherited the HLA haplotype from a considerably weaker-reacting parent (families 2, 4, 12). Thus, it may be concluded that the HLA haplotype does not per se determine the HLA reactivity of the RBC. The strength of the HLA reactivity of RBC may be influenced by both haplotypes, even if only one of them determines the presence of the HLA antigen investigated on RBC. There is no evidence from these family studies that any particular antigen determined by the other haplotype, or by the other locus on the same haplotype, should influence the reactivity, as a parallel to the 'position effect' in the Rh system. In five of our families, families 1, 3, 7, 10 and 11, we have apparently HLA-identical sibs, and in four of these the identical sibs show almost identical HLA reactivity of their RBC. In the fifth family, however (family 3), a relatively great difference was observed. This could mean that the strength of reactivity is not governed from the HLA locus only, but the difference found is not greater than the occasional variation seen
63
in some cases. This variation has occasionally been observed to exceed 20 ( A OD x 100) in rare cases (lo), but is usually less than 10. Unfortunately, we were not able to retest the members of family 3. If only one family had been examined, e.g. family 10, we would think that homozygosity is deciding reactivity, because all the homozygous individuals show a stronger reactivity than the heterozygous. However, many exceptions are seen in other families (families 6, 9, 11-13). In previous population studies, we have also noted that homozygosityheterozygosity does not seem to be important for the HLA reactivity of RBC [6, 8,91. If the reactivity were determined by a single dominant gene outside the HLA system, we would not expect any offspring tc. show greater reactivity than any of the parents, if both carry the antigen. We have no families in this study where both parents are non-reactors for the same antigen with reactor children (that is negative x negative matings with positive children), but family 11 is close to this pattern. Both parents carry HLA-B7 (medium and non-reactors, respectively). All the children are strong reactors, including 11.5, the heterozygous one. The members of this family were all examined with several antisera, all showing the same as the one listed in the table. Again, the difference might be explained by occasional variation. However, this is not seen very often. It is not evident after these family studies that the strength of HLA reactivity of RBC is inherited at all. We may see different variations of reactivity within the families, but it will require very extensive family studies to see if these variations are smaller than in population studies. We might, how-
64
Nordhagen
ever, note that a strong B8 reactivity, which is a rare trait, is found in two propositi (families 2 and 9, and the same strong B8 reactivity is also found in one of the parents in both families. Thus, if the strength of HLA reactivity of RBC is genetically determined, it is probably polygenic, and may possibly also be influenced by other, non genetic factors.
References 1 Fisher, Sir Ronald: The variation in strength
2
3
4
5
6
of the human blood group P. Heredity 7: 8189 (1953). Hart, M. van der; Szaloky, A.; Berg-Loonen, E. M. van den; Engelfriet, C. P. et Loghem, J. J. van: PrCsence d’antighnes HL-A sur les hematies d’un donneur normal. Noun Revue fr. HCmat. 14: 555-563 (1974). Ilenningsen, K.: On the heredity of blood factor P. Acta path. microbiol. scand. 26: 769785 (1949). Morton, J. A.; Pickles, M. M., and Sutton, L.: The correlation of the Bga blood group with the HL-A7 leucocyte group: demonstration of antigenic sites on red cells and leucocytes. Vox Sang. 21: 536-547 (1969). Morton, J. A.; Pickles, M. M.; Sutton, L., and Skov, F.: Identification of further antigens on red cells and lymphocytes. VOXSang. 21: 141153 (1971). Nordhagen, R. and Orjastzter, H.: Association between HL-A and red cell antigens. An AutoAnalyzer study. Vox Sang. 26: 97-106 (1974).
7 Nordhagen, R.: Association between HL-A and red cell antigens. 11. Absorption and titration analyses. Vox Sang. 27: 124-133 (1974). 8 Nordhagen, R.: Association between HL-A and red cell antigens. 111. Studies of haemagglutinins in cytotoxic anti-HL-A7- and anti-HL-AS-related sera. Vox Sang. 29: 23-35 (1975). 9 Nordhagen, R.: Association between HLA and red cell antigens. IV. Further studies of haemagglutinins in cytotoxic HLA antisera. Vox Sang. 32: 82-89 (1977). 10 Nordhagen, R.: Association between HLA and red cell antigens. V. A further study of the nature and behaviour of the HLA antigens on red blood cells and their corresponding haemagglutinins. Vox Sang. 35: 49-57 (1978). 11 Race, R. R.; Sanger, R., and Lawler, S. D.: The Rh antigen Du. Ann. Eugen. 14: 171-184 (1948). 12 Race, R.R. and Sanger, R.: Blood groups in man; 6th ed., p. 499 (Blackwell, Oxford 1975). 13 Renton, P. H. and Stratton, F.: Rhesus type Du. Ann. Eugen. 15: 189-209 (1950). 14 Seaman, M. J.; Benson, R.; Jones, M. N.; Morton, J. A., and Pickles, M. M.: The reactions of the Bennett-Goodspeed group of antibodies with the AutoAnalyzer. Br. J. Haemat. 13: 464-473 (1967).
Received: July 18, 1977 Accepted: October 8, 1977
Dr. Rannveig Nordhagen, National Institute of Public Health, SIFF, Postuttak Oslo 1 (Norway)
