Inherited Mosaicism Affecting the ABO Blood Groups W. L. MARSH,M. E. NICHOLS, R. @YEN, F. DECARY, C. WHITSETT, AND E. L. ETHRIDCE From the Serology and Genetics Laboratory. Lindsley F. Kimball Research Institute of the New York Blood Center,
New York and the Calijornia Transfusion Service, Los Angeles, Calijornia
Blood samples from two families and three other unrelated people contain a mixture of red blood cells of two different A B O types. Previously described causes of red blood cell mosaicism have been,excluded from responsibility. Other blood group systems are not involved. The phenotypes, which have been designated A B O mos, are inherited through a variant allele at the A B O locus and appear to arise from a change in regulation of an A B O gene of some somatic cells.
AN I N VIVO mixed population of red blood cells having different groups may result from a number of causes. If blood transfusion or transplacental hemorrhage can be excluded, then twin ~ h i m e r i s mdispermy? ,~ or somatic mutation may be responsible. In addition leukemia,* acquired A, or B antigen^,^.^ or may mixed-field polyaggl~tinability~~'~~~~ give rise to a serological picture in which cells of more than one blood type appear to be present. Mixtures of blood red cells of different ABO types, which cannot be explained by any of these phenomena, have been described by Furahata, et u I . , Kitahama,7 ~ and Ogita, et al." In each of these cases the mixed blood-cell population was shown to be an inherited characteristic. We have studied blood samples from members of two families and from three other unrelated people that contain a separable mixture of red blood cells of different ABO types. In both families, a parent and some of the children have a mixture of A2 and 0 red blood cells in their circulation. The other samples are mixtures of B and 0, A,B and A,, and A,B and B. The proportion of different cell types
varies in the unrelated bloods, but the proportions in the affected members within each family are the same. Although the serological details differ slightly from those described in previous reports of mixed ABO blood in broad outline they are comparable and we think it probable that they are examples of the same phenomenon. The ABO mosaic phenotype appears to arise by inheritance of a variant allele at the ABO locus and the term A mos or B mos is proposed to describe it. Family H Mr. H. is a 27-year-old healthy white blood donor of English parentage. Difficulty was encountered in determining his ABO blood type in routine tests. Tests with anti-A sera showed a few small agglutinates and it was assumed initially that he was of type A,. Further studies, made in parallel with known A, red blood cells, revealed that the serological reactions given by his cells differed from those given by A, and established that his blood contained a double population of A, and 0 red cells.
Received for publication March 3, 1975, accepted March 16, 1975. Supported in part by grant HL09011 from the National Heart and Lung Institute, N I H . Transfusion Nov.-Dec. 1975
589
Tests with Anti-A Reagents The red blood cells of Mr. H. reacted to give many firm, small clumps of agglutinated cells in a sea of unagglutinated red blood cells when they were tested with 26 anti-A testing sera, six potent anti-A,B (group 0) sera, two group 0 sera from which the anti-B component had been removed by absorption with B red blood cells, and with rabbit anti-A serum. The cells had weak absorptive capacity for anti-A and anti-A was recovered in a heat eluate subsequently prepared from them. His red blood cells were not agglutinated by Dolichos bijorus anti-A, reagent. In contrast, the A, control red blood cells showed a variable strength of agglutination with the anti-A sera, and gave greatly enhanced reactions in terms of Volume 15
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MARSH’. ET AL.
avidity and number of agglutinated cells when group 0 serum was used. Preliminary treatment of the red blood cell suspensions with ficin did not change the mixed agglutination result obtained with the cells of Mr. H. but greatly enhanced the strength of reaction obtained with A, cells. Similarly, use of a powerful immune anti-A serum by the indirect antiglobulin test gave almost complete agglutination of A, cells but did not increase the proportion of agglutinated red blood cells of the propositus. The mixed agglutination reaction obtained with anti-A was suppressed by prior addition of soluble group A substance to the testing serum, but addition of B o r H substances had no effect. Separation of the Two Cell Populations The A cells in a 10 per cent suspension of the donor’s red blood cells were agglutinated with anti-A serum and then removed by gravity sedimentation. Tests with a battery of potent anti-A sera on the supernatant free cells gave no further agglutination. The supernatant cells had no absorptive capacity for anti-A and an eluate prepared from them yielded only a trace of anti-A, no more than would be expected from the presence of an occasional residual A red blood cell. The mass of agglutinated A red blood cells was separated from the free cells by repeated resuspension in saline and sedimentation. An eluate prepared from a portion of the agglutinated cells yielded potent anti-A. ;The remainder of the agglutinated cells was heated in isotonic saliva from a group A secretor for ten minutes at 50 C with continuous agitation. This process released many of the cells, which remained in free suspension after subsequent washing with saline. These separated cells were strongly agglutinated by anti-A serum, with few nonreactive cells. The proportion of A red blood cells in Mr. H’s blood was determined by differential agglutination with anti-A serum and counting the unagglutinated type 0 cells in a hemocytometer. Five counts on different preparations gave a range of 12 to 15 per cent, with a mean of 14 per cent A cells and 86 per cent 0 cells. A count by the same technique on a ficinised cell preparation showed 15 per cent A cells. The proportion of A and 0 red blood cells is a constant feature of Mr. H’s blood. Repeated counts after 51 days and again nine months later showed the same result. An artificial mixture of A, and 0 red blood cells in the same proportion was compared with the cells of Mr. H. in a titration study with anti-A serum. The results given by the two samples were indistinguishable.
