A New Rh Antigen and Antibody BRUCE CHOWN, M.D., MARION LEWIS, B.A., HIROKO KAITA

RIi I-aboratory and Department of Paediatrics, University of Manitoba, Winnipeg. Canada

An antibody, anti-Wiel, defining an antigen, Wiel, in the Rh system, was found in the blood of a white woman who had been semitized by transfu- sion and possibly by pregnancy. The inheritance of the antigen was studied in two families, one white, the other Negro. In them it waa transmitted by cDvc or Wo. In one family this could be relined to cDac(d)c or Wodd). I t was also found in the blood of an unrelated, white woman whose phene type waa Rldr or Rhl%h and in that of an unre- lated Negro whose phenotype was Rlr. The antigen was not found on teating 2,600 random blooda, nearly all from Caucasians. I t is thought it may prove to be less rare in Negroes.

THIS ARTICLE reports the finding of an Rh antibody of a specificity not previously reported, the inheritance of the antigen de- fined by the antibody and the frequency of the antigen in a white population.

The Antibody and Its Production: The antibody was found in the serum of Mrs. Bill., a white woman of phenotype Rzr. In 1956 Mrs. Bill. was given four transfusions of Rh-positive blood in Chicago: it is prob- able that at least two of the donors were Negroes; one was of phenotype Ro.+ In 1958 Mrs. Bill.’s first pregnancy ended in the birth of a boy suffering from hemolytic disease of the newborn: his phenotype was RIWr.

Mrs. Bill.’s serum contained several Rh antibodies of known specificity as well as the antibody here reported. The antibody was first isolated by absorbing the serum with and then eluting the antibody from the cells of Mrs. Wiel., the propositus whose Rh type is described below; accordingly we are calling the antibody anti-Wiel.

Propositus of Pedigree I, Mrs. Wiel.: Mrs. Wiel. is a white woman whose blood

This work was supported by N.I.H. Grant R.C.- 7950. Based on a paper given at the 14th Annual Meeting AABB. Chicago, Oct. 27, 1961.

Address: 735 Notre Dame Ave.. Winnipeg, Canada. Received for publication December 12, 1961: ac-

cepted January 12, 1962. + We are indebted to Mrs. Carolyn Scott, Univ. of

Chicago Clinics Blood Bank, for this information.

was sent us as a routine specimen during her second pregnancy. Hers was the only example of her phenotype in about 3,000 pregnancy specimens selected as D-negative with an avid, saline-active anti-D screening serum (serum And of Table 1) . Her blood was remarkable in three rcspects: it had an unusual reaction pattern with anti-D sera; i t was Wiel-positive; and, as i t later turned out, i t did not react with anti-Rh* or anti- Rhc and weakly or not at all with anti- R h D . + * Because of the unusual Rh type of Mrs. Wiel.’s blood we studied the blood of as many of her relatives as we could. The results are set out in Pedigree I.

Propositus of Pedigree 11, Mr. Lang.: Following our study of the Wiel. family we reviewed records of several thousand bloods that had gone through our routine testing procedure, looking for any that had given reactions which suggested they might be Wiel-positive.? There were nine; the donors of five were traced. One proved to be Wiel- positive, Mr. Lang., the propositus of the Negro family set out in Pedigree I1 (see Fig. 1).

Inheritance of the Antigen Wiel.: In both Pedigrees the inheritance of the antigen Wiel is clear. The given genotypes are based on the following argument:

1. The phenotype of the propositus, Mrs. Wiel., of Pedigree I is RU

+ + Footnote opposite page. t As previously stated the serum “Bill” contained

several Rh antibodies of recognized specificity in addition to anti-Wiel. In routine testing the aerum had been used as a simple saline agglutinin and as a “papain altered” agglutinin for C typing. We had known that bloods containing the antigenic com- ponents CE or Cea reacted by the second method but weakly or not at all by the first. We now realized that this was also true for bloods that contained the antigen Wiel. It was the donors of bloods that had so reacted that we now sought.

