Anti-LW Specificity in Autoimmune Acquired Hemolytic Anemia

M. J. CELANO AND P. LEVINE From the Ortho Research Foundation, Raritan, New Jersey

Anti-LW has been demonstrated in sir unselected cases of acquired hemolytic anemia, either by direct testing or after aborption of the iera and cell eluates with Rh-pitive or Rh-negative LW- negative cells. Another antibody which is directed toward the Rh complex is also present as indicated by reactions with all bloods except Rh..,,. A third aalutinin, apparently nonspecific, is indicated by the reactions of some sera with all bloods including

THE specificity of antibodies of the “warm variety” on cells and in serum of acquired hemolytic anemia has been the subject of numerous investigattions. A fun- damental contribution was made by Weiner, Battey, Cleghorn, Marson and Meynell” who demonstrated anti-e in the serum and eluate from the coated cells of a patien’t who was of the probable genotype RIR1.

Since 1953 many specific anti-Rh anti- bodies have been demonstrated in the sera and cell eluates from patients with ac- quired hemolytic anemia, either as a single entity, or as mixed antibodies, or with nonspecific components. However, the non- specific antibody is the most prevalent, and Daciel wrote the follow,ing: “The exact nature of the apparently ‘nonspecific’ anti- body or antibodies of acquired hemolytic anemia awaits elucidation. The fact that, even when specific auto-antibodies can be idmentified as Rh antibodies, unidentifiable ‘nonspecific’ antibodies are almost always present at the same time, suggests rather strongly that the latter type of antibody is also related to the Rh system and recalls the sug,mestion of Wiener, Gordon and Gallop14 (1953) of an antibody directed against the ‘nucleus of the Rh-Hr sub- stance.’ ”

Rhmii.

Received for publication December 5, 1966; ac- cepted February 7, 1967.

Weiner and V O S ~ ~ in their elaborate study of 60 eluates from patients with acquired hemolytic anemia have shown that approxi- mately 70 per cent of the eluates reacted weakly or not at all with red cells of the original Rhnul, blood of Vos et a1.10 These results indicate a definite relationship of the auto-antibody to the Rh system. The fact that many eluates failed completely to agglutinate Rh,,,, blood led them to sus- pect that anti-LW could be present. How- ever, they excluded this possibility because they could not elute specific anti-LW from Rh-negative red cells; in fact, they obtained “the same specificity (or apparent lack of it) as before.”13

Pirofsky and Pratte tested eluates of anti. Rh and of erythrocyte auto-antibodies froin cases of acquired hemolytic anemia against primate and non-primate cells. Their re- sul~ts showed that both types of antibodies reacted with some primate cells known to contain the LW antigen,2 but not with the cells of non-primates.

Methods and Results A summary of our findings in six unselected

cases of autoimmune acquired hemolytic anemia referred to us from different hospitals during the past two years is given in Table 1. Among them were four men and two women. Their ages ranged from 41 to 73. Three were diag- nosed as acquired hemolytic anemia. Detailed information on case histories was scanty. One patient (By) was splenectomized and had a transfusion history; three were on steroid ther- apy. The cells of four patients were typed as group 0; of these three were Rh-positive and one was Rh-negative, type rh”rh. A fifth pa- tient was found to be group A, Rh-positive; the sixth was group B, Rh-positive. The direct antiglobulin test was strongly positive in all six cases. Specificity for these antiglobulin tests

265

266 CELANO AND LEVINE Transfusion July-August 1967

was not determined. Heat and ether9 eluates were made from the washed coated cells.

The sera and cell eluates, when first tested with a routine panel of red cells for atypical antibodies, showed nonspecific reactions. For the tests now reported the panel included at least one Rh-positive or Rh-negative LW- negative sample and one of the two Rh,,,, bloods. The mixtures were incubated at 37C for one hour, followed by the indirect anti- globulin test. One of the six sera (By) exhib- ited anti-LW specificity by direct tests, as did also an earlier specimen of another serum (Ha) which later became nonspecific.

Because the supply of the very rare LW negative bloods was limited, only two of the six sera were absorbed. Absorptions were car- ried out at 37 C for one hour and supernatants were tested as shown in the table.

Eluates prepared from the coated cells of five patients failed to react with Rh,,,, blood but did react with the LW-negative bloods (5 and/ or 6) . Anti-LW specificity became evident only after absorption with LW-negative cells con- taining Rh antigens. An eluate from the sixth case (By) was not made; specificity, however, was evident from direct tests of the serum.

