Antibody-mediated Pure Red Cell Aplasia Research Articles

Three-year safety observation of subcutaneous administration of epoetin-zeta in patients with chronic renal anemia: Results from PASCO II study.

Epoetin has been used to treat patients with renal anemia since 1988. -Anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA) has been associated with epoetin usage, and a PRCA incidence of 4.5per 10,000 patient-years was observed for epoetin-α (Eprex) in 2002. The PASCO II study (post-authorization safety cohort observation of Retacrit and Silapo (epoetin-ζ) administered subcutaneously for the treatment of renal anemia) followed 6,346 patients (4,501 Retacrit (group R); 1,845 Silapo (group S)) for up to 3 years of subcutaneous treatment with the biosimilar epoetin-ζ. One PRCA in 1 (0.02%) patient in group R who tested positive for neutralizing antibodies was reported. Overall, 527 adverse events of special interest (AESI) including PRCA occurred in 418 (6.60%) patients, lack of efficacy occurred in 34 (0.54%), and thromboembolic events in 389 (6.14%) patients. 41 adverse drug reactions other than AESIs were reported in 28 (0.44%) patients. The exposure-adjusted incident rate of PRCA was 0.84 per 10,000 patient-years. This real-world study showed that among patients with renal anemia receiving subcutaneous administration of the biosimilar product epoetin-ζ, the incidence rate of PRCA was substantially below the risk observed in 2002 for Eprex and that there was no immunogenicity concern or other new safety concern.

Roxadustat in the treatment of a hemodialysis patient with anti-erythropoietin antibody-mediated pure red cell aplasia

Roxadustat in the treatment of a hemodialysis patient with anti-erythropoietin antibody-mediated pure red cell aplasia

Diagnosis and management of pure red cell aplasia

Pure red cell aplasia (PRCA) is characterized by normocytic anemia with reticulocytopenia and marked reduction of the bone marrow erythroid precursors. PRCA may be congenital (Diamond-Blackfan anemia) or acquired. Acquired PRCA may present in the context of various backgrounds, the most common type in Japan being idiopathic, thymoma-associated, and large granular lymphocyte leukemia. Idiopathic and secondary PRCA that do not respond to the treatment of the underlying disease are generally treated using immunosuppressive agents. A retrospective study PRCA2004/2006 suggests that maintenance therapy and management of infectious complications is crucial for improving the prognosis in patients with PRCA. Recently, allogeneic stem cell transplantation has been considered as a potential option for the treatment of patients with PRCA who are refractory to immunosuppressive therapy. Sirolimus and roxadustat may be effective for relapsed/refractory PRCA with renal insufficiency and anti-erythropoietin antibody-mediated PRCA, respectively. Some gene mutations were detected in certain patients who had acquired PRCA, and the identification of STAT3 mutations may be useful in PRCA management. A prospective cohort study PRCA2016 has been ongoing in Japan, and novel discoveries provide hope for improving the outcome in patients with PRCA.

Remarkable response to roxadustat in a case of anti-erythropoietin antibody-mediated pure red cell aplasia

Remarkable response to roxadustat in a case of anti-erythropoietin antibody-mediated pure red cell aplasia

Antibody-mediated pure red cell aplasia related with epoetin-beta pegol (C.E.R.A.) as an erythropoietic agent: case report of a dialysis patient

BackgroundErythropoietin-stimulating agents (ESAs) are used to treat anemia in patients with chronic kidney disease, enabling maintenance of stable hemoglobin levels and eliminating the need for multiple transfusions. Epoetin-beta pegol (C.E.R.A.) is a continuous erythropoietin receptor activator created by integrating a large methoxy-polyethylene-glycol-polymer chain into the erythropoietin molecule, which provides it with a longer half-life. On rare occasions, cases of antibody-mediated pure red cell aplasia (PRCA) related to ESAs are reported. They are characterized by abrupt onset of severe transfusion-dependent anemia, despite ESA therapy. We herein report a case of antibody-mediated PRCA in a dialysis patient receiving C.E.R.A.Case presentationA 44-year-old man with end-stage renal failure had been receiving continuous ambulatory peritoneal dialysis for 2 years. C.E.R.A. was administered subcutaneously as a sole ESA once a month at the hospital since 4 years ago for the treatment of renal anemia and his hemoglobin level was well controlled at 12 g/dl. From 10 months before diagnosis, however, his hemoglobin level suddenly declined, necessitating frequent transfusions. Based on the results of a bone marrow examination and detection of anti-C.E.R.A. antibodies, the patient was diagnosed with antibody-mediated PRCA. After successful elimination of the antibodies using oral prednisolone plus cyclosporine, the patient was re-administrated C.E.R.A. intravenously, as there are few reports of antibody-mediated PRCA related to ESA using that administration route. He responded to the C.E.R.A., and his anemia dramatically improved, eliminating the need for blood transfusions.ConclusionsThis is the first reported case of recovery from an antibody-mediated PRCA with C.E.R.A. after its re-administration following a reversal of the antibody. It has been suggested that the additional large pegylation chain makes C.E.R.A. less likely to trigger antibody generation than other ESAs. Following successful treatment of antibody-mediated PRCA using immunosuppressive therapy, C.E.R.A. can be re-administered intravenously to treat renal anemia.

