Resistance to Erythropoietin: Immunohemolytic Anemia Induced by Residual Formaldehyde in Dialyzers

Resistance to Erythropoietin: Immunohemolytic Anemia Induced by Residual Formaldehyde in Dialyzers Yee-Yung Ng, MD, MPH, Mo-Ping Chow, MD, Jau-Yi Lyou, MT, Hui-Yu Hu, MT, Chien-Hui Yung, MD, Cherng-Der Fan, MD, and Tung-Po Huang, MD • Because of inadequate renal synthesis of erythropoietin, the anemia associated with chronic renal failure has been treated successfully in most patients on hemodialysis with recombinant human erythropoietin. Hemolysis due to antiN'onn antibody in dialysis patients with the reused dialyzer may be one of the factors that cause refractoriness to erythropoietin therapy. Patients who do not respond to erythropoietin administration should be screened for anti-N'onn antibody. © 1993 by the National Kidney Foundation, Inc. INDEX WORDS: Chronic renal failure; reused dialyzer; anti-N'orm antibody; erythropoietin.

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HRONIC RENAL FAILURE almost invariably results in anemia. The inadequate renal synthesis of erythropoietin has emerged as the major mechanism underlying the anemia associated with renal failure. In view of the serious complications of blood transfusion, recombinant human erythropoietin has been used successfully to correct anemia in most patients on hemodialysis. I-6 Hemolysis, iron and/or vitamin (B l2 and folic acid) deficiency, aluminum intoxication, and bacterial infection have been reported as common causes in uremic patients not responding to erythropoietin. However, anti-N form antibody has not been reported as a cause of resistance to erythropoietin therapy.2,6 Poor response to erythropoietin therapy in uremic patients complicating anti-N form antibody have not been reported. We report the case of a patient who resisted erythropoietin therapy due to anti-Nform antibody-induced hemolysis. CASE REPORT A 56-year-old woman had received regular conventional hemodialysis without dialyzer reuse three times per week because of chronic glomerular nephritis in uremic stage since December, 1986. Under medical and economic considerations, the conventional hemodialysis was shifted to high-flux hemodialysis with dialyzer reuse after October 10, 1990. The hematocrit had been maintained at approximately 20% with few blood transfusions until May 3, 1991. We had begun to treat our patients with erythropoietin therapy (1,500 U intravenous injection after each hemodialysis) on March 4, 1991 because of the successful correction of uremic anemia by erythropoietin reported in the literature. However, in this patient, there was no evidence of an increase in hematocrit (21 %) after 2 weeks of erythropoietin therapy and optimal iron supplement. The patient's hematocrit level decreased to 17%, and 2 U of packed red blood cells was transfused on May IS, 1991. Despite increasing the dose of erythropoietin to 3,000 U three times per week, the hematocrit decreased further to 15% on July 22, and another 2 U of packed red blood cells

was administered on July 24. The dose of erythropoietin was further increased to 4,000 U three times per week on July 31. However, the patient still needed another 2 U of packed red blood cells on August 5 due to a low hematocrit (15%). Erythropoietin was discontinued on August 30. She was maintained on repeated blood transfusions. She was transferred to our hospital because of severe anemia (hematocrit < 15%) and exertional dyspnea on October 7, 1991. During hospitalization, the serum levels of iron and ferritin were 189 IlgJdL (normal, 35 to 200 Ilg/dL) and 1,134 Ilg/L (normal, 10 to 300 Ilg/L), respectively. The serum vitamin B12 level was 2,033 pg/mL (normal, 171 to 953 pg/mL). The folate level was 19.5 ng/mL (normal, 2.6 to 18.8 ng/mL). The complete blood count was performed by automated counter and showed hemoglobin 5.9 g/dL (normal, 12 to 16 g/dL), hematocrit (14%) (normal, 37% to 47%), and mean corpuscular volume (MCV) 121 tL (normal, 81 to 99 tL). The reticulocyte count was 8.3% (normal, 0.5% to 1.5%). Direct and indirect Coombs' tests were positive. Peripheral blood smear showed agglutination of red blood cells. The bone marrow was slightly hypocellular with relative erythroid hyperplasia. The serum level of bilirubin (total/direct bilirubin, 1.0/0.2 mgJdL; normal, 0.2 to 1.6/0 to 0.2 mg/dL) and haptoglobin 106 mgJdL (normal, 70 to 379 mg/dL) were within normal range, but lactic dehydrogenase was 233 lUlL (normal, 95 to 213 lU/L). The cold agglutinin titer was I :256. It was further proved to be anti-N rorm antibody induced by formalin-treated reagent red blood cells, which enhances the serologic reactivity of antiN rorm , but does not potentiate the cold reactive anti-N (Table I). The patient was then switched back to conventional hemodialysis without dialyzer reuse. The dialysis machines was sterilized with hypochloride instead of formalin. Erythropoietin was reintroduced on October 30, 1991. Hemoglobin and