Further confirmation of anti-LW specificity was obtained when antibody was eluted from rr red cells of the two cases studied (Ha and

Results presented in Table I show that Rh- positive cells are more strongly agglutinated than Rh-negative, both by sera and by absorbed eluates. This is characteristic of human and animal anti-LW sera.6, 7

There are at least two other antibodies pres- ent, one of which is directed toward what might be regarded as the Rh complex: this could be removed by absorption of sera or eluates with blood which was LW-negative but had Rh antigens (bloods 5 and 6). The other antibody agglutinated the Rh,,,, blood as indicated by the reactions of sera Ro and Ba.

BY).

Discussion

The findings of anti-LW in acquired hemolytic anemia in the sera or elua.tes, either by direct testing or by specific ab- sorption, was quite surprising in view of the findings of Weiner and Vos.13 In their studies they used the procedure of re- elution of the original eluates from the absorbing cells (D - -/D- - and LW-nega-

tive), and they often obtained in the sec- ondary eluate the same activity as in the original eluate. This latter nonspecific effect was also observed by us in the ABO system when we obtained specific anti-A and anti-B in our secondary eluates after absorbing anti-A or anti-B primary eluates with group 0 cells.3 This nonspecific effect was avoided by absorbing the sera and eluates with LW-negative cells and testing as indicated in the table.

The patient’s cells were not tested for the presence of LW antigen since they were heavily coated. However, they would be expected to be LW-positive, because LW is a high-incidence blood factor present in all random Rh-positive and Rh-negative red cells.6, 7 The simultaneous presence of an antigen and its corresponding antibody on red cells or in serum, such as e and anti-e, I and anti-I, T ja and anti-Tja, is the usual situation in autoimmune dis-

Our main interest in this contribution lies in the specificity of the antibodies in acquired hemolytic anemia. No attempt was made to study the role of complement in the antiglobulin reactions on the red cells or in the indirect antiglobulin test with the sera or eluates. T h e antiglobulin re- agent employed was a mixture of gamma and non-gamma antibodies.

In view of the demonstration of anti-LW in the sera and/or ‘the absorbed eluates in cases of acquired hemolytic anemia, the observations of Pirofsky and Pratts on the reactions of their eluates on some primate red cells are not surprising. I t has been previously shown by us that antigen LW is present on the several monkey bloods tested.2

T h e reactions of some sera with Rh,,,, blood (Table 1) and the observation of Weiner and V0s13 that 30 per cent of their eluates also react with Rh,,,, blood sug- gest that this rare blood contains another “unspecific” or “universally distributed”

eze.4. 11, 12

Volume 7 Number 4 267 ANTI-LW IN HEMOLYTIC ANEMIA

TABLE 1 . Tests of Acquired Hemolytic Anemia Sera and Eluates

I 2 3 4 5 6 1 Rh+ Rh+ Rh- Rh- Rh+ Rh- Rhauii

Human anti-LW

Ha Serum, group B Serum absorbed with blood 6 Eluate Eluate absorbed with blood 6

Ro Serum, group 0 Eluate Eluate absorbed with bloods 5 and 6

Na Serum, group 0 Eluate Eluate absorbed with blood 5

Ba Serum, group A Eluate Eluated absorbed with blood 5

Mc Serum, group 0 Serum absorbed with blood 5 Eluate Eluate absorbed with blood 5

By+ Serum, group 0

+ ++++ ++++ ++++ +++ ++ ++++

+++ ++ ++++

+++ ++++ +++

+++ ++ + ++++

+++ ++++

+ ++++ ++++ ++++

+++ ++ ++++

+++ ++ ++++

+++ ++++ +++

+++ ++ + ++++

+++ ++++

+ ++ ++ ++++

+ +-c ++++ + + ++++

++ ++++ ++

++ ++

-t +++ +

++

+ 0

++ nt

++ 0 ++++ +*

+ nt

++ ++ ++++ ++ + 0

+ + ++++ ++++ ++ 0

+++ +++ ++ ++ ++ 0

++ ++ * 0

+++ ++ + 0

++ 0

0

+ * o ++

0

+* ++ 0

nt nt

nt

nt nt

nt

nt

nt nt

0

0

0

0

0 0

0

+* 0

0

0 0

0

+++ 0

0

0

0 0

0

nt

The tests were carried out by the indirect antiglobulin reaction with saline-suspended red cells. The absorptions were carried out with small quantities of either pooled or individual LW-negative bloods numbers 5 and 6.

