Autoimmune Hemolytic Anemia Associated with IgG Auto Anti-N

D. W. COHEN, G. GARRATTY, P. MOREL, and L. D. PETZ

From the Blood Bank of San Bernardino and Riverside Counties, Son Bernardino, California. the Hematology-Immunology Research Unit, Institutes of Medical Sciences, and the

Irnin Memorial Blood Bank, San Francisco, California

A second case of autoimmune hemolytic anemia mediated by an IgG auto Anti-N is described. The pa- tient's red blood cells were sensitized with both IgG and complement. The serum antibody was not inactivated by 2-mercaptoethanol treatment, and reacted by indirect antiglobulin test at 37 C with monospecific anti-IgG. The IgC antibody eluted from the red blood cells and in the serum showed anti-N specificity. The patient was thought to have systemic lupus erythematosus. Following steroid therapy, the hemolytic anemia resolved with disappear- ance of the anti-N.

THERE HAVE BEEN several reports of Anti-N in N positive Anti-N-like antibodies in N positive patients undergoing hemodialysis have also been re- p ~ r t e d . ~ . ~ . ~ . " In none of these reports was there evidence that the auto anti-N was IgG or associated with in v ivo hemolysis. Anti-N has been described as causing hemolytic dis- ease of the newborn.1H The antibody in this case, although not proven, was presumably IgG but was found in an M + , N-, S-, s-, U + individual. Anti-N formed by such in- dividuals shows different serologic char- acteristics from those formed by more com- mon MNSs

Only two examples of auto anti-N have been reported as being associated with hemolytic anemia. Bowman et al.' reported an IgM auto anti-N in a patient with infec- tious mononucleosis, and Dube et a / . 3 re- ported an IgG auto anti-N in a seven-year-old boy. This report describes another example of autoimmune hemolytic anemia associated with IgG auto anti-N.

Case Report A 21-year-old retarded white woman was

evaluated for a three-week history of pallor and

Received for publication May 26. 1978; accepted July 18, 1978.

easy bruisability. Physical examination revealed a spleen that was 10 cm below the left costal margin and nontender. Laboratory studies re- vealed the following: red blood cells 1.4 x IOVpI, hemoglobin 5.5 gldl, Hct 19 per cent, reticulocyte count 37 per cent, platelets 3000/pl and white blood cell count 7400/pI. There were 87 nucleated red blood cellsll00 WBCs. Serum bilirubin was 1.7 mgldl and LDH was 540 units. The L.E. test was negative, but ANA was posi- tive at 1:80 with a homogeneous pattern and anti- double stranded DNA was present to a titer of eight (normal: < 1.5); findings consistent with a diagnosis of systemic lupus erythematosus (S.L.E.). Complement levels were normal. Direct antiglobulin test was positive and anti-N was present in the serum.

The patient was placed on prednisone, 100 mg daily, with the hemoglobin increasing to 8.6 g/dl and the reticulocyte count falling to 16.6 per cent but the platelet count did not rise. A splenectomy was performed, and a spleen weighing 930 g was removed. Four N negative units were made avail- able but no blood was required for surgery. At discharge the patient's hemoglobin was 12.8 gldl, Hct 37 per cent, reticulocyte count 4.5 per cent and platelet count 77,OOO/pI. She remained asymp- tomatic and blood specimens tested 6 months post-splenectomy demonstrated no anti-N activity.

Results The patient's red blood cells were found to

agglutinate spontaneously following centrifuga- tion when suspended in AB serum, six per cent albumin, her own serum or saline. This occurred even when the cells were washed three times at 37 C with 37 C saline. Twelve washes with 45 C saline were required to obtain negative controls. ABO grouping on these well washed cells showed the patient's cells to be group AB. Using pro- longed sedimentation techniques ( i . ~ . , instead of centrifugation) and saline active Rh anti-sera, the patient was shown to be D+, C + , c + , E-, e + , with a probable genotype of R,r .

When tested with rabbit anti-N and anti-M, the patient's cells were found to be M negative,

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329 Volume 19 Number 3

COHEN ET AL. Transfusion May-June 1979

Table 1. Titration Studies on the MN Status of Patient's Cells

Red Blood Cells

Patient's Patient's Serum (Washed

Dilutions M MN N 12Times) _ _ _ ~

1 :1 0 3+ 4+ 4+ 1 :2 0 2+ 4+ 4+ 1 :4 0 1+ 2+ 2 + 1 :8 O ? 1+ 1 + 1:16 0 0 (m) ?

