Autoimmune hemolyt ic anemia associated with anti-Sc1 I. OWEN,V. CHOWDHURY, M.E. REID, J. POOLE, J.C.W. MARSH,AND J.M. Hows A case of autoimmune hemolytic anemia (AIHA) in a young child is described. The hemolysis was resistant to steroid therapy but responded to splenectomy and intravenous immunoglobulin. The autoantibody was shown to be anti-Scl by both serologic and immunoblotting techniques. This seems to be the first report of an autoantiScl detected b immunoblottin and the first example of AIHA in a child caused by autoanti-Scl . TLANSFUSION 8992;32:l73-176. Abbrevlatlons: AIHA = autolmmune hemolytlc anemla; DAT = direct antlglobulln test; IAT = lndlrect antlglobulln test: WIG = Intravenous Immunoglobulln; USS = low-lonlcstrength saline.
chromasia was strikingly absent and the reticulocyte count was only 2.5 percent (0.025) prior to transfusion. IgG and C3d were present on the red cell surface as demonstrated by the direct antiglobulin test (DAT) using monospecific antisera. The infant's blood was group AB, Rh-positive, and her serum reacted with 10 group 0 red cells of a standard panel. Additional features of intravascular hemolysis were present, including elevated unconjugated serum bilirubin, reduced serum haptoglobins, and macroscopic hemoglobinuria. A diagnosis of severe acute AIHA due to IgG autoantibody was established. Additional investigations failed to demonstrate evidence of recent infection or other underlying disease. A bone marrow aspirate was hypercellular, with erythroid hyperplasia, marked dyserythropoiesis, and mild megaloblastic changes. These findings were confirmed on trephine biopsy. There was no evidence of lymphoma. Vitamin B12 and folate levels were normal. Abdominal ultrasound revealed mild splenomegaly. Thymic enlargement was not seen on the chest roentgenogram. Treatment with high-dose prednisolone (2.5 mg/kg/day) and folic acid was begun, but it had no significant effect, as the patient remained relatively reticulocytopenic (maximum reticulocyte count, 4.5% [0.045]) and required blood transfusion five times during the first 6 weeks of the illness. At the end of this period, the dose of prednisolone was reduced because of the lack of response and the development of side effects, and the patient was transferred to our department for further management. At this stage she was jaundiced, cushinoid, and listless. Baseline hematologic and biochemical investigations confirmed the previous findings, and more-detailed serologic investigations were performed as described below. Further transfusions were given with very high dose methylprednisolone at 10 mg per kg per day intravenously on 3 consecutive days. Severe hemolysis and relative reticulocytopenia persisted and the hemoglobin level fell 1.5 to 2.0 g per dL (15-20 g/L) daily. The patient remained heavily transfusion-dependent. Urgent splenectomy with steroid and antibiotic cover (benzylpenicillin, cefuroxime, and metronidazole) was performed. %o 5-day courses of high-dose IVIG (0.4 kg/kg body weight/day) were given to attempt to control the hemolysi~.~-~* Splenectomy was uncomplicated, and spleen histologic examination showed no evidence of a lymphoma. After splenectomy, methylprednisolone was continued, in decreasing doses. The hemolysis was partially controlled immediately after splenectomy, but she remained reticulocytopenic and transfu-
AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA) is an uncommon disorder that has been reported in all age groups. In childhood, it often develops acutely, but, in most children, it runs a self-limiting course with rapid response to corticosteroids.1-3 In 26 to 70 percent of cases of warm AIHA, the autoantibody specificity can be defined, and it is usually anti-"Rh."4 We describe the case of a young West Indian girl w h o developed severe AIHA in infancy. The hemolysis proved resistant to steroid therapy but resolved following splenectomy and two perioperative courses of high-dose intravenous immunoglobulin (IVIG). Clear specificity of the autoantibody against the Scianna 1 antigen (Scl), a high-frequency antigen formerly referred to as Sm,5 was demonstrated by serologic techniques, including the use of panels of red cells with rare phenotypes, and by an immunoblot technique. Very few cases of autoanti-Scl have been reported previously,6-8 and those reports included only two definite examples of hemolytic anemia caused by autoanti-Scl.s
Case Report The patient was brought to her local hospital at age 10 months with sudden onset of jaundice, pallor, and fever. She was tachycardic but was otherwise not compromised hemodynamically. Mild hepatomegaly was present, but there were no other abnormal clinical findings. She had been born at full term following a normal delivery and had not previously been ill or taken medication. Initial investigations revealed a hemoglobin of 4.1 g per dL (41 g/L) and a mean corpuscular volume of 88 fL.The white cell count was 9 x lo9 per L (normal differential) and the platelet count was 400 x lo9 per L. Examination of the peripheral blood film revealed numerous spherocytes, but polyFrom the Dcpartmcnt of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK, and the International Blood Group Refercnce Laboratory, Bristol, UK. Rcccivcd for publication Decembcr 4, 1990; revision received June 1 1 , 1991, and accepted July 1, 1991.