150

NEW R h ANTIGEN

PEDIGREE I

151

PEDIGREE II LEGEND

Wiel - positive

00 Wiel - negative

Dead. Recorded in hospital m AB Rh+

r - t - l r - t l I I o m o o o o cDe cDUe cDe CDe cDE cDE cde cde cde cde cde cde

CAL FIG. 1. Pedigrees showing the inheritance of the antigen \Vie1 in two families, the first white,

the second Segro. .41I bloods were V (cel)-nc-gative and \'S (el)-ncgative.

++ MIS. WieL's blood was studied extensively in Dr. Ruth Sangcr's laboratory, in Dr. R. E. Rosenfield's and in our own. Omitting results with anti-Rh*, anti-RhB. anti-Rhc and anti-Rho and related antibodies, the exact specificities of which have not been determined, the combined results were: + - + -

Anti-D saline 5 14 Anti-c 5 0 Anti-D papain 28 2 Anti-E 0 9 Anti-CD 7 2 Anti-e 9 0 Anti-Ce f C 0 19 Anti-f 4 0 Anti-CE 0 1 Anti-\' (ces) 0 2 Anti-Cr 0 3 Anti-VS (em) 0 2 Anti-Cx 0 2 Anti-We1 1 0

Phenotype R,U.

Dr. Rosenfield found the blood to react with anti-RhB but not with anti-Rh* (Stollmack/lO) or with anti-Rhc, or with the serum "Fentry" which Wiener and Unger studied at his request and reported to have anti-RhD specificity. O n the other hand Dr. Sanger and Miss Tippett found it to react weakly with the sera "Mn. DS." and "Mrs. L.B.J." which they consider to be anti-RhD. For the moment we may refine Mn. \Yiel.'s phenotype to R.,.e(d)Wlel and, from family evidence, set out her genotype a5 R&d) Wic1.r.

Both Dr. Rosenfield and Dr. Sanger and Misa Tippett tested Mrs. Wiel.'a cells with a number of other "anti-D" sera from Rh-positive women. Dr. Rosenfield found the cells to be negative with the serum Mallor, doubtful with Correa, positive with Jenkins, Brent, Sorrentino, Nevins, Moran and Stollmack/l. Dr. Sanger and Miss Tippett found them to be negative with the sera of Mn. Kil and Mrs. Hig, weakly poaitive with that of Mrs. J.N.. positive with those of Mrs.E.J., Mrs. Rog, Mrs. B.B., Mrs. Cher, Mn. Lem, Mn. Jeff and Mrs. Nev (see Table 1. ref. 3). T h e meaning of these reactions is uncertain at present; they are placed on record for future reference.

152 CHOWN, ET AL.

TABLE 1 . Reaction Times in Minutes of Bloods in Pedigrees I and I I and that of Mrs. Bill,, the Producer of the Anti- Wicl, wi th Sewn Selected Saline-Active, Anti-D Sera

Sera Blood Sample 1 2 3 4 5 6 7

And Chi Pal Lei Fra Shoe Faw Undiluted

Shoe Faw

Pedigree I

Mrs. Wiel. cDUe.cde Wiel + Norman cDuE.cdc Wiel - - - - - - - -

Pedigree I I Cal cDc.cde Wiel + Producer of anti- Wiel M n . Bill. cDE.cdc Wiel - 4 4 4 4 6 6 4 2 4

1 1 C+ D+ samples 5-8 2-7 2-7 2-7 5-8 5-7 3-6 2-4 2-4

Timmy cDw.cDuE Wiel + _ _ -

Myrna cDuE.cde Wiel - - - - - - - -

21 14 4 7 - 37 21 12 5

- 27 - 21

_ - - _ -

40 30 15 4 - _ _ _

The 1 1 C+ D+ samoles are made UD of 3 CDe.CDc. 3 CDe.cde. 3 CDe.cDue Wiel + and

2.

3.

4.

5.

6.

2 CDc.cDuE ‘Wiel’ -. ?he tests were read to one hour and, if there was no clearly bisible agglutination by that time, then recorded as negative (-). A blank indicates no test was done.