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Other Blood Groups The red blood cells of Mr. H. were not agglutinated by any of eight examples of anti-B typing sera, by rabbit anti-B, by inert serum from an AB person, o r by anti-T extract of Arachis hypogea. Red blood cells of both the whole blood and the separated group 0 fraction were strongly agglutinated by both Ulex europeaus and eel anti-H. In titration studies with these reagents the reactions were comparable to those obtained with group 0 controls. Titration studies using anti-I and anti-i sera showed that all of the red blood cells possessed a normal I adult group status. Typing for antigens of the Rh, MN, P, Lewis, Lutheran, Kell, Duffy, and Kidd blood groups showed nothing unusual and no evidence of cell admixture. Tests for these antigens on separated group 0 red blood cells gave the same result as did tests on the whole blood sample. The whole blood and separated 0 and A cell populations were each tested for alkali-resistant fetal hemoglobin. None was detected. Saliva Studies Quantitative inhibition studies using both dilutions of saliva and dilutions of anti-serum showed that the propositus secreted Lea and Lebsubstances and a high concentration of H substance. Inhibition tests with his saliva against dilutions of anti-A sera showed trace inhibition but only marginally greater than that obtained with control A nonsecretor saliva (Table 1). Tests on the Serum of Mr. H . Strong anti-B agglutinins (titer: 32) were present. No anti-A or anti-A, activity could be detected in tests over the temperature range 0 to 37 C using saline suspended red blood cells, or by enzyme or antiglobulin techniques. No irregular blood group antibodies were found in tests with a panel of extensively typed group 0 red blood cells. No significant amounts of H, A, or B substances were detected by inhibition tests in which the serum was added to progressive dilutions of appropriate antisera. Hematological Studies Red and white blood cell counts, hematocrit, and hemoglobin levels were within the normal range. The reticulocyte count was 0.6 per cent. Examination of a blood film showed no morphological abnormality and no immature or atypical cells were found. Family Studies The investigation thus far had shown a mosaic phenomenon affecting the ABO blood group. The
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INHERITED ABO MOSAICISM Table 1. The Inhibitory Effect of Saliva from Donor H. on Anti-A Reciprocals of Dilutions of Anti-A Serum
Saliva Added: A1 secretor A2 secretor A1 nonsecretor Donor H Saline control
2
4
8
16
32
64
128
256
3
0
0
0
0
10
10
10
10
2 0 5
0 0 3
16 46
10
0 0 3 0 5
3
3 8 8 10
0 0 5 3 8
0
5 10
0 0 8 8 8
0
8
Score
39 59
All saliva samples diluted with an equal volume of saline.
propositus is not known to be a twin and has no unusual attributes in terms of eye color or skin pigmentation, but even so unsuspected twin chimerism seemed the most probable explanation. The family study immediately excluded this possibility, however, when it was found that the father and one sibling possessed the same mosaic blood type. The mother and a second sibling were type 0 (Fig. 1). Comparative tests on the blood of the three mosaic individuals showed not only that the serological reactions were the same but that the proportion of the two cell populations was approximately the same in the three people. Quantitative differential agglutination showed 12 per cent A, cells in the blood of the father and 15 per cent A, cells in the blood of the sister. Neither the father nor the sister showed evidence of cell admixture in tests for other blood group antigens.
Family Sc Miss Sc. is a 19-year-old healthy blood donor of Italian/Albanian descent. Difficulty in determining her ABO blood group led to the discovery that her blood contained a double population of A, and 0 red blood cells. Tests for red blood cell antigens of other blood group systems showed no evidence of admixture. The donor is not a twin and has no unusual physical attributes of eye color or skin pigmentation. Serological Findings The results of ABO typing tests were similar to those obtained with the blood of Mr. H. Scattered, firm agglutinates with many free red blood cells were found in tests with anti-A and anti-A,B sera. Anti-B and Dolichos biflorus anti-A, reagents were nonreactive. Strong reactions, comparable to group 0 controls, were obtained with human and Ulex europeaus anti-H reagents. Separation of the A cell population by differential agglutination with anti-A, left free red blood cells that could not be agglutinated by, and had no absorptive capacity for, other anti-A sera. Quan-
titative differential agglutination showed that 5 per cent of the red blood cells were A,. The donor’s saliva contained Lea and Leb substances and a high concentration of H substance. Trace inhibition of anti-A was obtained with her saliva. Her serum contains anti-B but has no anti-A activity. A repeat test after an interval of 90 days showed no change in the serological characteristics. Family study One of four siblings and the father possess the same A mos phenotype; three other siblings and mother are type 0. The serological characteristics of the A mos phenotype present in the three family members are the same. The blood of each contains a mixture of A, and 0 red blood cells; approximately 5 per cent are A,. That this small proportion of A, cells can be easily overlooked is evident from the fact that the father has been previously tested elsewhere and reported to be 0. Extensive typing for other red blood cell antigens show no further evidence of blood group mosaicism in any of the family. The saliva of the A mos individuals contains a high concentration of H substance. Each gives trace inhibition against anti-A serum, but on a level that cannot be considered conclusive evidence of soluble A substance.
w
12% 88%
A2
0
A?
FIG. I .
Pedigree of the H . family
c
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Table 2. Inhibitory Effect of Saliva from Donor W o n Anti-B Serum Reciprocals of Dilutions of Anti-B 1
2
4
Red cells Group B Donor W
10
10
5
5
10 5
Group B Donor W
10 5
10
Group B Donor W
8 0
5
3 0
10 2
3 0
8
16
32
64
128
Saline Added to Each Dilution 10 10 8 8 5 5 5 5 4 3 Donor W Saliva Added to Each Dilution 5 3 2 0 8 0 0 0 0 0 B Secretor Saliva Added to Each Dilution 0 0 0 0 0
0
Other Probable Examples of ABO mos Phenotype Blood samples from three other individuals have been studied which are believed to be further examples of the ABO mosaic phenotype. The serological characteristics in each case have remained constant over a period of time, but family studies have not been possible. None of them is a twin.
