NDT Advance Access originally published online on February 21, 2007
Nephrology Dialysis Transplantation 2007 22(5):1462-1464; doi:10.1093/ndt/gfl727
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Antibody-mediated acquired pure red cell aplasia (PRCA) after treatment with darbepoetin
1Department of Haemotology and 2Nephrology, Fremantle Hospital, Fremantle, Western Australia
Correspondence and offprint requests to: Dr Hemant A. Kulkarni, MD, MRCP, FRACP; Renal Physician, Fremantle Hospital, 2 Alma Street, Fremantle, Western Australia 6160. Email: hemant.kulkarni{at}health.wa.go.au
Keywords: antibody-mediated PRCA; darbepoetin; pure red cell aplasia
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Introduction |
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Recombinant erythropoietin (EPO) is used successfully as treatment for anaemia in chronic kidney disease (CKD), to maintain stable haemoglobin levels, avoiding the need for multiple transfusions. From 1998, epoetin formulations, in particular epoetin alfa (Eprex®, Ortho Biologics LLC), have been associated with a substantial increase in the incidence of antibody (Ab)-mediated pure red cell aplasia (PRCA) [1]. PRCA is a rare immunopathological disorder characterized by a severe reticulocytopenia, the absence of marrow erythroid precursors, normal granulocytic and megakaryocytic precursors, and the identification of circulating antibodies against EPO [2]. Patients typically develop an abrupt onset of severe transfusion-dependent anaemia. Consequently, EPO must be withdrawn and therapy initiated to decrease the production of the anti-EPO antibodies.
Darbepoetin alfa (Aranesp®, Amgen Inc., CA) is a second generation erythropoiesis stimulating agent with two extra carbohydrate chains and eight extra sialic acid residues as compared with epoetin alfa. It is suggested that the modifications to this molecule may make it more resistant to antibody development. There is only a single published case report of PRCA related to the exclusive use of darbepoetin alfa [3]; however, communication with Amgen Australia Pty Ltd indicate about four cases on their database. In a sole published case of PRCA due to darbepoetin alfa, the patient did not respond to treatment with steroids alone, intravenous immunoglobulin, cyclosporin or combination prednisolone and cyclophosphamide [3].
We report a second case of PRCA related to the exclusive use of darbepoetin alfa, where the patient responded to the first line treatment of oral cyclophosphamide and prednisolone, had non-neutralizing anti-EPO and anti-darbepoetin antibodies on bioassay, and died from severe pulmonary infection.
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Case |
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A 78-year-old man with stage 4 CKD secondary to hypertensive nephrosclerosis was started on subcutaneous darbepoetin alfa 40 mcg weekly for anaemia of CKD in April 2005. His medical history included hypertension, previous smoking with chronic obstructive pulmonary disease and a healed gastric ulcer. There was a good response to single agent darbepoetin alfa and he was not exposed to any other formulation of human recombinant EPO. In January 2006, his haemoglobin level fell from 112 g/l to 81 g/l without any obvious cause. In response, darbepoetin alpha was increased to 80mcg weekly and, given the past history of gastric ulceration, the proton pump inhibitor, Pantoprazole 40 mg daily, was initiated. Endoscopic examination of the upper gastrointestinal tract showed mild focal chronic gastritis with a colonoscopy showing haemorrhoids, diverticulosis and hyperplastic polyps, but no active site of gastrointestinal bleeding. Faecal occult blood examinations were negative on multiple occasions.
Further investigations showed abnormal iron studies with a raised ferritin of 1080 ng/l (normal 30–400 ng/l) and raised transferrin saturation of 91% (normal 14–53%). Serum cobalamin and red cell folate levels were normal and there was no evidence of haemolysis with normal haptoglobin and LDH levels. Interestingly, the direct antiglobulin test was positive (IgG specificity, C3d negative) with a panagglutinating autoantibody demonstrable in the serum. Absolute reticulocyte count was profoundly low at 3 x 109/l (normal 20–130 x 109/l). Bone marrow biopsy was normocellular with complete absence of erythroid precursors. Immunophenotyping showed normal lymphoid subsets and a clonal T cell population was not identified on molecular analysis. Anti-EPO antibodies tested by the Clinical Immunology Department, Amgen Inc., Thousand Oaks CA, USA, using the BIACORE 3000 Immunoassay, confirmed the presence of anti-darbepoetin alfa and anti-epoetin alfa antibodies. These antibodies were non-neutralizing, as demonstrated by bioassay. Serological investigations were negative for parvovirus B19, ANA and C3 levels were normal and a CT scan of the chest and abdomen excluded the possibility of thymoma or an underlying lymphoproliferative disorder. These results confirmed the diagnosis of Ab-mediated PRCA induced by darbepoetin alfa [2].