She has not transmitted the antigen Wiel to her cde.cde child, so Wiel. if i t is in the Rh system, must be part of cDUe (%to).

7. He has transmitted R , without Wiel to 2 of his children; reactions of their blood with anti-D sera are normal.

8. He has transmitted RU and Wiel to . . Her cells give a double dose reaction with antic and an average reaction with anti-e and anti-f,tt but an un- usual reaction with anti-D, and are negative with anti-.Rh*, anti-RhC and perhaps anti-RhD.

Similarly, Timmy’s Wiel-positive cells give a double dose reaction with anti-c, an average reaction with anti-e, anti-E and anti-f.77 but an unusual reaction with anti-D. The D of the cDUE that he has inherited from his father does not react with our saline-active anti-D sera whereas Timmy’s cells react with some of these (Table 1) and must have a second D, which D is an un- usual one.

The antigen Wiel was not transmitted by cde from Mrs. Wiel. to her first child or by CDe from her sister to the latter’s second child. Both transmitted i t by cD“e (Ro) or, more precisely CDac(d)e (8” MU)).

The phenotype of the propositus of Pedigree I1 is R, with a double dose reaction with antic.

one child, Cal. The reactions of his blood with anti-D are unusual, being similar to those of Mrs. Wiel. and Timmy of Pedigree I (Tables 1, 2).

9. In the three persons (Mrs. Wiel., Timmy and Cal) who are Wiel-posi- tive and who do not carry a “normal” D on their partnering chromosome the reactions with anti-D sera are unusual. It therefore seems to us likely that, if Wiel is related to D, c, e or f , i t is more closely related to D than to c, e or f.

The scores for linkage between Wiel and R; for the families marked A, B and C are 2-0, 2-0 and 3-0 respectively. According to Finney’s method of analysis the probability of linkage is greater than 1 in 20 but less than 1 in 100.

Wiel does not correspond with any rare or “family” antigen for which Drs. Race and Sanger or we have the antibody. Al- though Mrs. Wiel.’s blood has R h B and lacks R h A , R h C and possibly R h D , anti-Wiel cannot be anti-RhB; it does not react with “normal” Rh-positive cells, most of which are thought to contain RhB.

Reaction Pattern with Anti-D Sera: With sera that react in saline or papain we carry tt The anti-e. anti-f and anti-E used do not indi-

cate zygosity.

153 NEW Rh ANTIGEN

out blood grouping in capillary tubes. The time elapsing betwen the mixing of cells and serum and the development of readily visible agglutination is recorded as the “re- action time.” For the present study seven, avid, saline-active, anti-D sera with reaction times of two to eight minutes with the average D-positive blood were first used. T h e reaction times of selected samples from Pedigrees I and I1 with these sera, as we use them for routine screening and for the delineation of the “D pattern,” are set out in Table 1. Sera No. 6, Shoe, and No. 7, Faw, are diluted 1:20 and 1:2 respectively for this purpose because, if used undiluted they fail to make the distinction in which we are interested. The reaction times with undiluted Shoe and Faw are given in the last two columns. It can be seen that Mrs. IYiel., Timmy and Cal now have essentially the same reaction times as the C+ D+ bloods; the singular quality of their D is masked. Norman and Myrna still fail to react with Shoe but do react with Faw, al- though their reaction times are well out- side the normal range. Dr. R. E. Rosen- field examined Norman’s blood and found that i t did not react with anti-Rh*, anti- R h B , anti-Rhc or anti-RhD. Dr. Sanger and Miss Tippett examined Myrna’s which appears genotypically identical with Nor- man’s, and classified it simply as a low grade Du; the latter would also be our classifica- tion based on reactions in Tables 1. 2.

Blood samples from Mrs. Wiel., Timmy.

Norman and Myrna of Pedigree I and from Cal of Pedigree I1 were tested with incom- plete anti-D sera by indirect Coombs or papain technics; results are seen in Table 2.