B mos Phcnorype The blood of a black man, who is a blood donor, is a mixture of 15 per cent B cells and 85 per cent 0 cells. The partial agglutination with anti-B serum is specifically inhibited by prior addition of a low concentration of group B specific substance. A higher level of B substance must be added to the same antiserum to inhibit the agglutination of an artificial B:O cell mixture. It a p pears that the B antigen present on a minority of the donor’s red cells is weaker than normal. Enzyme treatment o r use of a powerful immune anti-B serum does not increase the proportion of reactive cells. Separated free cells have no absorptive capacity for anti-B. The donor’s scrum contains strong anti-A, but tests for anti-B using saline agglutination, enzyme, and antiglobulin methods were negative. Extensive typing for different red blood cell antigens shows no evidence of other blood group involvement. Blood transfusions have not been given. Repeat tests on the donor after an interval of three months showed the same serological result. No saliva sample was available. A , B mos Phenotype Mr. W. is a healthy donor whose blood is a mosaic of A,B and A , cells. He has donated blood at intervals for more than five years. While his red blood cells have a normal A , antigen and can be completely agglutinated by anti-A or ’ anti-A,
0
0
0
0
256
Score
5
76
0
37
0
48
0
10
0
16
0
0
sera, he has always posed problems in the interpretation of tests with anti-B. Tests with ten anti-B sera all show a scattering of small firm agglutinates in a background of unagglutinated cells. Use of a powerful hyperimmune anti-B serum or prior treatment of the cells with papain does not change this result. Tests on separated unagglutinated cells with other anti-B sera do not result in further agglutination. Extensive typing tests show no other blood group involvement, and the donor.’s red blood cells are not polyagglutinable. No anti-A o r anti-B can be detected in his serum. Differential agglutination studies using anti-B serum show the A,B and A , cells to be in the proportions of 15 per cent to 85 per cent, respectively. The donor’s cells were compared with an artificial mixture of A , B and A , red blood cells in a titration experiment with anti-B serum. Both titration end points were the same, but the reaction given by the cells of the propositus is less avid. Further studies, in which graded amounts of B secretor saliva were added to different dilutions of anti-B serum, revealed that the agglutination of the propositus’s red blood cells could be suppressed by one quarter of the concentration of B substance needed to inhibit agglutination of the artificial mixture. It was concluded from these results that the B antigen present on 15 per cent of the donor’s red blood cells is more weakly reactive than is the antigen of normal B cells. The donor’s saliva contains H and A substances and also causes modest inhibition of the activity of anti-B serum against group B red blood cells, and more striking inhibition of activity of the same serum against his own red blood cells (Table 2). A mos Bphenorypc
A 66-year-old white man gave difficulty in establishing his ABO blood type during routine laboratory tests prior to a transurethral resection of the prostate. He has never been given a blood
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transfusion. His blood shows no hematological abnormality. His red blood cells react strongly with anti-B, but show mixed agglutination in tests with anti-A sera. The reaction with anti-A is not enhanced by prior treatment of the cells with papain. Free red blood cells, separated after agglutination of the A cell population, cannot be agglutinated by other anti-A sera. All of his red blood cells are agglutinated by Ulex europeaus anti-H, but they are nonreactive with Dolichos bijlorous extract and with serum from group AB persons. Quantitative differential agglutination tests with anti-A sera show the cell populations to be in the proportion 14 per cent A,B and 86 per cent B. The red blood cells have a normal I adult status and extensive typing for other blood group antigens shows nothing unusual and no other evidence of mosaicism. The cells aggregate normally when they are suspended in I per cent Polybrene solution, evidence that the membrane sialic acid level is within the normal range. No anti-A or anti-B is present in the patient’s serum. His saliva contains B and H substances and clearly demonstrable A substance.
Cytogenetic Studies
Chromosome studies were made on the A mos propositus of family Sc. and on A,B mos donor W. Phytohemagglutinin-stimulated lymphocytes were studied by G and C banding technique^.'^ The chromosome complement of both individuals was normal with no evidence of structural rearrangement or mosaicism. Cells of the A,B mos donor were examined specifically for the presence of chromosome quadriradial configurations and none was found in 150 cells examined.
Discussion
The ABO mos phenotypes represent a well-defined phenomenon of the ABO blood groups and are a permanent characteristic of the individual. The red blood cells are not polyagglutinable, nor do they have the characteristics of cells with acquired A or B antigens. Blood transfusion, transplacental hemorrhage, chimerism, dispermy, leukemia, somatic mutation and recognizable chromosome deletion or translocation have all been excluded as possible causes. The differentiation of A mos from A, is not easy, for in direct tests with anti-A serum both types give small agglutinates with many free cells. Enzyme treatment of the red blood cells greatly increases the strength of agglutination given by A, but causes no significant change in the mixed agglutination reaction given by A mos. We think that this procedure is the easiest means of distinguishing the two phenotypes. The similarities and differences between the A, and A mos phenotypes are summarized in Table 3. The propositus of family H was first studied in 1966. Numerous blood samples of apparent A,, A,, or weak B type have been examined during the succeeding years in our laboratory. The great majority have proved to be weak ABO sub types and the ABO mos phenotype is rare. The serological results show that expression of H on the red blood cells is not affected by the mosaicism and only A or B is modified. In both of the families, each time
Table 3. Serological Characteristics of the A and A mos Phenotypes ~~~~~~
~~~~~
Tests with: Anti-A Anti-A, enzyme-treated red cells Anti-A, B Anti-AB, enzyme-treated red cells
~
~
A3 Phenotype Weak mixed field agglutination Stronger agglutination, fewer free cells Stronger agglutination Good agglutination with few free cells After agglutination with anti-A, separated free cells have A antigen
~~
~~~~~
~~
~~
A rnos Phenotype Mixed field agglutination Mixed field unchanged Mixed field unchanged Mixed field unchanged After agglutination with anti-A, separated free cells lack A antigen
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MARSH, ET AL.