Acquired Antibody-Mediated Pure Red Cell Aplasia Following Treatment With Darbepoetin

Acquired Antibody-Mediated Pure Red Cell Aplasia Following Treatment With Darbepoetin

Antibody-mediated pure red cell aplasia after treatment with darbepoetin-alpha postrenal transplantation

Pure red cell aplasia (PRCA) is a rare normochromic, normocytic anemia with a near-to-complete absence of erythroblasts in the bone marrow. Erythropoiesis-stimulating agents have been used widely to improve anemia in patients with chronic kidney disease. We present a case of a 38-year-old woman who was detected to have anemia (hemoglobin: 10 g/dl) and renal dysfunction (creatinine: 3 mg/dl) on a routine health check in December 2016. Her kidney biopsy showed chronic interstitial nephritis. She received intravenous (IV) iron and was started on darbepoetin-alpha. Her hemoglobin after 3 months of treatment was 12 mg/dl. She underwent preemptive one haplomatch renal transplantation on June 2017. She was discharged with serum creatinine of 0.9 mg/dl and hemoglobin of 8 g/dl. She was not on any Erthropoietin stimulating agents (ESA) at discharge. In the 4th week after transplant, her hemoglobin dropped to 7 g/dl, and she was restarted on darbepoetin-alpha 40 μg subcutaneously once a week. In the 6th week posttransplant, her hemoglobin dropped to 6 g/dl. Her peripheral smear showed normocytic normochromic anemia with no evidence of hemolysis. Transferrin saturation was 81%, serum ferritin levels were 1812 ng/ml, and reticulocyte count was 0.1%. Bone marrow biopsy showed suppression of erythroid precursors with myeloid-to-erythroid ratio of 8:1, adequate iron stores, and normal white blood cell and platelet precursors with no dysplastic cells. Paroxysmal Nocturnal hemoglobinuria (PNH) workup; antinuclear antibody; and screening for hepatitis B, hepatitis C, HIV, CMV, and human parvovirus B19 were negative. Anti-erythropoietin (EPO) antibody by enzyme-linked immunosorbent assay was positive. She was diagnosed to have PRCA secondary to anti-EPO antibodies. She was managed with pulse doses of methylprednisolone, adequate dose of tacrolimus, IV immunoglobulin of 125 g (2 g/kg) over 5 days, and two packed red cell transfusions. Darbepoetin-alpha was stopped. At present, her hemoglobin is 12.5 g/dl with serum creatinine of 1 mg/dl on triple immunosuppression with no requirement of ESA.

Investigating Serious Adverse Drug Reactions in Patients Receiving Erythropoiesis-Stimulating Agents: A Root Cause Analysis Using the "ANTICIPATE" Framework.