A powerful, incomplete anti-D serum was adsorbed on Mrs. Wiel.’s cells and the anti- body then eluted. The eluate reacted in papain with 17 “standard” D-positive sam- ples, with 12 Dus of various grades, and with Mrs. Wiel., Timmy and Cal, but not with 33 D-negative samples. Tke same serum was adsorbed on CDe.CDe Wiel- negative cells; the antibody then eluted re- acted with “standard” D-positive bloods, with DU bloods of various grades and with Mrs. Wiel.’s blood.

Frequency of Wiel-Positive Bloods: We have tested 2,600 random blood samples with anti-Wiel; no Wiel-positive was found. These samples were drawn from a popula- tion which is almost entirely Caucasian. In contrast Dr. E. R. Giblettl and Miss Lucy Brooks have found one Wiel-Fosi tive blood (phenotype R,r) among 96 specimens from

Negroes. While this may be a matter of chance, this finding, in addition to the good possibility that the stimulus that led to the production of anti-Wiel by Mrs. Bill. was transfusion of Negro blood, and the fact that our second example was found in one of the few Negro families in this area, all seem to point to the probability that the antigen is more common in Negroes than in whites.

TABLE 2. Tests a~itli Incomplete Anti-D Sera by the Indirect Coombs or Papain Teclrnics on Selected Bloods from Pedigrees I and I1

Method

Blood sample

Mra. \Viel. cDwe.cde Wiel + Myrna cDuE.cde Wiel- Norman cL)uE.cde Wiel - Timmy cDUE.cDUe Wiel + Cal cDwerde Wiel +

Indirect Coomhs + 15 4 19 0 3 0 3 0

- Papain +

4 0 4 0 4 0 4 0 4 0

-

Of the four aera that did not react with M n . Wiel. by the indirect Coomba technic three reacted by the papain technic.2 These were the three aera used with Norman and Timmy. The four aera shown in the table as used by the papain technic are not included in thoae uaed by indirect Coomba. These four are used routinely to test all blood8 that do not react with our tint m e e n anti-D serum. No. 1 of Table 1. They have been aelected from among many anti-D aera berauae of their high degree of reactivity by this method.

154 CHOWN, E T AL.

Antigen Wiel in a Woman of Phenotype R,r: The following is based on information supplied by Dr. Sanger. She and Miss T i p pett found that, among twelve Rh-positive women who had produced some sort of anti-D, the red cells of only one reacted with anti-Wiel. This last was Mrs. J.N., a white woman, who had been reported by Wiener, Unger and Jack4 to have the genotype Rlcdr. Although the data published by Wiener, Unger and Jack left i t open to question whether Mrs. J.N.’s genotype was Rkdr or R’Rocd, unpublished observations by Dr. Sanger on samples of blood supplied her by Mr. J. A. Jack from additional mem- bers of the family prove that Mrs. J.N. carries a kind of R, and r, so that her genotype might indeed be written Rlodr.

Combining the observations of Dr. Sanger and Miss Tippett with those of Wiener, Unger and Jack one might now tentatively extend Mrs. J.N.’s genotypic description to R l c d WW-. But since other members of the family have not been tested with anti-Wiel we do not know for certain whether the an- tigen Wiel is associated in Mrs. J.N.’s blood with R, or with r. If Wiel were shown to be transmitted by Rz in Mrs. J.N.’s family, this would prove that it is not associated with c or f and make it still more likely that it is related to D. If Wiel should later be found associated with R2, the name of the antigen may be appropriately changed to D W or rhw3 and that of the antibody to anti-DW or anti-rhW3.