Darbepoetin alfa was discontinued and the patient started on cyclophosphamide 100 mg and prednisolone 50 mg daily on 22 May 2006. Eight units of packed cells were transfused over an 8-week period to maintain adequate haemoglobin levels (Figure 1). Following the introduction of immunosuppression, his haemoglobin level remained stable, with a reticulocyte count of 72 x 109/l, and no further transfusions were required. His recovery occurred within 4 weeks of the start of therapy. Prednisolone was reduced to 37.5 mg on 13 June 2006 due to steroid-related tremors and weight gain. Three weeks later, doses were further tapered to cyclophosphamide 50 mg daily and prednisolone 25 mg daily.
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The patient was admitted on 7 July 2006 with acute shortness of breath, calf pain and new onset atrial fibrillation that was initially treated with broad-spectrum intravenous antibiotics for suspected sepsis. A ventilation perfusion scan showed mismatched perfusion abnormalities in the anterior and apical-posterior segment of the left upper lobe, the lateral accessory segment of the right upper lobe and the posterior basal segment of the right lower lobe. In addition, there was matched loss of ventilation and perfusion at the right apex and in the inferior lingula. Anticoagulation was commenced with symptomatic improvement prior to discharge on 14 July 2006. Immunosuppression was continued at unaltered doses throughout this admission.
The patient was readmitted within a few days with worsening dyspnoea, fever, a C-reactive protein of 400 mg/l and new bilateral pulmonary interstitial infiltrates. Broad-spectrum antibiotics were started and cyclophosphamide was ceased, due to probable infection and possibility of cyclophosphamide-related pulmonary thrombosis. Blood cultures were sterile and urine cultures grew Escherichia coli in absence of pyuria. The chest X-ray showed extensive bilateral pulmonary infiltrates with no change in the ventilation perfusion scan. Sputum cultures did not identify a causative organism and bronchoalveolar washings were negative for pneumocystis carinii, legionella, influenza, para-influenza, metapneumovirus, respiratory syncitial virus and varicella zoster. PCR on bronchoalveolar washings was subsequently positive for Epstein Barr Virus, although its role as a causative agent was not proven. Despite treatment with broad-spectrum intravenous antibiotics and non-ventilatory support, the patient died on 3 August 2006, with death presumed secondary to pneumonia.
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Discussion |
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We report the first case of Ab-mediated PRCA in a non-dialysis patient receiving darbepoetin alfa as the sole erythropoietic agent, with clinical response to the treatment, and treatment complicated by fatal pulmonary infection. Our patient developed PRCA nine months after starting darbepoetin alfa.
To date, there appears to be no consensus on the definition of Ab-mediated PRCA. Published criteria require exclusion of the most frequent causes of PRCA, bone marrow examination showing <5% erythroid precursors and the presence of EPO-binding Abs confirmed with a validated assay [2]. A number of validated assays are available for measuring EPO Abs and these include radioimmunoprecipitation (RIP), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (BIAcore) and bioassays [4]. These assays show varying levels of sensitivity and versatility [4]. Typically the antibodies in Ab-mediated PRCA inhibit erythropoietic activity of endogenous EPO and recombinant erythropoetic stimulating agents in vivo and in vitro. The neutralizing effect of these antibodies can be demonstrated using a bioassay in which antibodies in the patient serum inhibit EPO-dependent proliferation of bone marrow-derived erythroid cells or of certain cell lines. Whilst bioassays are the only assays which measure the functional activity of EPO antibodies, they are known to be less sensitive than immunoassays [5] and are difficult to standardize [2,4]. In a case series of 12 patients with Ab-mediated PRCA, only five patients had neutralizing antibodies as measured by the bioassay [6]. This may in part be related to the epitope recognized by specific anti-EPO antibodies [7]. Our patient meets criteria for Ab-mediated PRCA, with a sudden fall in haemoglobin levels associated with an absolute reticulocytopaenia, absence of erythroid precursors in the marrow, exclusion of other possible causes including a lymphoproliferative disorder, thymoma, parvovirus B19 infection or an autoimmune condition, and the presence of anti-EPO antibody by the validated BIAcore immunoassay. The absence of neutralizing activity in this patient does not negate the diagnosis of Ab-mediated PRCA.