Unexpected serious adverse drug reactions (sADRs) affecting patients with chronic kidney disease (CKD) who received erythropoiesis-stimulating agents were identified by study co-authors. These included pure red cell aplasia (PRCA) after administration of the Eprex formulation of epoetin or the epoetin biosimilar HX575 and fatal anaphylaxis associated with peginesatide, an erythropoietin receptor agonist. We developed and applied a structured framework to describe these sADRs, including root cause analyses and eradication efforts. A 10-step framework termed "ANTICIPATE," focusing on signal identification, incidence, causality, and eradication guided our evaluations. Initial cases were identified by a hematologist (Eprex), clinical study monitors (HX575), and 4 nurses (peginesatide). The number of persons with individual ADRs was 13 PRCA cases for epoetin, 2 antibody-mediated PRCA cases for HX575, and 5 fatal anaphylaxis cases for peginesatide. Initial incidence estimates per 1000 treated persons were 0.27 for Eprex-associated PRCA, 11 for HX575-associated PRCA, and 0.38 for peginesatide fatalities. Likely causes were subcutaneous administration of epoetin formulated with polysorbate 80 (Eprex), tungsten leaching from pins included in product syringes (HX575), and inclusion of a phenol stabilizer (peginesatide). Eradication strategies included restricting Eprex administration to the intravenous route, excluding tungsten from HX575 syringes, and for peginesatide, proposed eradication was to return to single-dose vials without preservatives. Although the number of cases of each sADR was small, eradication was successful for 2 sADRs, and a proposed eradication was developed for a third sADR. The structured framework used to describe the above 3 sADRs in patients with CKD can also be used in other clinical settings.

Preventing or Eradicating Factor VIII Antibody Formation in Patients with Hemophilia A: What Can We Learn from Other Disorders?

Eradication of factor VIII (FVIII) specific neutralizing antibodies (also known as inhibitors) by the traditional method of immune tolerance induction (ITI) is costly and unsuccessful in one out of three patients. Furthermore, effective inhibitor prevention strategies are presently lacking. An overview is given in this narrative review of antidrug antibody prevention or eradication strategies that have been used in disorders beyond hemophilia A, with the aim of analyzing what we can learn from these strategies for hemophilia A. Prevention of antidrug antibody formation using rituximab, methotrexate, and intravenous immunoglobulins in patients with Pompe's disease seems effective but carries a high risk of adverse events. Based on studies in patients with rheumatoid arthritis and inflammatory bowel disease, it seems likely that treatment with methotrexate alone would also be able to prevent inhibitor formation in hemophilia A patients. Besides side effects, it is unclear whether immune tolerance to FVIII would persist after cessation of immunomodulatory therapy with methotrexate. A combination of cyclophosphamide and corticosteroids, used to treat antibody-mediated pure red cell aplasia, could be further investigated to eradicate inhibitors in hemophilia A patients who are refractory to ITI. In summary, insights gained from research on antidrug antibody formation in other diseases could be helpful in devising alternative treatment strategies for inhibitor development.

Development of an anti-EPO antibody detection kit based on lab-on-a-chip and bridging antibody technologies

Immunogenicity is a major concern in the use of biological drugs. In particular, antibody-mediated pure red cell aplasia (PRCA) is a rare condition that is caused by administration of recombinant erythropoietin. There are numerous assay platforms for detect EPO anti-drug antibody (ADA), and most have appropriate assay sensitivity, but in need of improvement in terms of assay turnaround time and user accessibility. Here, the new method was developed based on lab-on-a-chip technology and bridging ELISA. The FREND™ Cartridge is equipped with a microfluidic lateral flow channel, enabling easy, fast and accurate immunoassays with small sample volumes. Biotinylated EPO was immobilized on the avidin-coated solid phase of the test zone in the FREND™ cartridge. Initially, ADA in the serum sample binds to the detector conjugate (EPO-HRP-anti HRP antibody-FL bead) in the conjugation zone, and it flows into the test zone prepared with capture complex (avidin-biotinylated EPO). Unbound detector complexes are captured in the reference zone. The FREND™ system detects and quantifies the fluorescence signals in each zone and then calculates the concentration of EPO ADA in the sample. The FREND™ EPO ADA kit may be useful in local clinics as a rapid method for monitoring patients administered recombinant erythropoietin.

T-cell assays confirm immunogenicity of tungsten-induced erythropoietin aggregates associated with pure red cell aplasia

AbstractImmunogenicity of biotherapeutics and the elicitation of anti-drug antibodies are a key concern for their efficacy, pharmacokinetics, and safety. A particularly severe consequence of immunogenicity of a biotherapeutic is the rare development of antibody-mediated pure red cell aplasia (PRCA) in anemic patients treated with aggregated forms of recombinant human erythropoietin (rhEPO). Here, we investigated in vitro T-cell responses to experimentally heat-induced rhEPO aggregates, and to tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA. Heat-stressed rhEPO elicited T-cell responses only in blood obtained from healthy individuals identified as responders, whereas nonstressed rhEPO overall did not induce reactions neither in responders nor nonresponders. Tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA could induce in vitro T-cell responses in blood obtained from healthy donors, in contrast to rhEPO from low tungsten syringes. Importantly, ex vivo T-cell recall responses of patients treated with rhEPO without PRCA showed no T-cell responses, whereas T cells of a patient who developed PRCA after treatment with a clinical batch with elevated levels of tungsten and rhEPO aggregates showed a clear response to rhEPO from that clinical batch. To our knowledge, this is the first time that T-cell assays confirm the root cause of increased rhEPO immunogenicity associated with PRCA.

