Brief Report

Vox Sang. 21: 284-287 (1971)

An Example of an Anti-Antibody Causing Difficulty in ABO Grouping

M. SIMMONS, H. 13. STAPLETON, DIANA M. BRAZIER and K. L. G. GOLDSMITH

Rcgionnl Blood Transfusion Centre, Slieffield and MRC Blood Group Reference Laboratory, London

KONUGRES et a!. [3] reported false-positive agglutination, by 2 anti-Rh sera contaminated with anti-Gm(x), of coated red cells, sensitized in vim. This potential cause of error in red cell grouping had been recognised a t the MRC Blood Group Reference Laboratory since 1964 and precautions had already been taken to overcome the problem. Nevertheless, as the following report shows, the difficulty had not been completely resolved, and further steps had t o be taken.

At the Regional Blood Transfusion Centre Sheffield, the red cells of a n infant, baby D, suffering from haemolytic disease of the new- born, were shown t o be agglutinated by a pool of anti-B, batch 1947, processed at the MRC Blood Group Reference Laboratory. At the same time i t was shown that 2 more batches of anti-I3 and 2 batches of anti-(AfB), group 0 serum, failed to react with these cells. In- vestigations performed a t Sheffield Regional Blood Transfusion Centre suggested that the unexplained reaction with the anti-B might be due to the presence of an anti-y-globulin in the serum reacting with the coated foetal red cells. It was known that the blood group of the mother was group 0 Rh negative cdelcde and that of her husband group A Rh positive, probably CDelCDe. Subsequent in- vestigations showed baby D t o be group 0 CDelcde.

Anti-B batch 1947 had been prepared from 12 donations of human whole serum one of which was known t o contain a non-inhibitable antibody of the type described by ANDRESEN [l]. The latter antibody had a titre of 1 in 4 against group 0 CDelcDE cells sensitized with anti-D known to coat red cells with the Gm(4) antigen. The other

Received: January 4, 1971; accepted: April 19, 1971.

An Example of an Anti-Antibody Causing Difficulty in ABO Grouping 285

eleven donations of anti-B which made up the pool had each been tested individually and showed no evidence of containing antibodies to serum antigens. Before issue, the final pool had been tested by a tile technique [2] for antibodies against serum antigens, with negative results.

The sample of the anti-B returned from Sheffield and a reference sample stored at -25°C at the MRC Blood Group Reference Labora- tory were tested against group 0 CDelcDE cells sensitized with a panel of high titre incomplete anti-D sera. Two techniques were used, one being the tile technique referred to above and the other, the tube technique recommended for ABO typing. The latter was employed because an ABO grouping reagent was being studied. The sensitized cells were not agglutinated by the anti-B pool by the tile technique but, by the tube technique, cells sensitized with certain batches of anti-D were agglutinated.

Serum from Mrs. D., mother of the infant, was used to sensitize Croup 0 CDelcDE cells which were then tested by the tube and tile techniques against a panel of sera containing antibodies to the antigens Gm (1, 2, 4, 5, 6, 10, 11, 14), Inv (1, 2) ISf (1) and against anti-B 1947. By both tube and tile techniques, cells sensitized with Mrs. D’s serum were strongly agglutinated by sera containing anti- bodies to Gm (4, 5, 10, 11, 14) and by anti-B batch 1947. The Gm phenotype of Mrs. D’s serum was Gm (-1, -2, 4, 5, 10, 11, 14), Inv (-1, -2).

Tests were also performed t o see whether the sensitized cells were agglutinated by any of a series of hitherto unidentified antibodies detected at the MRC Blood Croup Reference Laboratory. The coated cells were not agglutinated by any of these unknown antibodies. The specificity of these antibodies is unknown because their reactions with sensitized cells are inhibited only by the anti-D serum used to sensitize the red cells. Some of these antibodies may fall into the category described by ROPARTZ et al. [5].