Addondnm: Since this article was written Dr. H. V. Bell at the request of Dr. Amos Cahan, generous1 ’obtained a sample of Mrs. J.N.’s blood for us. fn addition to being Wiel-positive we found it to react as follows with the anti-D sera of Table 1: sera 1 2 3 and 4 negative. serum 6 34 minutes. serum’6 ’12 minutes. Shoe ’undiluted’ 3 minutes; ’Faw., unhiluted, not b s t e k ’ It reacted with the 4 papain sera of Table 2. Its reactions with antl-D are therefore similar to those of Mrs. Wiel Timmy and Cal so that Mrs. J.N.’s D appears t; be similar to theirs and the antigen Wiel is again associated with their sort of D. This Increases the probability that Wiel is asso- ciated with or a part of D, but since we do not know whether Wiel is traveling with Rl or with r in this family, the proof that it is a part of D is not absolute. Dr. Giblett writes in reference to the Wiel-positive R g blood that she and Miss Brooks found that ”with some anti-D sera [it] would be called I)Y. [It is] definitely weak with our saline anti-D, but very strong by ICT with incomolete anti-D.” Aaain Wiel is associated with a variant D. but since this was the blood of a Negro the association with a variant does not

have great signiflcance. Mrs. Wiel has had an- other baby; phenotype R g Wiel-negative with normal reactions with anti-D sera.

Blood Croups of Key Members of Pedigree I 11-2. A, MsNs Fy(a-b+) Jk(a+b-)

111-4. A, MSNa Fy(a-b+) Jk(a+b+) 111-3 0 MsMs Fy (a+b+) Jk (a+b+) IV-1 A1 MSMs Fy (a-b+) Jk (a+b+) IV-2 A, MSMs Fy (a-b+) Jk (a+b+) 111-6 A, MSMs Fy (a-b+) j k (a-b+) 111-7. A1 MSNs Fy (a-b+) Jk (a-b+) IV-3 A1 MSMS Fy (a-b+) Jk (a-b+) IV-4. A1 MSMS Jk (a-b+) 111-1 B MSMs Fy (a+b+) Jk (b+) 111-2 A2B MSMs Fy (a-b+) Jk (b+) 111-5. A1 MSNs Fy (a+b+) Jk (a+b+) Not recorded on the Pedigree: Sister of 11-1

Fy (a+b+) Jk (b+) Wiel-negative. Father of 111-1 and 2 A2 MsMs CDe.cde

Fy (a-b+) Jk (a+b-) Wiel-negative. Brother of 111-1 and 2 B MSMs CDe.CDe

Fy (a+b+) Jk (b+) Wiel-negative. 111-3 and IV-1

others K-k+Kp (a-b+); all Lu (a-b+).

AIB MSMs CDe.CDe

K+k+Kp (a-b+), all

Tested and negative with anti-Mg, -Vw, -Mil, -He, -V, -el, - B e n , -BYE, -Bin. -WF. 111-4 also tested and negative in Dr. Sanger’s laboratory with other low-frequency and “private” antibodies.

Acknowledgment We are most grateful to Dr. Ruth Sanger and

Miss Patricia Tippett and to Dr. R. E. Rosenfield for the work they did, the observations they made and their permission to publish thoae observations here, and for their criticism and advice. We are also grateful to Dr. E. R. Giblett and Miss Lucy Brooks for testing Negro samples and to Dr. Fred H. Allen, Jr., and Ortho Research Laboratories for each sup- plying us with an anti-C serum.

Drs. A. H. Smithen and H. McNicol of Flin Flon, Manitoba, brought us into friendly contact with the IViel. connection, obtained numerous blood speci- mens for us, and showed us many other kindnesses. Dr. L. A. Jentz of Brantford, Ontario, obtained the blood sample from MIS. Wiel.’s father, and Miss Catherine Anderson of the Manitoba Division of the Canadian Red Cross Blood Transfusion Service supplied us with the majority of the random blood specimens tested. T o all and to members of the two families we owe our sincere thanks.

References 1. Giblett, E. R.: Personal communication. 2. Lewis, M., H. Kaita and B. Chown: Kell typing

in the capillary tube. J. Lab. Clin. Med. 52: 163, 1958.

3. Tippett, P. and R. Sanger: Observations on sub- divisions of the R h antigen D. Vox Sang. In press.

4. Wiener, A. S., L. J. Unger and J. A. Jack: Proof of the existcnre of a new R h gene allele: R W Rev. IIemat. 15:286, 1960.