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sion-dependent for the next 4 weeks. After this period, her hemoglobin level stabilized and then rose spontaneously, along with a marked reticulocytosis to 12 percent (0.12). Seven months after splenectomy, steroids were discontinued without any sign of hematologic relapse. At the time of writing, the child is 4 years old, in good health, and developing normally. Her blood count is normal, the DAT is negative, and there is no evidence of hemolysis. She continues on long-term prophylactic penicillin, which is her only regular medication.
Materials and Methods Serologic methods A standard centrifugation technique was used to perform the DAT. We tested the washed cells with monospecific anti-IgG, -IgA, -IgM, and -C3d, and a saline control was included. For the indirect antiglobulin test (IAT), cells and serum were incubated for 1 hour at 37”C, washed, and tested after centrifugation with anti-IgG and anti-C3d; a saline control was also performed. The reagents were supplied by the Blood Products Laboratory (Radcliffe Infirmary, Oxford, UK) and by the Scottish Antibody Production Unit (Glasgow, UK). We treated the cells with papain made by the North London Blood Transfusion Service (London, UK), the International Blood Group Reference Laboratory (Bristol, UK), and the Immunohematology Laboratories (Duke University, Durham, NC), following the technique recommended by the manufacturers. Serum and eluate were tested against papain-treated cells in one of three ways. 1. We incubated equal volumes of serum or eluate and papain-treated cells for 1 hour at 37°C and then read the mixture for agglutination. 2. We performed a standard low-ionic-strength saline (LISS) IAT, that is, an IAT performed in the normal way but using cells suspended in LISS. 3. We performed a standard normal-ionic-strength saline IAT, that is, IAT using cells suspended in phosphate-buffered saline of normal ionic strength. Eluates were made by the ether technique following Issitt’s modification of Rubin’s method.13 We carried out antibody screening on the serum.14Titrations for specificity in both serum and eluate were performed against a standard panel of group 0 cells as supplied by the Oxford Blood Transfusion Service. We carried out two sequential alloabsorptions of serum and eluate using both papain-treated and untreated normal group 0 R,R,, R2R2,and rr cells.” In addition to being selected for Rh phenotype, at least one of the three cells lacked each of the following significant blood group antigens: S , s, K, Fy’, Fyb, Jk”, and Jkb.2
Immunoblot method We performed immunoblotting as described by Mallinson et except that we used 5-percent (wt/vol) bovine milk powder in phosphate-buffered saline at pH 7.4 as the blocking agent. Briefly, the components of red cell membranes were separated by sodium dodecyl sulfate-polyacryalamide gel electrophoresis and then transferred to nitrocellulose paper. The nitrocellulose paper was blocked and incubated in eluate prepared from the patient’s red cells or after incubation of patient’s serum with reactive red cells.*7 The binding of antibody to the nitrocellulose paper was detected with peroxidase-conjugated goat anti-human Ig (BioRad Laboratories, Richmond, CA). al.,I6
Serologic testing On the child’s admission to our unit in September 1987, the DAT was strongly positive with IgG (4+) and to a lesser
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extent with C3d (1 +) on the red cell surface. The serum antibody reacted strongly with the 10 group 0 red cells of the standard panel in the IAT using anti-IgG (titer 16) but only with neat serum as an agglutinin with papain-treated cells. The eluted antibody also reacted strongly in the IAT using anti-IgG (titer 32) but failed to react as an agglutinin with papain-treated cells. No antibody specificity was initially found in the serum or the eluate by titration or absorption techniques using standard red cell panels. We carried out absorptions using both papain-treated and normal cells, but they were not done simultaneously, so strict comparisons between the methods are not possible. After two absorptions, both