the A gene was transmitted to a child it was expressed as an A mos phenotype, and neither A mos parent has produced a child of normal A type. The mosaic phenotype must, therefore, be an effect of an allele at the ABO locus. The A mos B and A,B mos individuals have normal expression of their respective concomitant B and A , genes. It follows that the modified gene expression cannot result from a defect in an ABO precursor substance. The A mos and B mos antigens are weaker than the antigens of normal red blood cells of the same type. However, the bloods contain two separable populations of weakly antigenic and negative red blood cells and not a single population of cells having a continuous gradient of antigenic strength. The reactive red blood cells in the A mos phenotype are classified as A, on the basis of nonreactivity with lectin antiA,, but it might be more appropriate to view them as “weak A.” When normal blood samples are tested with anti-A or anti-B, small numbers o f inagglutinable red blood cells are found not infreq~ent1y.l~ The reason why a few cells are nonreactive is not known.’, It is possible that they arise by somatic mutation of an ABO gene, although Winkelstein and Mollison’6 have shown that unagglutinated B cells do have B antigen. The mosaic phenotype differs sharply from this inagglutinable cell phenomenon, for the aberrant cells of the ABO mos phenotype lack antigen and are present in large numbers. The reactive cells of the ABO mos type have a weakened form of the affected antigen, and the characteristic is inherited. Our experience of the red blood cell differential agglutination technique using anti-A and anti-B sera indicates that it gives accurate and. reproducible results. Five separate counts on the blood of Mr. H. for example, all fell within the range of 12 to 15 per cent type A cells. The finding that most of the mosaic individuals have about 15 per cent reactive red blood cells is unlikely to be a reflection of technical imprecision. This
Nov.-Dec. 1975
consistency may be a coincidence, but it may also be an effect of a precise genetic mechanism that determines a change in somatic gene expression in specific cell clones. The results of tests for soluble A or B group substances in saliva of the mosaic individuals were not always conclusive but we think it probable that a low concentration is present. It is possible, therefore, that the mosaicism is not restricted to erythropoietic cells but also affects secretory tissues and possibly other cellular elements. The genetic background to the ABO mos phenotype cannot yet be understood. The phenomenon is inherited, and yet it affects only a portion of the somatic cells. It is unlikely to be caused by inheritance of a modifying gene, for such a gene would have to be closely linked to the ABO locus and activated in some unknown manner in only a portion of the cells. A striking feature of the mosaic phenotype is the precision of its inheritance, for not only are the antigenic characteristics of parent and child the same, but the proportion of the two-cell populations is closely comparable in different affected members of the same family. In a broad sense, people with A, and B, phenotypes may also be viewed as mosaics, for an A or B gene is expressed in one cell line, the secretory tissues, but not in the red blood cells. The A mos and B mos phenotypes go a step further in that there are differences in expression of an A or B gene in the same cell line. The biological mechanism by which genes are activated is complex, but it must be assumed that the ABO mos phenotypes arise by germ-cell controlled changes in the background regulation of an ABO gene in some somatic cells. References Berman, H. J., J. Srnarto, C. H. Issitt, P. D. Issitt, W. L. Marsh, and L. Jensen: Tn-activation with acquired A-like antigen. Transfusion 2:35, 1972. 2. Bird, G. W. G., N. K. Shinton, and J. Wingharn: Persistent mixed-field polyagglutination. Br. J. Haematol. 21:443, 1971. 3. Cameron, C., F. Graham, I. Dunsford, G . Sickles,
I.
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5.
6.
7.
8. 9.
10.
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12.
13.
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C. R. Macpherson, A. Cahan, R. Sanger, and R. R. Race. Acquisition of a B-like antigen by red blood cells. Br. Med. J. 11:29, 1959. Dunsford, I., C. C. Bowley, A. M. Hutchison, J. S. Thompson, R. Sanger, and R. R. Race: A human blood-group chimera. Br. Med. J. 11231. 1953. Furuhata, T., M. Kitahama, and T. Nozawa: A family study of the so-called blood group chimera. Proc. Jap. Acad. 35:55, 1959. Gartler, S. M., S. H. Waxman, and E. Giblett: An X X / X Y human hermaphrodite resulting from double fertilization. Proc. Nat. Acad. Sci. U.S.A.48:332, 1962. Kitahama, M.: Two family studies of the erythrocyte antigen mosaicism. Shinshu Med. J . 12:641, 1963. Lophem. J. J. van. H. Dorfmeier. and M. V. D. i a r t : T w o A antigens with abnormal serologic properties. Vox Sang. 2:16, 1957. Marsh, W. L., W. J . Jenkins, and W. W. Walther: Pseudo B: an acquired group antigen. Br. Med. J. 11:63, 1959. Myllyla, G., U. Furuhjelm, S. Nordling, A. Pirkola, P. Tippett, J . Gavin, and R. Sanger. Persistent mixed field polyagglutinability. Electrokinetic and serological aspects. Vox Sang. 20:7, 1971. Ogita, Z., H. Kikkawa, K. Yarnamoto, and F. Murakami: Erythrocyte antigen mosaicism in a Japanese family. Jap. J . Hum. Genet. 13:264, 1969. Race, R. R., and R. Sanger: Blood Groups in Man. 5th ed. Oxford, Blackwell, p. 38. Solomon, J . M., M. B. Gibbs, and A. J. Bowdler: Methods in quantitative hemagglutination. Part I . VoxSang. 10:54, 1965.
14. Sturgeon, P., D. T. McQuiston, H. F. Taswell, and C. J . Allan: Permanent mixed-field polyagglutinability (PMFP). Vox Sang. 25:481, 1973. 15. The National Foundation: Standardization in human cytogenetics. Birth Defects. Paris Conference, I97 I . Original article series, No. VIII, p. 7, (National Foundation, New York 1972). 16. Winkelstein, J . A,, and P. L. Mollison: The antigen content of ‘inagglutinable’ group B erythrocytes. Vox Sang. 10:614, 1965.
Acknowledgments We are grateful to the Humphreys and Sciarretta families for their enthusiastic cooperation, to Dr. J. L. German for performing the cytogenetic studies, and to Mr. P. Ferrau for his technical assistance. W. Laurence Marsh, F.I.M.L.S., M.I. Biol., Immunohematologist, Serology and Genetics Laboratory, Lindsley F. Kimball Research Institute of the New York Blood Center, 310 East 67th Street, New York, N.Y. 10021. Margaret E. Nichols, F.I.M.L.S., Supervisor. Serology and Genetics Laboratory, New York Blood Center. Ragnhild @yen, Chief Technologist, Serology and Genetics Laboratory, New York Blood Center. Francine Decary, M.D., Trainee in Blood Banking, New York Blood Center. Carolyn Whitsett, M.D., Trainee in Blood Banking, New York Blood Center. Emma L. Etheridge, Staff Technologist, California Transfusion Service, California.