Reports available from the manufacturers of erythropoiesis-stimulating agents indicate that more than 200 patients have developed PRCA since 1998, the vast majority of which were receiving the epoetin alfa formation, Eprex® [1]. The exact cause of the increased immunogenicity of EPO formulations is unknown. It is thought that Eprex® is more commonly implicated, as the increased incidence coincided with a change of formulation [1]. In 1998, human serum albumin was removed as a stabilizer, due to concerns in Europe over the risk of Creutzfeldt-Jakob prions in blood-derived products and replaced with Polysorbate 80. Polysorbate 80 is now known to cause hypersensitivity reactions [8]. In addition, all patients who developed PRCA had received the drug subcutaneously. Immunogenicity of medication is usually highest after subcutaneous administration, and few cases of PRCA have been described following intravenous administration of EPO [9]. It is important to note that Aranesp® also contains Polysorbate 80 as the stabilizing agent and is given by subcutaneous injection.
PRCA can be difficult to treat and simply withdrawing erythropoiesis-stimulating agents is not sufficient to reverse the condition. Due to the small numbers of affected patients, only retrospective data can be used to establish the most effective therapy. The largest retrospective series of patients with PRCA included 47 patients, with the most effective therapies being renal transplantation, corticosteroids (alone or in combination with intravenous immunoglobulin), and corticosteroids in combination with Cyclophosphamide [10]. Our case demonstrates a clear temporal association between recovery and the introduction of combination Prednisolone and Cyclophosphamide. It also highlights the risk of life-threatening infections associated with the use of immunosuppression.
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Acknowledgement |
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We acknowledge Amgen Australia Pty Ltd for performing tests for anti-erythropoietin and anti-darbepoetin antibodies; Dr Andrew McQuillian for haematology advice and Staff South Metropolitan Area Health Services for patient care and support.
Conflict of interest statement. H.K. has attended Investigator meeting sponsored by Amgen Australia Pty Ltd.
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References |
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- Macdougall IC. (2005) Antibody-mediated pure red cell aplasia (PRCA): epidemiology, immunogenicity and risks. Nephrol Dial Transplant 20:Suppl 4, iv9–iv15.[Abstract]
- Rossert J. (2005) Pure red cell aplasia global scientific advisory board (GSAB). Erythropoietin-induced, antibody-mediated pure red cell aplasia. Eur J Clin Invest 35:Suppl 3, 95–99.[CrossRef][Web of Science][Medline]
- Jacob A, Sandu K, Nicholas J, et al. (2006) Antibody-mediated pure red cell aplasia in a dialysis patient receiving darbepoetin alfa as the sole erythropoietic agent. Nephrol Dial Transplant 21:2963–2965.
[Free Full Text] - Thorpe R and Swanson SJ. (2005) Assays for detecting and diagnosing antibody-mediated pure red cell aplasia (PRCA): an assessment of available procedures. Nephrol Dial Transplant 20:Suppl 4, iv16–iv22.[Abstract]
- Swanson SJ, Ferbas J, Mayeux P, Casadevall N. (2004) Evaluation of methods to detect and characterize antibodies against recombinant human erythropoietin. Nephrol Clin Pract 96:c88–c95.[CrossRef]
- Madueno JA, Ramirez R, Martin-Malo A, de Francisco ALM. (2005) Aljama. Antibody-mediated pure red-cell aplasia (PRCA): the spanish experience. J Nephrol 18:382–387.[Web of Science][Medline]
- Fibi MR, Aslan M, Hintz-Obertreis P, et al. (1993) Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization. Blood 81:670–675.
[Abstract/Free Full Text] - Steele RH, Limaye S, Cleland B, et al. (2005) Hypersensitivity reactions to the polysorbate contained in recombinant erythropoietin and darbepoetin. Nephrology 10:317–320.[CrossRef][Medline]
- Cournoyer D, Toffelmire EB, Wells GA, et al. (2004) Anti-erythropoietin antibody-mediated pure red cell aplasia after treatment with recombinant erythropoietin products: recommendations for minimization of risk. J Am Soc Nephrol 15:2728–2734.
[Abstract/Free Full Text] - Verhelst D, Rosser J, Casadevall N, et al. (2004) Treatment of erythropoietin-induced pure red cell aplasia: a retrospective study. Lancet 363:1768–1771.[CrossRef][Web of Science][Medline]
Accepted in revised form: 6.11.06
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