papain-treated and normal red cells partially removed the antibody; however, more antibody remained when papain-treated cells were used for absorption. We sent the serum and eluate to two reference laboratories, where panels of rare cells lacking high-frequency red cell antigens were used to define the specificity of the autoantibodies present. Anti-Scl was detected in both serum and eluate by the IAT and enzyme techniques. At one reference laboratory, the serum antibody was found to react on the IAT with both ficin- and trypsin-treated red cells as well as with untreated cells, but it did not react with enzyme-treated cells on disect agglutination. Sc:-1 cells did not react, and anti-Scl was suspected. At the second reference laboratory, the eluate was shown to be anti-Scl, reacting on IAT with either untreated, papain-treated, or trypsin-treated cells. Titrations clearly showed stronger reactions with homozygous Scl cells than with heterozygous cells. With Sc:l, - 2 cells, the autoanti-Scl gave a titer of 32 and a score of 27; with Sc:1,2 cells, the titer was 4 (score 6 ) , and there was no reaction with two examples of Sc: - 1,2 cells.
Immunoblot testing An ether eluate prepared from the patient’s DAT-positive red cells consistently reacted weakly on immunoblotting, giving a band with a relative molecular weight of 60 to 65 kDa, which was the same as that recognized by the alloantiSc1 control sample. A digitonin eluate prepared from RBCs that had been incubated with the patient’s serum failed to react by immunoblotting, although it reacted well on the IAT, showing anti-Scl specificity.
Discussion AIHA in children is often acute and associated with severe hemolysis, but it is usually self-limiting. A link with infections is found more frequently in children than in adults, but less often in association with other disorders such as collagen disease or In patients treated with corticosteroids, a rapid response to treatment is usually seen. However, previous reports suggested that some childhood cases of AIHA can be extremely difficult to treat, proving refractory to conventional therapy, including IVIG.9 Children who recover from the first hemolytic episode are less likely to relapse than adult patients. The overall mortality rate is lower in children than in adults: in the report by Sokol et al.,3 it was found to be less than 5 percent. The hemolysis in our case was unusually severe, despite highdose steroid therapy; it prompted urgent splenectomy and the use of high-dose M G given in two 5-day courses.
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AUTOANTI-Scl BY IMMUNOBLOT AND SEROLOGIC TESTS
The time course from splenectomy to stabilization and cessation of hemolysis, with accompanying reticulocytosis, supports the idea that the spleen was important in the pathogenesis of this patient's hemolysis. The spleen may have been the site of autoantibody production as well as the major site of red cell destruction. Initially, milder hemolysis continued after splenectomy, probably as a result of circulating autoantibody that sensitized red cells that were subsequently destroyed elsewhere in the reticuloendothelial system. The contribution of high-dose WIG therapy to recovery is unclear, but it may have partially controlled hemolysis early after splenectomy. A possible mechanism is the blockade of Fc receptors on macrophages in the liver and other parts of the reticuloendothelial system, which results in prolonged survival of sensitized red cells.'* Reticulocytopenia is uncommon but well recognized in AIHA.'9,20 It is usually seen in the severest cases, which raises the possibility that the antigen against which the autoantibody is directed is also present on immature red cells. Scl was originally described as a very high-frequency antigen, Sm, by Schmidt et al. in 1962.' Sc2 was described a year later as a low-frequency antigen, Busy2' and a year after that, the two were shown to be antitheThe phenotype Sc: - 1, - 2 is extremely rare. In 1988, Devine et al.23 described three separate individuals with the phenotype Sc:l, - 2 who formed antibodies to a high-frequency antigen that appeared to be absent only on