New York and the Calijornia Transfusion Service, Los Angeles, Calijornia
Blood samples from two families and three other unrelated people contain a mixture of red blood cells of two different A B O types. Previously described causes of red blood cell mosaicism have been,excluded from responsibility. Other blood group systems are not involved. The phenotypes, which have been designated A B O mos, are inherited through a variant allele at the A B O locus and appear to arise from a change in regulation of an A B O gene of some somatic cells.
AN I N VIVO mixed population of red blood cells having different groups may result from a number of causes. If blood transfusion or transplacental hemorrhage can be excluded, then twin ~ h i m e r i s mdispermy? ,~ or somatic mutation may be responsible. In addition leukemia,* acquired A, or B antigen^,^.^ or may mixed-field polyaggl~tinability~~'~~~~ give rise to a serological picture in which cells of more than one blood type appear to be present. Mixtures of blood red cells of different ABO types, which cannot be explained by any of these phenomena, have been described by Furahata, et u I . , Kitahama,7 ~ and Ogita, et al." In each of these cases the mixed blood-cell population was shown to be an inherited characteristic. We have studied blood samples from members of two families and from three other unrelated people that contain a separable mixture of red blood cells of different ABO types. In both families, a parent and some of the children have a mixture of A2 and 0 red blood cells in their circulation. The other samples are mixtures of B and 0, A,B and A,, and A,B and B. The proportion of different cell types
varies in the unrelated bloods, but the proportions in the affected members within each family are the same. Although the serological details differ slightly from those described in previous reports of mixed ABO blood in broad outline they are comparable and we think it probable that they are examples of the same phenomenon. The ABO mosaic phenotype appears to arise by inheritance of a variant allele at the ABO locus and the term A mos or B mos is proposed to describe it. Family H Mr. H. is a 27-year-old healthy white blood donor of English parentage. Difficulty was encountered in determining his ABO blood type in routine tests. Tests with anti-A sera showed a few small agglutinates and it was assumed initially that he was of type A,. Further studies, made in parallel with known A, red blood cells, revealed that the serological reactions given by his cells differed from those given by A, and established that his blood contained a double population of A, and 0 red cells.
Received for publication March 3, 1975, accepted March 16, 1975. Supported in part by grant HL09011 from the National Heart and Lung Institute, N I H . Transfusion Nov.-Dec. 1975
589
Tests with Anti-A Reagents The red blood cells of Mr. H. reacted to give many firm, small clumps of agglutinated cells in a sea of unagglutinated red blood cells when they were tested with 26 anti-A testing sera, six potent anti-A,B (group 0) sera, two group 0 sera from which the anti-B component had been removed by absorption with B red blood cells, and with rabbit anti-A serum. The cells had weak absorptive capacity for anti-A and anti-A was recovered in a heat eluate subsequently prepared from them. His red blood cells were not agglutinated by Dolichos bijorus anti-A, reagent. In contrast, the A, control red blood cells showed a variable strength of agglutination with the anti-A sera, and gave greatly enhanced reactions in terms of Volume 15
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MARSH’. ET AL.
avidity and number of agglutinated cells when group 0 serum was used. Preliminary treatment of the red blood cell suspensions with ficin did not change the mixed agglutination result obtained with the cells of Mr. H. but greatly enhanced the strength of reaction obtained with A, cells. Similarly, use of a powerful immune anti-A serum by the indirect antiglobulin test gave almost complete agglutination of A, cells but did not increase the proportion of agglutinated red blood cells of the propositus. The mixed agglutination reaction obtained with anti-A was suppressed by prior addition of soluble group A substance to the testing serum, but addition of B o r H substances had no effect. Separation of the Two Cell Populations The A cells in a 10 per cent suspension of the donor’s red blood cells were agglutinated with anti-A serum and then removed by gravity sedimentation. Tests with a battery of potent anti-A sera on the supernatant free cells gave no further agglutination. The supernatant cells had no absorptive capacity for anti-A and an eluate prepared from them yielded only a trace of anti-A, no more than would be expected from the presence of an occasional residual A red blood cell. The mass of agglutinated A red blood cells was separated from the free cells by repeated resuspension in saline and sedimentation. An eluate prepared from a portion of the agglutinated cells yielded potent anti-A. ;The remainder of the agglutinated cells was heated in isotonic saliva from a group A secretor for ten minutes at 50 C with continuous agitation. This process released many of the cells, which remained in free suspension after subsequent washing with saline. These separated cells were strongly agglutinated by anti-A serum, with few nonreactive cells. The proportion of A red blood cells in Mr. H’s blood was determined by differential agglutination with anti-A serum and counting the unagglutinated type 0 cells in a hemocytometer. Five counts on different preparations gave a range of 12 to 15 per cent, with a mean of 14 per cent A cells and 86 per cent 0 cells. A count by the same technique on a ficinised cell preparation showed 15 per cent A cells. The proportion of A and 0 red blood cells is a constant feature of Mr. H’s blood. Repeated counts after 51 days and again nine months later showed the same result. An artificial mixture of A, and 0 red blood cells in the same proportion was compared with the cells of Mr. H. in a titration study with anti-A serum. The results given by the two samples were indistinguishable.