Score 0 27 37 37 - - - __

(m) = Microscopic positive.

N positive. The agglutination score of the pa- tient's well washed cells versus her own serum was identical to that of M negative N positive cells (Table I ) .

Due to the spontaneous agglutination, the direct antiglobulin test was initially difficult to interpret. Using well washed cells which gave negative control results for spontaneous aggluti- nation, the patient's cells were confirmed to be strongly sensitized with IgG and less strongly with complement.

The patient's serum strongly agglutinated A, B and 0 reagent red blood cells at room tem- perature. Panel results showed an anti-N pattern. The antibody reacted strongest as an agglutinin at room temperature; slightly weaker agglutina- tion occurred at 37 C and weaker reactions still were obtained by the indirect antiglobulin test. Some N negative cells reacted weakly. Some typical results with various MN phenotypes, selected from many such cells tested, are shown in Table 2. A panel of N negative red blood cells was tested against the patient's serum to deter- mine whether any alloantibody activity was being masked by the auto anti-N. None was detected. Anti-N was not detectable in the patient's serum six months following the initial testing.

An ether eluate prepared from the patient's cells caused strong direct agglutination of saline suspended N positive red blood cells; rarely, N negative red blood cells reacted weakly. Stronger reactions were obtained by the indirect antiglob- ulin technique. Weak variable activity occurred with N negative red blood cells (Table 2). The weak reactions of the serum and eluate with some M red blood cells were thought probably to be due to thetresidual N activity possessed by M cells."

Treatment of the patient's serum and eluate with 2-mercaptoethanol showed no significant re- duction in antibody strength, suggesting that the anti-N was IgG. The immunoglobulin nature of the antibody was confirmed by obtaining positive

results at the indirect antiglobulin stage using monospecific anti-lgG when testing both the pa- tient's serum and eluate.

Discussion

This case represents an example of warm autoimmune hemolytic anemia (AIHA) as- sociated with an IgG anti-N. In the case reported by Dube el ~ 1 . ~ no significant ac- tivity occurred at room temperature with the patient's serum, nor was the phenomenon of spontaneous agglutination of the patient's cells observed. Bowman ef al.' reported an IgM auto anti-N with a broad thermal range which was detectable at room temperature and at 37 C by the indirect antiglobulin only using anti-complement (C3/C4) antiglobulin serum. Although complement components were detected on our patient's cells by the direct antiglobulin test, no complement binding was detected by the indirect anti- globulin test against N positive red blood cells. As it is relatively common to detect red blood cell complement sensitization in patients with systemic lupus erythematosus,I6 and anti-N does not usually activate com- plement,I3 we suspect that the complement sensitization of the patient's cells was prob- ably related to the condition and not to the presence of the auto anti-N. Hinz and Boyer' described a case of AIHA associated with anti-N. Their patient was N negative, but the authors suggested that the anti-N may have cross reacted with the patient's M cells to cause AIHA.'

Spontaneous agglutination of red blood cells is sometimes associated with immune hemolytic anemia. Commonly agglutination

Table 2. Testing of Patient's Serum and Eluate Prepared from Patient's Red Blood Cells

Serum MN Eluate

type Pheno- RT 37C AGT

(high protein) 37C AGT ~

N 4+ 4+ 2+ 3+ 4+ N 4+ 3+ 2+ 3+ 4+ MN 4+ 2+ 1 + 2+ 3+ MN 4+ 2+ 1+ 2+ 3+ M 1 + 0 1+ ? 1 + M ? O ? 0 0 M 0 0 0 0 0

Volume 19 Number 3 IgG AUTO ANTI-N 33 1

occurs with cells suspended in a medium containing protein. The degree of spontane- ous agglutination observed is often propor- tional to the amount of IgG sensitizing the red blood cells. Less often, the red blood cells will spontaneously agglutinate when centrifuged in saline. This phenomenon is unrelated to the presence or absence of cold agglutinins and in our experience has been unrelated to the amount of IgG on the pa- tient’s red blood cells (i.e., we have seen it associated with weakly and strongly positive direct antiglobulin tests). It is tempting to suggest that removal of sialic acid from the red blood cell by an agent such as a virus, causes an alteration of membrane charge, leading to aggregation of the cells. The ob- servation that extensive washing of the pa- tient’s red blood cells at 45 C prevented the spontaneous agglutination suggests that the phenomenon is due to the antibody mole- cules sensitizing the red blood cells rather than an altered cell membrane.