Chronic preclinical safety evaluation of EPO-018B, a pegylated peptidic erythropoiesis-stimulating agent in monkeys and rats

EPO-018B, a synthetic peptide-based erythropoiesis stimulating agent (ESA), is mainly designed for treatment of anemia caused by chronic renal failure and chemotherapy against cancer. It overcomes the deficiencies of currently approved ESA, including the frequent administration of temperature-sensitive recombinant protein and anti-EPO antibody-mediated pure red cell aplasia (PRCA). This study was designed to evaluate the potential chronic toxicity of EPO-018B. Subcutaneous administration doses were designed as 0, 0.2, 1 and 10mg/kg for six months for 160 rats (20/gender/group) and 0, 0.3, 3 and 20mg/kg for nine months for 32 monkeys (4/gender/group) once every three weeks. The vehicles received the same volume of physiological saline injection. All animals survived to the scheduled necropsies after six weeks (for rats) and fourteen weeks (for monkeys) recovery period, except for the two high-dose female rats and two high-dose male monkeys, which were considered related to the increased RBCs, chronic blood hyperviscosity and chronic cardiac injury. EPO-018B is supposed to be subcutaneously injected once every month and the intended human therapeutic dose is 0.025mg/kg. The study findings at 0.2mg/kg for rats and 0.3mg/kg for monkeys were considered to be the study NOAEL (the no observed adverse effect level), which were more than ten times the intended human therapeutic dose. Higher doses caused adverse effects related to the liver toxicity, cardiotoxicity, appearance of neutralizing antibodies of EPO-018B and the decrease of serum glucose and cholesterol. Most treatment-induced effects were reversible or revealed ongoing recovery upon the discontinuation of treatment. The sequelae occurred in rats and monkeys were considered secondary to exaggerated pharmacology and would less likely occur in the intended patient population. As to the differences between human beings and animals, the safety of EPO-018B need to be further confirmed in the future clinical studies.

A cluster of Epoetin-associated pure red cell aplasia: clinical features and the possible association of HLA-DRB1*12:02.

Antibody-mediated pure red cell aplasia is a rare but serious complication in chronic kidney disease patients receiving recombinant human erythropoietin (r-HuEpo). Between April 2012 and May 2013, eight such cases were reported in our institution. Their clinical features were reviewed and their HLA alleles were compared with those of healthy controls. All patients were exposed to epoetin alfa (Eprex®) with polysorbate-80 as stabilizer via subcutaneous route with a mean age of 61.9 years and mean exposure of 11.2 months of r-HuEpo before loss of efficacy. 87.5% of the cases were male and Chinese and received immunosuppression as treatment for pure red cell aplasia. All three of the successfully treated patients are alive compared with only 40% of the transfusion-dependent patients. DRB1*12:02 was more frequently expressed among the cases than healthy controls suggesting a plausible molecular link.

Establishment of the first WHO Erythropoietin antibody reference panel: Report of an international collaborative study

A panel of 9 fully human monoclonal antibodies against human erythropoietin (EPO) with defined characteristics (non-neutralizing, neutralizing, various isotypes, affinities) representative of those evident in antibody-mediated pure red cell aplasia (PRCA) and non-PRCA patients were formulated and lyophilized. The panel was evaluated in a multi-centre international collaborative study comprising eighteen different laboratories using different assay platforms including those in routine use. These included binding assays, some based on use of novel technologies and neutralization assays predominantly employing EPO responsive cell-lines. Results showed that detection and titre varied depending on antibody characteristics and the method used. Only selective assay platforms were capable of detecting the diverse repertoire of EPO antibodies in the panel indicating that some clinically relevant antibodies are likely to be missed in some assays. Importantly, the clinical samples from PRCA patients were distinguished as antibody-positive and the healthy donor serum as antibody negative across all different platforms tested. For neutralization, data was generally consistent across the assays for the different samples regardless of the cell-line and the assay conditions.The heterogeneity in data from the study clearly indicated the need for reference standards for consistency in detecting and measuring EPO antibodies across different assay platforms for monitoring the safety and efficacy of erythropoiesis stimulating agents. Therefore, the WHO ECBS at its meeting in October’15 established the EPO antibody panel, available from NIBSC, to facilitate decision-making on assay selection for testing antibodies against human EPO, for evaluating assay performance of antibody assays for clinical use, for assay validation and for standardization.