Sc: - 1, - 2 cells. There was no compatibility among the three persons, but an eluate made from one of the sera failed to react with the Sc:l,-2 cells of a sibling. It would appear therefore that these antibodies must be detecting an antigen at the Sc locus. Devine et al. suggested that the three red cell antigens determined by these antibodies are part of an antigen complex similar to the Kell or Rh antigen systems. This theory suggests that Sc: - 1, - 2 cells correspond to K, or Rh,,,,. A few examples of autoanti-Scl have been described in the literature. Tregellas et al.7 reported an autoantiScl found in a healthy donor, Steane et aL6 described a patient with Evans syndrome who had a possible autoanti-Scl, and McDowell et a1.* reported two examples of autoanti-Scl causing AIHA. In one of these cases, the antibody was formed after a transfusion, when the patient's red cells appeared to have transiently depressed Scl. There have also been two examples of autoanti-Sc3 causing AIHA, one of which occurred following a transfusion.24 In both patients, weakened Scl and Sc3 were demonstrated, and the antibody failed to react with Sc: - 1, - 2 cells, but it reacted weakly with Sc: - 1,2 cells and strongly with all other (Sc:l) cells. One example also failed to react autologously, while the other gave a weak positive reaction. One of the patients was
followed: within 70 days, the serum antibody could no longer be detected but Scl and Sc3 remained weakened. Previous examples of antibodies within the Scianna system gave variable reactions with enzyme-treated cells. The autoanti-Scl described by Tregellas et al.' reacted only on the IAT, while the three alloantibodiesdescribed by Devine et al.23 all reacted well with enzyme-treated cells as well as on the IAT. Our own example will react only with enzyme-treated cells on the IAT and not when tested by direct agglutination. Our case clearly demonstrated that severe AIHA may be associated with auto anti-Scl. The Scl specificity was demonstrated both by immunoblotting and by standard serologic techniques. The immunoblot of the patient's red cell eluate corresponded to a 60- to 65-kDa red cell membrane antigen, which has the same immunoblot characteristics as the antigen that reacts with known Scl alloantibodies.= In the past, the detection of autoanti-Scl and other autoantibodies specific for high-frequency red cell antigens has been restricted by the availability of the very rare antigen-negative red cells necessary for serologic identification. Although data are still very limited, immunoblotting may be a useful alternative technique once the bands corresponding to the various high-frequency red cell antigens have been characterized. To our knowledge, this is the first report of an autoanti-Scl being detected by immunoblotting and the first instance of Scl autoantibody being associated with severe acute AIHA in childhood. Acknowledgments The authors thank Dr. Peter Issitt for confirming the specificity of autoanti-Scl and for helpful discussion and Kate Druce and Maria Mukheqee for secretarial help.
References 1. Habibi B, Homberg JC, Schaison G, Salmon C. Autoimmune hemolytic anemia in children. A review of 80 cases. Am J Med 1974;56:61-9. 2. Zupanska B, Lawkowicz W,Gorska B, et al. Autoimmune haemolytic anaemia in children. Br J Haematol 1976;34:511-20. 3. Sokol RJ, Hewitt S, Stamps BK, Hitchen PA. Autoimmune haemolysis in childhood and adolescence. Acta Haematol (Basel) 1984;72245-57. 4. Petz LD, Garratty G.Acquired immune hemolytic anemias. New York: Churchill Livingstone, 1980. 5. Schmidt RP, Griffitts JJ, Northman FF. A new antibody, antiSm, reacting with a high incidence antigen. Transfusion 1%2;2:33840. 6. Steane EA, Sheehan RG, Brooks BA, Frenkel EP. Therapeutic plasmapheresis in patients with antibodies to high-frequency red cell antigens. Prog Clin Biol Res 1982;106:347-53. 7. Tregellas WM, Holub MP, Moulds JJ, Lacey PA. An example of autoanti-Scl demonstrable in serum but not in plasma (abstract). Transfusion 1979;19:650. 8. McDowell MA, Stocker I, Nance S, Garratty G. Auto anti-Scl associated with autoimmune hemolytic anemia (abstract). Transfusion 1986;26:578.