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Nov:Dec. 1975
Other Blood Groups The red blood cells of Mr. H. were not agglutinated by any of eight examples of anti-B typing sera, by rabbit anti-B, by inert serum from an AB person, o r by anti-T extract of Arachis hypogea. Red blood cells of both the whole blood and the separated group 0 fraction were strongly agglutinated by both Ulex europeaus and eel anti-H. In titration studies with these reagents the reactions were comparable to those obtained with group 0 controls. Titration studies using anti-I and anti-i sera showed that all of the red blood cells possessed a normal I adult group status. Typing for antigens of the Rh, MN, P, Lewis, Lutheran, Kell, Duffy, and Kidd blood groups showed nothing unusual and no evidence of cell admixture. Tests for these antigens on separated group 0 red blood cells gave the same result as did tests on the whole blood sample. The whole blood and separated 0 and A cell populations were each tested for alkali-resistant fetal hemoglobin. None was detected. Saliva Studies Quantitative inhibition studies using both dilutions of saliva and dilutions of anti-serum showed that the propositus secreted Lea and Lebsubstances and a high concentration of H substance. Inhibition tests with his saliva against dilutions of anti-A sera showed trace inhibition but only marginally greater than that obtained with control A nonsecretor saliva (Table 1). Tests on the Serum of Mr. H . Strong anti-B agglutinins (titer: 32) were present. No anti-A or anti-A, activity could be detected in tests over the temperature range 0 to 37 C using saline suspended red blood cells, or by enzyme or antiglobulin techniques. No irregular blood group antibodies were found in tests with a panel of extensively typed group 0 red blood cells. No significant amounts of H, A, or B substances were detected by inhibition tests in which the serum was added to progressive dilutions of appropriate antisera. Hematological Studies Red and white blood cell counts, hematocrit, and hemoglobin levels were within the normal range. The reticulocyte count was 0.6 per cent. Examination of a blood film showed no morphological abnormality and no immature or atypical cells were found. Family Studies The investigation thus far had shown a mosaic phenomenon affecting the ABO blood group. The
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59 1
INHERITED ABO MOSAICISM Table 1. The Inhibitory Effect of Saliva from Donor H. on Anti-A Reciprocals of Dilutions of Anti-A Serum
Saliva Added: A1 secretor A2 secretor A1 nonsecretor Donor H Saline control
2
4
8
16
32
64
128
256
3
0
0
0
0
10
10
10
10
2 0 5
0 0 3
16 46
10
0 0 3 0 5
3
3 8 8 10
0 0 5 3 8
0
5 10
0 0 8 8 8
0
8
Score
39 59
All saliva samples diluted with an equal volume of saline.
propositus is not known to be a twin and has no unusual attributes in terms of eye color or skin pigmentation, but even so unsuspected twin chimerism seemed the most probable explanation. The family study immediately excluded this possibility, however, when it was found that the father and one sibling possessed the same mosaic blood type. The mother and a second sibling were type 0 (Fig. 1). Comparative tests on the blood of the three mosaic individuals showed not only that the serological reactions were the same but that the proportion of the two cell populations was approximately the same in the three people. Quantitative differential agglutination showed 12 per cent A, cells in the blood of the father and 15 per cent A, cells in the blood of the sister. Neither the father nor the sister showed evidence of cell admixture in tests for other blood group antigens.
Family Sc Miss Sc. is a 19-year-old healthy blood donor of Italian/Albanian descent. Difficulty in determining her ABO blood group led to the discovery that her blood contained a double population of A, and 0 red blood cells. Tests for red blood cell antigens of other blood group systems showed no evidence of admixture. The donor is not a twin and has no unusual physical attributes of eye color or skin pigmentation. Serological Findings The results of ABO typing tests were similar to those obtained with the blood of Mr. H. Scattered, firm agglutinates with many free red blood cells were found in tests with anti-A and anti-A,B sera. Anti-B and Dolichos biflorus anti-A, reagents were nonreactive. Strong reactions, comparable to group 0 controls, were obtained with human and Ulex europeaus anti-H reagents. Separation of the A cell population by differential agglutination with anti-A, left free red blood cells that could not be agglutinated by, and had no absorptive capacity for, other anti-A sera. Quan-
titative differential agglutination showed that 5 per cent of the red blood cells were A,. The donor’s saliva contained Lea and Leb substances and a high concentration of H substance. Trace inhibition of anti-A was obtained with her saliva. Her serum contains anti-B but has no anti-A activity. A repeat test after an interval of 90 days showed no change in the serological characteristics. Family study One of four siblings and the father possess the same A mos phenotype; three other siblings and mother are type 0. The serological characteristics of the A mos phenotype present in the three family members are the same. The blood of each contains a mixture of A, and 0 red blood cells; approximately 5 per cent are A,. That this small proportion of A, cells can be easily overlooked is evident from the fact that the father has been previously tested elsewhere and reported to be 0. Extensive typing for other red blood cell antigens show no further evidence of blood group mosaicism in any of the family. The saliva of the A mos individuals contains a high concentration of H substance. Each gives trace inhibition against anti-A serum, but on a level that cannot be considered conclusive evidence of soluble A substance.
w
12% 88%
A2
0
A?
FIG. I .
Pedigree of the H . family
c
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Nov.-Occ. 1975
Table 2. Inhibitory Effect of Saliva from Donor W o n Anti-B Serum Reciprocals of Dilutions of Anti-B 1
2
4
Red cells Group B Donor W
10
10
5
5
10 5
Group B Donor W
10 5
10
Group B Donor W
8 0
5
3 0
10 2
3 0
8
16
32
64
128
Saline Added to Each Dilution 10 10 8 8 5 5 5 5 4 3 Donor W Saliva Added to Each Dilution 5 3 2 0 8 0 0 0 0 0 B Secretor Saliva Added to Each Dilution 0 0 0 0 0
0
Other Probable Examples of ABO mos Phenotype Blood samples from three other individuals have been studied which are believed to be further examples of the ABO mosaic phenotype. The serological characteristics in each case have remained constant over a period of time, but family studies have not been possible. None of them is a twin.