Both viral transformation and exposure to foreign substances that cross react with N substance have been invoked as possible etiologic factors in the development of anti- N-induced autoimmune hemolytic anemia.3 Perhaps, by the cleaving of sialic acid re- sidues from the red cell membrane, as is known to occur with neuraminidase-produc- ing organisms, precursor N antigen could be e x p ~ s e d ’ ~ ” ~ and subsequently not recog- nized as self, resulting in the production of anti-N-like antibodies. The anti-N-like anti- bodies associated with dialysis seem to be formed as a result of red blood cell mem- brane modifications caused by the formalin used in the sterilization p r o ~ e s s . * * ~ * ~ . ~ ~ It is possible that other substances (e .g . , drugs or chemicals) could affect the red blood cells of nondialysis patients in a similar way lead- ing to the formation of clinically significant auto anti-N.

References I . Bowman, H. S., W. L. Marsh. H. R. Schumacher,

R. Oyen, and J. Reihard: Auto anti-N immuno- hemolytic anemia in infectious mononucleosis. Am. J. Clin. Pathol. 61:465, 1974.

2. Crosson. J. T., J. J. Moulds, C. M. Comty, and H. F. Polesky: A clinical study of anti-N in the sera of patients in a large repetitive hemodial- ysis program. Kidney Int. 10463, 1978.

3. Dube, V. E., R. F. House, J. J. Moulds, and H. F. Polesky: Hemolytic anemia caused by auto anti-N. Am. J. C h . Pathol. 63:828, 1975.

4. Francis, B. J., and D. E. Hatcher: MN Blood Types. The S-s-U+ and the M, phenotype. Vox Sang. 11:213, 1966.

5 . Greenwalt, T. J., T. Sasaki, and E. A. Steane: Second example of anti-N in a blood donor of group MN. Vox Sang. 11:184, 1966.

6. Harrison, P. B., K. Jansson, H. Kronenberg, J. F. Mahony, and D. Tiller: Cold agglutinin form- ation in patients undergoing haemodialysis-A possible relationship to dialyzer re-use. Aust. N. Z. J . Med. 5195, 1975.

7. Hinz, C. F. Jr., and J. P. Boyer: Dysgamma- globulinemia in the adult manifested as auto im-

’ mune hemolytic anemia. N. Engl. J. Med. 269: 1329, 1963.

8. Hirsh, W., P. Moores, R. Sanger, and R. R. Race: Notes on some reactions of human anti-M and anti-N sera. Br. J. Hematol. 3:134, 1957.

9. Howell, E. D., and H. A. Perkins: Anti-N-like antibodies in the sera of patients undergoing chronic hemodialysis. Vox Sang. 23:291, 1972.

LO. Hysell, J. K., J. M. Gray, and M. L. Beck: Auto- anti-N- An additional example. Transfusion 14: 72, 1974.

1 1 . McLeish, W. A., A. F. Brathwaite, and P. M. Peterson: Anti-N antibodies in hemodialysis pa- tients. Transfusion 1543, 1975.

12. Metaxas-Biihler, M., E. W. Ikin. and J. Romanske: Anti-N in the serum of a healthy blood donor. Vox Sang. 6574, 1961.

13. Mollison, P. L.: Blood Transfusion in Clinical Medicine, 5th ed. Oxford, Blackwell, 1972, p. 478.

14. Moores, P., M. C. Botha. and S. Brink: Anti-N in the serum of a healthy type MN person-A fur- ther example. Am. J. Clin. Pathol. 5 4 9 , 1970.

15. Perrault, R.: Naturally-occurring anti-M and anti-N with a special case: IgG anti-N in a NN Donor. Vox Sang. 24:134, 1973.

16. Pirofsky, B.: Autoimmunization and the Autoim- mune Hemolytic Anemias. Baltimore, Williams and Wilkins, 1969.

17. Springer, G. F., H. Tegtmeyer, and S. V. Huprikar: Anti-N reagents in elucidation of the genetic basis of human blood group MN specificities. Vox Sang. 22:325, 1972.

18. Telischi, M., 0. Behzad, P. D. lssitt, and B. G. Pavone: Hemolytic disease of the newborn due to anti-N. Vox Sang. 31:109, 1976.

19. Uhlenbruck, G.: Possible genetical pathways in the MNSSUu system. Vox Sang. 16200, 1%9.

20. Winn, L. C., P. L. Eska, and A. J. Grindon: ABO discrepancy caused by an auto anti-N. Trans- fusion 19612, 1975.