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9. Hilgartner MW, Bussel J. Use of intravenous gamma globulin for the trcatment of autoimmune neutropenia of childhood and autoimmune hemolytic anemia. Am J Med 1987;83:25-9. 10. Oda H, Honda A, Sugita D, Nakamura A, Nakajima H. Highdose intravcnous intact IgG infusion in refractory autoimmune hemolytic anemia (Evans syndrome). J Pediatr 1985;107:744-6. 11. Bussel JB, Cunningham-Rundlcs C, Abraham C. Intravenous treatment of autoimmune hemolytic anemia with very high dose gammaglobulin. Vox Sang 1986;51:264-9. 12. Macintyre EA, Linch DC, Macey MG, Newland AC. Successful response to intravenous immunoglobulin in autoimmune haemolytic anaemia. Br J Haematol 1985;60:387-8. 13. lssitt PD, lssitt CH. Applied blood group serology. 2nd ed. Malvern, PA: Cooper Biomedical, 1975. 14. Dacie JV, Lewis SM. Practical haematology. 6th ed. New York: Churchill Livingstone, 1984. 15. Laine ML. Beattie KM. Frequency of alloantibodies accompanying autoantibodies. Transfusion 1985;25:545-6. 16. Mallinson G, Martin PG, Anstee DJ, Tanner MJA, Tills D, Sonneborn HH. Identification and partial characterization of the human erythrocyte membrane component(s) that express the antigens of the LW blood-group system. Biochem J 1986;234:649-52. 17. Reid ME, Anstee DJ, Tanner MJA, Ridgwell K, Nurse GT. Structural relationships between human erythrocyte sialoglycoproteins p and y and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking in Gerbich blood-group antigcn(s). Biochem J 1987;244:123-8. 18. Imbach P, Muller B, Imholz B, Wagner HP. Intravenous immunoglobulin therapy in immune thrombocytopenic purpura (ITP) and other immune related hemorrhagic disorders in childhood. In: Morel1 A, Nydegger VE, eds. Clinical use of intravenous immunoglobulins. London: Academic Press, 1986. 19. Greenburg J, Curtis-Cohen M, Gill FM, Cohen A. Prolonged reticulocytopenia in autoimmune hemolytic anemia of childhood. J Pediatr 1980;97:784-6.
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20. Conley CL, Lippman SM, Ness P. Autoimmune hemolytic anemia with reticulocytopenia. A medical emergency. JAMA 1980;244:1688-90. 21. Anderson C, Hunter J, Zipursky A, Lewis M, Chown B. An antibody defining a new blood group antigen, Bu'. Transfusion 1963;3:30-3. 22. Lewis M, Chown B, Schmidt RP, Griffitts JJ. A possible relationship behveen the blood group antigens Sm and Bu'. Am J Hum Genet 1964;16:254-5. 23. Devine P, Dawson FE, Motschman TL, et al. Serologic evidence that Scianna null (Sc: - 1- 2) red cells lack multiple high-frequency antigens. Transfusion 1988;28:346-9. 24. Peloquin P, Moulds M, Keenan J, Kennedy M. Anti-Sc3 as an apparent autoantibody in two patients (abstract). Transfusion 1989;29(Suppl):49S. 25. Spring FA, Herron R, Rowe G. An erythrocyte glycoprotein of apparent M, 60,000 expresses the Scl and Sc2 antigens. Vox Sang 1990;58:122-5. Irene M. Owen, FIMLS, Senior MLSO, Blood Transfusion, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. [No reprints available] Vijoy Chowdhury, MBBS, MRCP, Senior Registrar in Haematology, Department of Haematology, John Radcliffe Hospital, Oxford, UK. Marion E. Reid, PhD, Reference Division Manager, International Blood Group Reference Laboratory, South West Regional Transfusion Centre, Southmead Road, Bristol, UK. Joyce Poole. FIMLS, Serological Red Cell Reference Manager, International Blood Group Reference Laboratory. Judith E.W. Marsh, MRCP, Senior Registrar, Department of haematology, Hammersmith Hospital. Jill M. Hows, MD, FRCP, Consultant Haematologist, Department of Haematology, Hammersmith Hospital.