B mos Phcnorype The blood of a black man, who is a blood donor, is a mixture of 15 per cent B cells and 85 per cent 0 cells. The partial agglutination with anti-B serum is specifically inhibited by prior addition of a low concentration of group B specific substance. A higher level of B substance must be added to the same antiserum to inhibit the agglutination of an artificial B:O cell mixture. It a p pears that the B antigen present on a minority of the donor’s red cells is weaker than normal. Enzyme treatment o r use of a powerful immune anti-B serum does not increase the proportion of reactive cells. Separated free cells have no absorptive capacity for anti-B. The donor’s scrum contains strong anti-A, but tests for anti-B using saline agglutination, enzyme, and antiglobulin methods were negative. Extensive typing for different red blood cell antigens shows no evidence of other blood group involvement. Blood transfusions have not been given. Repeat tests on the donor after an interval of three months showed the same serological result. No saliva sample was available. A , B mos Phenotype Mr. W. is a healthy donor whose blood is a mosaic of A,B and A , cells. He has donated blood at intervals for more than five years. While his red blood cells have a normal A , antigen and can be completely agglutinated by anti-A or ’ anti-A,
0
0
0
0
256
Score
5
76
0
37
0
48
0
10
0
16
0
0
sera, he has always posed problems in the interpretation of tests with anti-B. Tests with ten anti-B sera all show a scattering of small firm agglutinates in a background of unagglutinated cells. Use of a powerful hyperimmune anti-B serum or prior treatment of the cells with papain does not change this result. Tests on separated unagglutinated cells with other anti-B sera do not result in further agglutination. Extensive typing tests show no other blood group involvement, and the donor.’s red blood cells are not polyagglutinable. No anti-A o r anti-B can be detected in his serum. Differential agglutination studies using anti-B serum show the A,B and A , cells to be in the proportions of 15 per cent to 85 per cent, respectively. The donor’s cells were compared with an artificial mixture of A , B and A , red blood cells in a titration experiment with anti-B serum. Both titration end points were the same, but the reaction given by the cells of the propositus is less avid. Further studies, in which graded amounts of B secretor saliva were added to different dilutions of anti-B serum, revealed that the agglutination of the propositus’s red blood cells could be suppressed by one quarter of the concentration of B substance needed to inhibit agglutination of the artificial mixture. It was concluded from these results that the B antigen present on 15 per cent of the donor’s red blood cells is more weakly reactive than is the antigen of normal B cells. The donor’s saliva contains H and A substances and also causes modest inhibition of the activity of anti-B serum against group B red blood cells, and more striking inhibition of activity of the same serum against his own red blood cells (Table 2). A mos Bphenorypc
A 66-year-old white man gave difficulty in establishing his ABO blood type during routine laboratory tests prior to a transurethral resection of the prostate. He has never been given a blood
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INHERITED ABO MOSAICISM
Number6
transfusion. His blood shows no hematological abnormality. His red blood cells react strongly with anti-B, but show mixed agglutination in tests with anti-A sera. The reaction with anti-A is not enhanced by prior treatment of the cells with papain. Free red blood cells, separated after agglutination of the A cell population, cannot be agglutinated by other anti-A sera. All of his red blood cells are agglutinated by Ulex europeaus anti-H, but they are nonreactive with Dolichos bijlorous extract and with serum from group AB persons. Quantitative differential agglutination tests with anti-A sera show the cell populations to be in the proportion 14 per cent A,B and 86 per cent B. The red blood cells have a normal I adult status and extensive typing for other blood group antigens shows nothing unusual and no other evidence of mosaicism. The cells aggregate normally when they are suspended in I per cent Polybrene solution, evidence that the membrane sialic acid level is within the normal range. No anti-A or anti-B is present in the patient’s serum. His saliva contains B and H substances and clearly demonstrable A substance.
Cytogenetic Studies
Chromosome studies were made on the A mos propositus of family Sc. and on A,B mos donor W. Phytohemagglutinin-stimulated lymphocytes were studied by G and C banding technique^.'^ The chromosome complement of both individuals was normal with no evidence of structural rearrangement or mosaicism. Cells of the A,B mos donor were examined specifically for the presence of chromosome quadriradial configurations and none was found in 150 cells examined.
Discussion
The ABO mos phenotypes represent a well-defined phenomenon of the ABO blood groups and are a permanent characteristic of the individual. The red blood cells are not polyagglutinable, nor do they have the characteristics of cells with acquired A or B antigens. Blood transfusion, transplacental hemorrhage, chimerism, dispermy, leukemia, somatic mutation and recognizable chromosome deletion or translocation have all been excluded as possible causes. The differentiation of A mos from A, is not easy, for in direct tests with anti-A serum both types give small agglutinates with many free cells. Enzyme treatment of the red blood cells greatly increases the strength of agglutination given by A, but causes no significant change in the mixed agglutination reaction given by A mos. We think that this procedure is the easiest means of distinguishing the two phenotypes. The similarities and differences between the A, and A mos phenotypes are summarized in Table 3. The propositus of family H was first studied in 1966. Numerous blood samples of apparent A,, A,, or weak B type have been examined during the succeeding years in our laboratory. The great majority have proved to be weak ABO sub types and the ABO mos phenotype is rare. The serological results show that expression of H on the red blood cells is not affected by the mosaicism and only A or B is modified. In both of the families, each time
Table 3. Serological Characteristics of the A and A mos Phenotypes ~~~~~~
~~~~~
Tests with: Anti-A Anti-A, enzyme-treated red cells Anti-A, B Anti-AB, enzyme-treated red cells
~
~
A3 Phenotype Weak mixed field agglutination Stronger agglutination, fewer free cells Stronger agglutination Good agglutination with few free cells After agglutination with anti-A, separated free cells have A antigen
~~
~~~~~
~~
~~
A rnos Phenotype Mixed field agglutination Mixed field unchanged Mixed field unchanged Mixed field unchanged After agglutination with anti-A, separated free cells lack A antigen
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MARSH, ET AL.
