Pure red cell aplasia and anti-erythropoietin antibodies in patients on hemodialysis: a report of two cases and a literature review / Aplasia pura de células vermelhas e anticorpo antieritropoietina em pacientes de hemodiálise: relato de dois casos e revisão da literatura

ABSTRACT Introduction: Anemia is a frequent multifactorial complication of CKD seen in patients on dialysis derived mainly from impaired erythropoietin (EPO) production. A less common cause of anemia in individuals with CKD is pure red cell aplasia (PRCA) secondary to the production of anti-EPO antibodies. Objective: This paper aimed two describe two cases of PRCA secondary to the production of anti-EPO antibodies including choice of treatment, patient progression, and a literature review. Materials: This study included the cases of two patients with CKD on hemodialysis with severe anemia in need of specific investigation and management. Results: Patient 1 with CKD secondary to hypertension treated with EPO for 7 months showed persistent decreases in hemoglobin (Hb) levels despite the subcutaneous administration of increasing doses of EPO; the patient required recurring blood transfusions. Workup and imaging tests were negative for the main causes of anemia in individuals with CKD on dialysis. Patient 2 with CKD secondary to adult polycystic kidney disease had been taking EPO for 2 years. The patient developed severe abrupt anemia the month he was started on HD, and required recurring transfusions to treat the symptoms of anemia. Workup and imaging findings were inconclusive. Specific laboratory tests confirmed the patients had anti-EPO antibodies. After six months of immunosuppressant therapy (corticosteroids + cyclosporine) the patients were stable with Hb > 9.0 g/dl. Conclusion: PRCA is a rare condition among patients on dialysis treated with rhEPO and should be considered as a possible cause of refractory anemia. Treating patients with PRCA may be challenging, since the specific management and diagnostic procedures needed in this condition are not always readily available.

RESUMO Introdução: Anemia é complicação frequente da Doença Renal Crônica (DRC) em pacientes dialíticos. Apresenta caráter multifatorial principalmente pela insuficiente produção de eritropoietina (EPO). Situação rara causadora de anemia na DRC é Aplasia Pura de Células Vermelhas (APCV), em decorrência da produção de anticorpos anti-EPO. Objetivo: Descrever 2 casos de APCV com formação de anticorpos anti-EPO, sua abordagem clínica, evolução e revisão de literatura. Métodos: Dois pacientes em hemodiálise que desenvolveram anemia grave, necessitando investigação e manejo específico. Resultados: Paciente nº 1: feminina, 75 anos, DRC secundária à hipertensão arterial. Após 7 meses com EPO desenvolveu queda persistente em valores de hemoglobina (Hb) mesmo com incremento em doses EPO SC, necessitando transfusões de sangue recorrentes. Extensa investigação laboratorial e de imagem resultou negativa para principais causas de anemia. Paciente nº 2: masculino, 66 anos, DRC secundária à DRPA, há 2 anos em uso de EPO. No mês de entrada em HD desenvolveu anemia severa, também exigindo transfusões recorrentes para tratamento da anemia sintomática. Extensa investigação laboratorial e por imagem, sem chegar a uma conclusão definitiva. Em ambos os casos a presença de anticorpos anti-EPO foi confirmada por exames laboratoriais específicos. Terapia imunossupressora resultou em estabilização do quadro e Hb > 9,0 g/dl em ambos os pacientes, 6 meses após início do tratamento. Conclusão: APCV é condição rara entre pacientes dialíticos que recebem EPOHuR e deve ser lembrada como causa de anemia refratária. Seu manejo específico e diagnóstico laboratorial nem sempre acessível, tornando desafiadora a condução dos casos para o nefrologista.

Production of erythropoietin-specific polyclonal antibodies

Background: Erythropoietin, as a principal hormone promotes red blood cell production in bone marrow. Varieties of erythropoietin biosimilar are being produced by recombinant DNA technology in cell cultures. The detection or quantification of these molecules are being performed by different methods which some of theme such as Western blot and enzymelinked immunosorbent assay [ELISA] require specific antibodies. High cost, inappropriate shipping [cold chain failures], reduced sensitivity and thus poor detection performance are common pitfalls of using commercial kits for performing immunological tests

Objectives: To produce in-house polyclonal antibody against active pharmaceutical ingredient [API] of recombinant human erythropoietin [rh-EPO] was the aim of this study

Materials and Methods: Two healthy female albino rabbits were injected four times in 14 days interval using rh-EPO API as antigen. The produced antibody was separated from plasma via either caprylic acid or saturated ammonium sulfate precipitation and the results were compared from each purification methodologies. The antibody was further purified by ion exchange chromatography. Acceptable purity and good immunogenicity were detected respectively by SDS-PAGE and western blot analysis. The purified antibody was compared with a commercial kit to determine rh-EPO concentration in different steps of production batches via ELISA

Results: The purity of antibodies after ion exchange chromatography, obtained from caprylic acid and ammonium sulfate precipitation were 97 and 80%, respectively

Conclusions: As producing in house kits is one of the important challenges of bio- pharmaceutical manufacturers, a simple, cost- and time-effective, and easy to scale up strategy for making in-house polyclonal antibody was set up. Caprylic acid precipitation resulted higher purity than ammonium sulfate and finally purified antibody [97% purity] used as a capture antibody in sandwich ELISA test was able to detect erythropoietin antigen as sensitive [100%] and specific [100%] as commercial kits

Serum erithropoietin levels in normal population of Mexico City

La ciudad de México está localizada a una gran altitud sobre el nivel del mar (2240 m), es una de las metrópolis más contaminadas del mundo, y dado que la presión barométrica afecta la concentración de oxígeno en la sangre, asumimos que esto se podría ver reflejado en los niveles séricos de eritropoyetina. Para establecer circulante de determinó en 100 sujetos sanos, 52 hombres y 48 mujeres, utilizando un ensayo inmunoenzimático. El valor promedio obtenido fue de 18.67 ñ 7.6 mU/mL, que resulta similar al reportado previamente en ciudades al nivel del mar (18.0 ñ 5.8 mU/mL). El hábito de fumar no modifica en forma significativa las concentraciones séricas de eritropoyetina (21.03 ñ 10 mU/mL). No encontramos correlación entre los valores de hematocrito y las concentraciones séricas de eritropoyetina. Estos hallazgos sugieren la existencia de otro u otros factores que pudieran tener un papel en la regulación de la cantidad de eritrocitos circulantes