From the Division a/Nephrology and the Blood Bank, Department 0/ Medicine, Veterans General Hospital, and the Division a/Nephrology, Department a/Medicine, Far Eastern Memorial Hospital, Taipei, Taiwan, Republic a/China. Received March 9, 1992; accepted in revised/arm August 18, 1992. Address reprint requests to Yee-Yung Ng, MD, MPH, Division a/Nephrology, Department a/Medicine, Veterans General Hospital, Taipei, Taiwan 11217, Republic a/China. © 1993 by the National Kidney Foundation, Inc. 0272-6386/93/2102-0015$3.00;0

American Journal of Kidney Diseases, Vol 21, No 2 (February), 1993: pp 213-216

213

NG ET AL

214 Table 1. Titration Scores of Untreated and Formaldehyde-Treated Red Blood Cells Reagent Red Blood Cell M+N-, M+N+, M-N+, M+N-, M+N+, M-N+,

untreated untreated untreated formaldehyde-treated formaldehyde-treated formaldehyde-treated

METHOD

Titer

Score'

1:4 1 :8 1:64 1:256 1:256

5 15 23 48 81 83

Following each high-flux hemodialysis session, the blood and dialysate compartment of the dialyzer (F-60) were flushed with 5 to 20 L oftap water and filled with 2.5% formaldehyde. Before the next dialysis (-40 hours later), the blood compartment was flushed with 2,000 mL normal saline. The blood line effluent was then tested for residual formaldehyde by Fast Formalert (Organon Teknika, Durham, NC). The dialyzer was used only if a negative Formalert was obtained. The dialysate compartment was flushed with a minimum of 10 L of sterile dialysate. The serum of anti-Nrorm antibody was titrated and tested with M+N-, M+N+, and M-N+ untreated and formaldehyde-treated red blood cells7•8 (reagent red blood cells, 2% to 4% suspension, Ortho Diagnostic System, Raritan, NJ). M and N are the minor antigens of the red blood cell grouping system; N + and N - represent the presence and absence of the corresponding antigens, respectively.

Abbreviations: M+, presence of M antigen; M-, absence of M antigen; N+, presence of N antigen; N-, absence of N antigen. * The sum of the grading of different titers. The corresponding grades and scores for agglutination are: 4+ = 12, 3+' = 11, 3+ = 10, 2+' = 9, 2+ = 8, 1 +' = 6, 1 + = 5, 1 +w = 3, microscopic agglutination = 2, 0 = O. Data from Widmann. s

DISCUSSION

The anti-N form antibody is commonly produced by renal dialysis patients who are treated regularly on hemodialysis with reuse of a dialyzer sterilized with formaldehyde 9 - ll and may be produced in N+ and N- individuals. 11 ,12 It is thought that formaldehyde alters the Nand 'N' antigens on the individual's red blood cells suf-

hematocrit were increased from 64 g/dL and 18% to 105 g/ Land 31%, respectively, in 10 weeks (Table 2). The patient had no symptoms of anemia. Throughout the treatment course, the patient was receiving aluminum-containing (AI(OH)3) phosphate binders. The patient did not undergo any operations or deferoxamine therapy. There were no episodes of bacterial infection.