the A gene was transmitted to a child it was expressed as an A mos phenotype, and neither A mos parent has produced a child of normal A type. The mosaic phenotype must, therefore, be an effect of an allele at the ABO locus. The A mos B and A,B mos individuals have normal expression of their respective concomitant B and A , genes. It follows that the modified gene expression cannot result from a defect in an ABO precursor substance. The A mos and B mos antigens are weaker than the antigens of normal red blood cells of the same type. However, the bloods contain two separable populations of weakly antigenic and negative red blood cells and not a single population of cells having a continuous gradient of antigenic strength. The reactive red blood cells in the A mos phenotype are classified as A, on the basis of nonreactivity with lectin antiA,, but it might be more appropriate to view them as “weak A.” When normal blood samples are tested with anti-A or anti-B, small numbers o f inagglutinable red blood cells are found not infreq~ent1y.l~ The reason why a few cells are nonreactive is not known.’, It is possible that they arise by somatic mutation of an ABO gene, although Winkelstein and Mollison’6 have shown that unagglutinated B cells do have B antigen. The mosaic phenotype differs sharply from this inagglutinable cell phenomenon, for the aberrant cells of the ABO mos phenotype lack antigen and are present in large numbers. The reactive cells of the ABO mos type have a weakened form of the affected antigen, and the characteristic is inherited. Our experience of the red blood cell differential agglutination technique using anti-A and anti-B sera indicates that it gives accurate and. reproducible results. Five separate counts on the blood of Mr. H. for example, all fell within the range of 12 to 15 per cent type A cells. The finding that most of the mosaic individuals have about 15 per cent reactive red blood cells is unlikely to be a reflection of technical imprecision. This
Nov.-Dec. 1975
consistency may be a coincidence, but it may also be an effect of a precise genetic mechanism that determines a change in somatic gene expression in specific cell clones. The results of tests for soluble A or B group substances in saliva of the mosaic individuals were not always conclusive but we think it probable that a low concentration is present. It is possible, therefore, that the mosaicism is not restricted to erythropoietic cells but also affects secretory tissues and possibly other cellular elements. The genetic background to the ABO mos phenotype cannot yet be understood. The phenomenon is inherited, and yet it affects only a portion of the somatic cells. It is unlikely to be caused by inheritance of a modifying gene, for such a gene would have to be closely linked to the ABO locus and activated in some unknown manner in only a portion of the cells. A striking feature of the mosaic phenotype is the precision of its inheritance, for not only are the antigenic characteristics of parent and child the same, but the proportion of the two-cell populations is closely comparable in different affected members of the same family. In a broad sense, people with A, and B, phenotypes may also be viewed as mosaics, for an A or B gene is expressed in one cell line, the secretory tissues, but not in the red blood cells. The A mos and B mos phenotypes go a step further in that there are differences in expression of an A or B gene in the same cell line. The biological mechanism by which genes are activated is complex, but it must be assumed that the ABO mos phenotypes arise by germ-cell controlled changes in the background regulation of an ABO gene in some somatic cells. References Berman, H. J., J. Srnarto, C. H. Issitt, P. D. Issitt, W. L. Marsh, and L. Jensen: Tn-activation with acquired A-like antigen. Transfusion 2:35, 1972. 2. Bird, G. W. G., N. K. Shinton, and J. Wingharn: Persistent mixed-field polyagglutination. Br. J. Haematol. 21:443, 1971. 3. Cameron, C., F. Graham, I. Dunsford, G . Sickles,
I.
Volume 15 Numbcr6
4.
5.
6.
7.
8. 9.
10.
I I.
12.
13.
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INHERITED ABO MOSAICISM
C. R. Macpherson, A. Cahan, R. Sanger, and R. R. Race. Acquisition of a B-like antigen by red blood cells. Br. Med. J. 11:29, 1959. Dunsford, I., C. C. Bowley, A. M. Hutchison, J. S. Thompson, R. Sanger, and R. R. Race: A human blood-group chimera. Br. Med. J. 11231. 1953. Furuhata, T., M. Kitahama, and T. Nozawa: A family study of the so-called blood group chimera. Proc. Jap. Acad. 35:55, 1959. Gartler, S. M., S. H. Waxman, and E. Giblett: An X X / X Y human hermaphrodite resulting from double fertilization. Proc. Nat. Acad. Sci. U.S.A.48:332, 1962. Kitahama, M.: Two family studies of the erythrocyte antigen mosaicism. Shinshu Med. J . 12:641, 1963. Lophem. J. J. van. H. Dorfmeier. and M. V. D. i a r t : T w o A antigens with abnormal serologic properties. Vox Sang. 2:16, 1957. Marsh, W. L., W. J . Jenkins, and W. W. Walther: Pseudo B: an acquired group antigen. Br. Med. J. 11:63, 1959. Myllyla, G., U. Furuhjelm, S. Nordling, A. Pirkola, P. Tippett, J . Gavin, and R. Sanger. Persistent mixed field polyagglutinability. Electrokinetic and serological aspects. Vox Sang. 20:7, 1971. Ogita, Z., H. Kikkawa, K. Yarnamoto, and F. Murakami: Erythrocyte antigen mosaicism in a Japanese family. Jap. J . Hum. Genet. 13:264, 1969. Race, R. R., and R. Sanger: Blood Groups in Man. 5th ed. Oxford, Blackwell, p. 38. Solomon, J . M., M. B. Gibbs, and A. J. Bowdler: Methods in quantitative hemagglutination. Part I . VoxSang. 10:54, 1965.
14. Sturgeon, P., D. T. McQuiston, H. F. Taswell, and C. J . Allan: Permanent mixed-field polyagglutinability (PMFP). Vox Sang. 25:481, 1973. 15. The National Foundation: Standardization in human cytogenetics. Birth Defects. Paris Conference, I97 I . Original article series, No. VIII, p. 7, (National Foundation, New York 1972). 16. Winkelstein, J . A,, and P. L. Mollison: The antigen content of ‘inagglutinable’ group B erythrocytes. Vox Sang. 10:614, 1965.
Acknowledgments We are grateful to the Humphreys and Sciarretta families for their enthusiastic cooperation, to Dr. J. L. German for performing the cytogenetic studies, and to Mr. P. Ferrau for his technical assistance. W. Laurence Marsh, F.I.M.L.S., M.I. Biol., Immunohematologist, Serology and Genetics Laboratory, Lindsley F. Kimball Research Institute of the New York Blood Center, 310 East 67th Street, New York, N.Y. 10021. Margaret E. Nichols, F.I.M.L.S., Supervisor. Serology and Genetics Laboratory, New York Blood Center. Ragnhild @yen, Chief Technologist, Serology and Genetics Laboratory, New York Blood Center. Francine Decary, M.D., Trainee in Blood Banking, New York Blood Center. Carolyn Whitsett, M.D., Trainee in Blood Banking, New York Blood Center. Emma L. Etheridge, Staff Technologist, California Transfusion Service, California.