The eluates were prepared from the coated cells by heat or Rubin’s ether method? Blood number 7, Rh.,,,, was either the original blood of Vos et al.10 (E.N.) or the second example of Levine et al.5 (L.M.) , nt = not tested.

+ Absorption and elution were not made because anti-LW specificity was evident in direct tests.

antigen, which is not necessarily associated with Rh, as postulated by these authors.

Acknowledgments The authors are greatly indebted to our Con-

sultation Service for screening the blood specimens, to Mr. G. H. Vos, of the King Edward Memorial Hospital for Women, Subiaco, Australia, for his supply of the original Rh..,, blood, and Dr. 0. B. Hunter, Jr.. of Washington, D. C., for obtaining the second Rh., blood. Dr. J. Wallace of Carluke, Lanarkshire, provided us with Rh-positive and Rh- negative LW-negative bloods.

For submitting specimens from cases of acquired

Dr. W. Kuhns, New York University Medical

Mr. T. Owens, Veterans Administration Hospital,

Mr. R. Luxton, St. Luke’s Hospital, Pasadena,

Dr. M. Bergnes, Sacred Heart Hospital, Norris-

Mr. L. Tice, Hospital of St. Raphael, New Haven,

Dr. W. Hoops. Salinas Valley Memorial Hos-

hemolytic anemia, we also thank:

School

East Orange, N. J.

Calif.

town, Pa.

Conn.

pital, Salinas, Calif.

268 CELANO AND LEVINE Transfusion July-August 1967

References I . Dacie, J. V.: The Hemolytic Anemias, Part 11:

The autoimmune hemolytic anemias. New York, Grune and Stratton, Inc., 1962, p. 449.

2. Levine, P., and M. J. Celano: Presence of “D-like” antigens on various monkey red blood cells. Nature 193: 184, 1962.

3. Levine, P., and M. J. Celano: Unpublished observations, 1955.

4. Levine, P., M. J. Celano, and F. Falkowski: The specificity of the antibody in paroxysmal cold hemaglobinuria (P.C.H.). Ann. N. Y. Acad. Sci. 124:456, 1965.

5. Levine, P., M. J. Celano, F. Falkowski, Jane White Chambers, 0. B. Hunter, Jr.. and Carol T. English: A second example of - - -1- - - or Rh,,.,, blood. Transfusion 6 492, 1965.

6. Levine, P., M. J. Celano, R. Fenichel, W. Pol- lack, and H. Singher: A “D-like” antigen in rhesus monkey, human Rh positive and hu- man Rh negative red blood cells. J. Im- munol. 87: 747, 1961.

7. Levine, P., M. J. Celano, J. Wallace, and Ruth Sanger: A human “D-like” antibody. Nature 198:596, 1963.

8. Pirofsky, B., and Katherine Pratt: The antigen in autoimmune hemolytic anemia. Am. J. Clin. Path. 45: 75, 1966.

9. Rubin, H.: Antibody elution from red blood cells. J. Clin. Path. 16: 70, 1963.

10. Vos, G. H., Dell Vos, R. L. Kirk, and Ruth Sanger: A sample of blood with no detectable Rh antigens. Lancet 1: 14, 1961.

11. Weiner, W., D. A. Battey, T. E. Cleghorn, F. G. W. Marson, and M. J. Meynell: Sero- logical findings in a case of haemolytic ane- mia, with some general observations on the pathogenesis of this syndrome. Brit. Med. J. 2: 125, 1953.

12. Weiner, W., N. K. Shinton, and I. R. Gray: Antibody of blood group specificity in simple (“cold”) hemolytic anemias. J. Clin. Path. 13: 232, 1960.

13. Weiner, W., and G. H. Vos: Serology of ac- quired hemolytic anemia. Blood 22: 606, 1969.

14. Wiener, A. S., Eve B. Gordon, and C. Gallop: Studies on auto-antibodies in human sera. J. Immunol. 71:58, 1953.

M. J. Celano, B.S., Ortho Research Foundation,

Philip Levine, M.D., F.A.C.P., Ortho Research Raritan, New Jersey.

Foundation, Raritan, New Jersey.