Table 2. Series Data of Peripheral Blood Date

Hemoglobin (normal, 12-16 g/dL)

Hematocrit (normal,37%-47%)

MCV (normal, 81-99 fL)

10/07/91 10/08/91 10/10/91 10/11/91 10/12/91 10/16/91 10/17/91 10/18/91 10/19/91 10/21/91 10/22/91 10/23/91 10/24/91 10/28/91 10/29/91 11/27/91 12/11/91 12/25/91 01/08/92 01/22/92

5.9 5.8 5.5 4.7 4.9 4.3 4.8 4.2 5.4 5.3 7.0 5.8 5.8 5.5 6.4 7.3 8.3 8.9 10.5 10.7

14.2% 14.6% 11.8% 13.1% 12.0% 12.2% 11.5% 11.8% 16.0% 14.2% 17.4% 15.8% 15.4% 16.3% 18.4% 21.2% 24.8% 26.3% 30.9% 31.4%

121 123 120 110 123 130 126 123 105 111 107 112 111 106 108 109 110 107 106 106

MCH (normal, 21-31 pg)

MCHC (normal, 33-37 g/dL)

48.6

39.5

50.4

41.0

53.0 44.0

42.0 35.8

41.4 42.9 41.0 41.7

37.4 40.3 36.4 37.7

37.7 36.8 36.0 36.1 36.1

34.5 33.6 33.7 34.0 34.1

Remark Admission Ret 8.3%

BT

EPO·

EPO b

Abbreviations: Ret, reticulocyte; BT, blood transfusion with N-packed RBCs, 2 U; EPO·, erythropoietin, 3,000 U subcutaneous injection three times per week after each hemodialysis; EPO b , erythropoietin, 3,000 U subcutaneous injection twice per week after hemodialysis.

215

ERYTHROPOIETIN WITH ANTI-NFORM ANTIBODY

ficiently to render them foreign and stimulate antibody production. 13-16 Although the presence of anti-Nform antibody in dialysis patients is usually not manifested clinically, it has been implicated as a cause of hemolytic anemia. ll ,12 In our case, there was no evidence of hemolysis and change of hematocrit after high-flux hemodialysis with a formaldehyde-resterilized dialyzer for 5 months. Erythropoietin was administered to correct uremic anemia. However, our patient did not response to erythropoietin. Although iron supplement was given under the suspicion of functional iron deficiency, which is the most common adverse effect of recombinant human erythropoietin therapy,17.18 the patient still remained anemic. The increased MCV value read by automated counter had been interpreted and treated as vitamin BI2 or folic acid deficiency. In fact, the morphology of red blood cells under microscope was microcytic and hypochromic. The inappropriate value ofMCV was due to the cold agglutination of red blood cells. In our case, the occult hemolysis occurred 5 months after high-flux hemodialysis. It might have been caused by the agglutinated red blood cells and fragments that adhered to the fibers of dialyzers prepared as iffor reuse. 19,20 Thus, reuse sterilization provides the opportunity for lengthy exposure of red blood cell antigenic surfaces to formaldehyde during the 40 hours of sterilization before the next dialysis session. Subsequent hemodialysis may then wash some of the altered antigens into the patient's circulating blood. If these substances are sufficiently antigenic, and if the patient's immune system is competent, antiN-like antibody will be formed and may induce hemolysis. II Koch et afl found that formaldehyde levels less than 10 JLg/mL did not alter the red blood cell membrane, which makes the erythrocyte more susceptible to anti-N-like antibodies. Lewis et al 22 showed that residual formaldehyde levels less than 1 JLg/mL were not associated with the formation ofanti-N-like antibodies. Lewis et al23 also showed that rinsing of the dialyzer before use could lower the level of formaldehyde infused to the patient; formaldehyde remained in the membrane and, when isolated sections of membrane were continuously stirred in a flask, the formaldehyde levels observed continued to increase even after 2 hours, which suggests that it

is held, at least in part, in chemical combination by the membrane. It was impossible by the technique used to render a dialyzer completely free of formaldehyde within an acceptable period of time. This finding suggests some extent of formaldehyde binding to plastic material or dialyzer membranes during the sterilization phase and then slow dissociation during the washout phase. With reuse of dialyzers, it might be necessary to screen patients for anti-Nform antibody if erythropoietin administration fails to correct the anemIa. ACKNOWLEDGMENT The authors wish to thank Suh-Chen Yang for secretarial assistance, and Jill Storry and Delores Mallory at the National Reference Laboratory of the American Red Cross for the antiN fonn antibody confirmation. The authors also thank Chugai Pharmaceutical Corporation for the free erythropoietin supply.