Incidence of erythropoietin antibody-mediated pure red cell aplasia: the Prospective Immunogenicity Surveillance Registry (PRIMS)

BackgroundSubcutaneous administration of Eprex® (epoetin alfa) in patients with chronic kidney disease (CKD) was contraindicated in the European Union between 2002 and 2006 after increased reports of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA). The Prospective Immunogenicity Surveillance Registry (PRIMS) was conducted to estimate the incidence of antibody-mediated PRCA with subcutaneous administration of a new coated-stopper syringe presentation of Eprex® and to compare this with the PRCA incidence with subcutaneous NeoRecormon® (epoetin beta) and Aranesp® (darbepoetin alfa).MethodsPRIMS was a multicentre, multinational, non-interventional, parallel-group, immunogenicity surveillance registry. Adults with CKD receiving or about to initiate subcutaneous Eprex®, NeoRecormon® or Aranesp® for anaemia were enrolled and followed for up to 3 years. Unexplained loss or lack of effect (LOE), including suspected PRCA, was reported, with antibody testing for confirmation of PRCA.ResultsOf the 15 333 patients enrolled, 5948 received Eprex® (8377 patient-years) and 9356 received NeoRecormon®/Aranesp® (14 286 patient-years). No treatment data were available for 29 patients. Among 23 patients with LOE, five cases of PRCA were confirmed (Eprex®, n = 3; NeoRecormon®, n = 1; Aranesp®, n = 1). Based on exposed time, PRCA incidence was 35.8/100 000 patient-years (95% CI 7.4–104.7) for Eprex® versus 14.0/100 000 patient-years (95% CI 1.7–50.6) for NeoRecormon®/Aranesp®. The incidence of PRCA with Eprex® was not significantly different versus comparator ESAs (rate ratio: 2.56; 95% CI 0.43–15.31). An analysis based on observed time produced similar findings.ConclusionThis large, prospective registry demonstrates that PRCA is rare with subcutaneous administration of either the new coated-stopper syringe presentation of Eprex®, or NeoRecormon® or Aranesp®.

Risk of Pure Red Cell Aplasia in Patients With Hepatitis C Receiving Antiviral Therapy and an Erythropoiesis-Stimulating Agent

Antibody-mediated pure red cell aplasia (PRCA) has been primarily observed in patients with chronic kidney disease treated with an erythropoiesis-stimulating agent (ESA); only a few anecdotal cases have been reported in other patient populations. We searched the Amgen Global Safety Adverse Event Database and identified 14 patients with hepatitis C who developed severe anemia, anti-erythropoietin antibodies, and bone marrow biopsy-proven PRCA, while receiving interferon therapy (with or without ribavirin) and an ESA. During the follow-up period and after ESA treatment stopped, 11 patients no longer required transfusions and 3 did. Analysis of antibody isotypes showed that, contrary to reports of patients with chronic kidney disease, immunoglobulin G1 was the predominant isotype rather than immunoglobulin G4 (immunoglobulin G4 was detected in only 1 of 6 patients). Epitope mapping showed the anti-erythropoietin antibodies bound domains required for receptor binding. Therefore, the potential benefits of ESA therapy must be weighed against the risk for PRCA in patients with hepatitis C who are receiving treatment with interferon and ribavirin.

A simple screening test for the detection of erythropoietin antibodies

Antibody-mediated pure red cell aplasia (PRCA) is a rare complication of erythropoietin (EPO) therapy. Identification and demonstration of functional activity of EPO antibodies required to diagnose this condition is difficult and only performed in selected laboratories worldwide. In this article we report a recent cluster of three cases of antibody-mediated PRCA over a 16-month period in a single center associated with EPREX use. We also describe the use of a simple low-cost inhibitor assay that can be used to screen for PRCA in local laboratories.