Anti-D serum capable of coating red cells with a serum antigen must contain the latter in solution and may be used to inhibit the corresponding antibody. The only exception occurs when an antibody is non-inhibitable as described by ANDRESEN [l]. This principle was applied to the present investigation. Cells coated with Mrs. D’s serum were still agglutinated by anti-B batch 1947 even in the presence of an excess of Mrs. D’s serum, showing that the antibody in the pool was of the type described by ANDRESEN.

286 SIMMONS et al.

Anti-B batch 1947 was tested against Group 0 CDelcDE cells sensitized with 92 high titre incomplete Rh-antibodies suitable for use as red cell grouping reagents. Three sera sensitized the cells so that they were agglutinated by anti-B 1947 whereas 89 gave negative results.

Changes Introduced in Processing Techniques for Red Cell Antibodies

All ABO grouping sera prepared a t the MRC Blood Group Ref- erence Laboratory are screened for the presence of antibodies to serum antigens and not infrequently traces are found of non-specific, non-inhibitable antibodies against y-globulin as described by MIL- GROM [4] and ANDRESEN [l]. Until the discovery of the unwanted antibody in anti-B batch 1947, i t was assumed that if the titre of these antibodies was low, they would be diluted out in a pool. Sera of this type were then included with other normal antibody-con- taining reagents in the preparation of pools of grouping sera. The finished product was tested to ensure that no trace of activity against sensitized red cells could be detected by a tile technique. In the case of this particular anti-B pool, although the activity of the anti-anti- body had been diluted out sufficiently to give negative results by the tile method, i t could still be detected after incubation with sensitized cells for 2 hrs by the tube technique. This is obviously yet another source of error in red cell grouping. In view of the trouble that oc- curred with this particular anti-B pool i t was decided that, in future, red cell grouping sera which also contained antibodies against serum antigens detected by haemagglutination techniques would not be used. Furthermore, all reagents are now tested for antibodies to serum antigens at all stages of preparation by both tube and tile techniques and any shown to contain these antibodies are not used for ABO typing.

Summary

Coated red cells of a Laby suffering from haemolytic disease of the newborn were agglutinated by a red-cell typing serum due to the presence in the latter of a non-inhibitable anti-antibody. The need to exclude the presence of antibodies to sernm antigens in red-cell grouping reagents is stressed and the changes in the control of these reagents at the Blood Group Reference Laboratory are described.

An Example of an Anti-Antibody Causing Difficulty in ABO Grouping 287

Acknowledgements

The authors wish to thank Dr. C. C. BOWLEY for his helpful advice in the prep- aration of this paper.

References

1. ANDRESEN, P. H. : Relation between anti-antibodies and Grn agglutinins. Transfusion, Philad. 3: 21 1 (1963).

2. BRAZIER, DIANA M.: A study of serum antibodies which can be detected by haemagglutination - inhibition techniques. Thesis for Institute of Medical Laboratory Technology (1969).

3. KONUGRES, A. A.; HOLBROOK, E. R. and CORCORAN, P. 8.: Another source of error in red cell typing. Transfusion, Philad. 6: 80 (1966).

4. MILGROM, G.; UUBISRI, S. and W~ZNICZKO, G.: Human sera with ‘anti-anti- body’. Vox Sang. 1: 172 (1956).

5. ROPARTZ, C.; RIVAT, LILIANE; RIVAT, C. et BRAZIER, DIANA: Anti-idiotypes d’origine humaine. Nouv. Rev. franq. Hbmat. 10: 363 (1970).

Authors’ addresses: Mr. M. SIMMONS, F.I.M.L.T. and Mr. R. R. STAPLETON, L.I. Biol., Regional Blood Transfusion Centre, Northfield Road, Shefield, SZO IQT; Miss DIANA M. BRAZIER, F.I.M.L.T. and Dr. K. L. G. GOLDSMITH, MRC Blood Group Reference Laboratory, Gntliff Road, off Ebury Bridge Road, London, S.W. 1 (England)