REFERENCES I. Winearls CG, Oliver DO, Pippard MJ, Reid C, Downing MR, Cotes PM: Effect of human erythropoietin derived from recombinant DNA on the anaemia of patients maintained by chronic haemodialysis. Lancet 2:1175-1178, 1986 2. Eschback JW, Egrie JC, Downing MR, Browne JK, Adamson JW: Correction of the anemia of end-stage renal disease with recombinant human erythropoietin: Results of a combined phase I and II clinical trial. N Eng! J Med 316: 73-78, 1987 3. Bommer J, Alexiou C, Muller-Buhl U, Eifert J, Ritz E: Recombinant human erythropoietin therapy in haemodialysis patients-Dose determination and clinical experience. Nephrol Dial Transplant 2:238-242, 1987 4. Casati S, Passerini P, Campise MR, Graziani G, Cesana B, Perisic M, Ponti celli C: Benefits and risks of protracted treatment with human recombinant erythropoietin in patients having haemodialysis. Br Med J 295: 1017-1020, 1987 5. Eschbach JW, Adamson JW: Correction of the anemia of hemodialysis (HD) patients with recombinant human erythropoietin (rHuEpo): Results of a multicenter study. Kidney Int 33:189, 1988 (abstr) 6. Eschbach JW, Adamson JW: Recombinant human erythropoietin: Implications for nephrology. Am J Kidney Dis 11:203-209, 1988 7. Judd WJ: Methods in Immunohematology. Miami, FL, Montgomery, 1988, pp 234-236 8. Widmann FK: Technical Manual (ed 10). Arlington, VA, American Association of Blood Banks, 1990, p 529 9. Howell ED, Perkins HA: Anti-N like antibodies in the sera of patients undergoing chronic hemodialysis. Vox Sang 23:291-299, 1972 10. Harrison PB, Jansson K, Kronenberg H, Mahony JF, Tiller D: Cold agglutinin formation in patients undergoing hemodialysis: A possible relationship to dialyzer re-use. Aust NZ J Med 5:195-197, 1975

216 11. Kaehny WD, Miller GE, White WL: Relationship between dialyzer reuse and the presence of anti-N-like antibodies in chronic hemodialysis patients. Kidney Int 12:59-65, 1977 12. Crosson JT, Moulds J, Comty CM, Polesky HF: A clinical study of anti-N DP in the sera of patients in a large repetitive hemodialysis program. Kidney Int 10:463-470, 1976 13. Springer GF, Huprikar SU: On the biochemical and genetic basis of the human blood-group MN specificities. Haematologia (Budap) 6:81-92, 1972 14. Springer GF, Tegtmeyer H, Huprikar SU: Anti-N reagents in elucidation of the genetical basis of human blood group MN specificities. Vox Sang 22:325-43, 1972 15. White WL, Miller GE, Kuehny WD: Formaldehyde in the pathogenesis of hemodialysis-related anti-N antibodies. Transfusion 17:443-447, 1977 16. French D, Edsall JT: The reactions offormaldehyde with amino acids and proteins. Adv Protein Chern 2:277-335, 1945 17. Zehnder C, Blumberg A: Human recombinant erythropoietin treatment in transfusion dependent anemic patients on maintenance hemodialysis. C1in Nephrol 31:55-59, 1989

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18. Van Wyck DB, Stivelman JC, Ruiz J, Kirlin LF, Katz MA, Ogden DA: Iron status in patients receiving erythropoietin for dialysis-associated anemia. Kidney Int 35:712-716, 1989 19. Marshall JW, Ahearn DJ, Nothum RJ, Esterly J, Nolph KD, Maher JF: Adherence of blood components to dialyzer membranes: Morphological studies. Nephron 12: 157-170, 1974 20. Zucker WH, Shinoda BA; Mason RG: Experimental interactions of components of hemodialysis units with human blood. Am J Pathol 75:139-160, 1974 21. Koch KM, Frei U, Fassbinder W: Hemolysis and anemia in anti-N-like antibody positive hemodialysis patients. Trans Am Soc Artif Intern Organs 24:709-713, 1978 22. Lewis KJ, Dewar PJ, Ward MK, Kerr DNS: Formation of anti-N-like antibodies in dialysis patients: Effect of different methods of dialyzer rinsing to remove formaldehyde. Clin Nephrol 15:39-43, 1981 23. Lewis KJ, Ward MK, Kerr DNS: Residual formaldehyde in dialyzers: Quantity, location and the effect of different methods of rinsing. ArtifOrgans 5:269-277, 1981