Successful treatment of anti-erythropoietin antibody-mediated pure red cell aplasia with low-dose prednisolone

The standard therapy for anti-erythropoietin (EPO) antibody-mediated pure red cell aplasia (PRCA) is cyclosporine (CyA) or prednisolone (PSL) 0.5-1.0 mg/kg. However, many patients with severe chronic kidney disease (CKD) and chronic heart failure cannot tolerate such an immunosuppressive regimen. An 86-year-old man with anemia related to CKD and chronic heart failure, who had received recombinant human erythropoietin subcutaneously, developed anti-EPO antibody-mediated PRCA. The patient was treated with CyA followed by PSL (1.0 mg/kg); however, he was unable to tolerate this drug regimen. The PSL dose was reduced to 0.2 mg/kg. Surprisingly, his reticulocyte count increased 3 months later, and RBC transfusion was no longer required. Low-dose PSL is a treatment option for patients with anti-EPO antibody-mediated PRCA who cannot tolerate CyA and PSL (0.5-1.0 mg/kg).

Detection of neutralizing antibodies to erythropoietin by inhibition of rHuEPO-stimulated EGR1 gene expression in the UT-7/EPO cell line

Recombinant erythropoietin (rHuEPO) is used extensively to treat anaemia associated with chronic kidney disease. However, the development of neutralizing antibodies (NAbs) to rHuEPO can result in the development of antibody-mediated pure red cell aplasia (PRCA). The detection of NAb in patient sera by in vitro bioassay relies on the inhibition of a cellular response to rHuEPO. Current bioassays for rHuEPO measure proliferation in responsive cell lines such as the erythroleukaemic cell lines, UT-7 and UT-7/EPO, the latter sensitized to EPO. Using these cell lines, we show the dose-responsive induction of both PIM1 and EGR1 gene expression in UT-7 cells and of EGR1 in UT-7/EPO cells. The expression of EGR1 in UT-7/EPO cells in response to rHuEPO was comparable to the proliferative response measured by 3H-thymidine incorporation and could be inhibited by serum from a patient with NAb-mediated PRCA in a dilution-dependent manner. Bioassays based on the induction of endogenous gene expression are comparable to current bioassays but are considerably quicker given that incubation time is decreased from 2–3days to 50min. Measurement of EGR1 gene expression in response to rHuEPO in UT-7/EPO cells offers a rapid, non-radioactive and automatable alternative to current assays for the detection of rHuEPO NAbs.

Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia

Patients treated with erythropoietin-based erythropoiesis-stimulating agents (ESAs) can develop a rare but life-threatening condition called antibody-mediated pure red cell aplasia (amPRCA). The antibody characteristics in a nephrology patient with amPRCA include high antibody concentrations with neutralizing activity and a mixed IgG subclass including anti-ESA IgG4 antibodies. In contrast, anti-ESA IgG4 antibody is generally not detected in baseline samples and antibody-positive non-PRCA patients. Therefore, we validated a highly sensitive immunoassay on the ImmunoCAP 100 instrument to quantitate anti-ESA IgG4 antibodies using a human recombinant anti-epoetin alfa (EPO) IgG4 antibody as a calibrator. The biotinylated ESA was applied to a streptavidin ImmunoCAP, and bound anti-ESA IgG4 antibodies were detected using a β-galactosidase-conjugated mouse anti-human IgG4 antibody. The validated assay was used to detect anti-ESA IgG4 in amPRCA and non-PRCA patients. The immunoassay detected 15 ng/ml of human anti-EPO IgG4 antibody in the presence of a 200 M excess of human anti-ESA IgG1, IgG2, or IgM antibody and tolerated 2 μg/ml of soluble erythropoietin. All patient samples with confirmed amPRCA had measurable anti-ESA IgG4 antibodies. In addition, 94% (17/18) of non-PRCA patient samples were antibody negative or had below 15 ng/ml of anti-ESA IgG4 antibodies. This novel immunoassay can measure low-nanogram quantities of human anti-ESA IgG4 antibodies in the presence of other anti-ESA antibodies. An increased concentration of anti-ESA IgG4 antibody is associated with the development of amPRCA. We propose that the measurement of anti-ESA specific IgG4 antibodies may facilitate early detection of amPRCA in patients receiving all ESAs structurally related to human erythropoietin.