Four cases of pure red cell aplasia secondary to epoetin ß, with strong temporal relationships

Cae Tolman1, Shams Duja1, Donald Richardson1, Ramina Rashid2, Anthony McVerry2, Andrew Mooney1, Richard Baker1 and Eric Will1

1 Departments of Renal Medicine and 2 Haematology, St James's University Hospital, Leeds, UK

Correspondence and offprint requests to: Dr E. J. Will, Department of Renal Medicine, St James's University Hospital, Leeds, UK. Email: Eric.Will{at}leedsth.nhs.uk

Keywords: epoetin ß; pure red cell aplasia; temporal relationship



   Introduction
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 Introduction
 Cases
 Discussion
 References
 
Recombinant human erythropoietin (epoetin) has been widely and safely used in patients with chronic renal failure for over 10 years, with few complications. Recently, the use of epoetin has been associated with induction of formation of antibodies to erythropoietin (anti-erythropoietin antibodies) and the development of pure red cell aplasia (PRCA) [1,2]. Until September 2002, this syndrome had been reported in 155 patients worldwide, almost all associated with subcutaneous (s.c.) epoetin-{alpha} (Eprex®) [3]. In December 2002, the UK Committee on Safety of Medicines (CSM) distributed an urgent communication advising against s.c. use of Eprex in patients with chronic renal disease [4]. This stimulated a reappraisal of erythropoietin prescribing habits, with the reconsideration of epoetin-ß (Neorecormon®) and darbepoetin-{alpha} (Aranesp®), which rarely have been associated with PRCA [5]. Here, we describe the only patients in the UK who have developed PRCA during administration of epoetin-ß (personal communication from Christian Freitag, UK Drug Surveillance, Roche Pharmaceuticals), with a previously unpublished and noteworthy co-incidence in timing.



   Cases
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 Introduction
 Cases
 Discussion
 References
 
The first patient, a 65-year-old man with end-stage renal failure (ESRF) secondary to obstructive uropathy, began haemodialysis in June 2000. S.c. epoetin-ß was administered from June 2000 until February 2001. In November 2001, coronary artery bypass surgery was associated with a fall in haemoglobin (Hb), and epoetin-ß was restarted with relief of anaemia. Between May and June 2002, haemoglobin dropped precipitously (12.1 to 7.4 g/dl), requiring a blood transfusion. Investigations failed to demonstrate a cause for anaemia, other than the reticulocyte count, which reached a nadir of 3 x 109/l (normal range 20–80). In August 2002, bone marrow examination revealed PRCA. Subsequently, anti-erythropoietin antibodies were demonstrated in his serum at a high titre by radioimmunoprecipitation (RIP) assay (binding 2.5 U of [125I]epoetin/ml serum). Epoetin was stopped and he was treated with prednisolone for 3 months (1 mg/kg body weight for 1 month, halved each month, to stop in 3 months). After 6 weeks of treatment, a reticulocytosis developed. He currently is transfusion independent, maintaining Hb at 11 g/dl, with a reticulocyte count of 84 x 109/l. Repeat antibodies after treatment remained faintly positive by RIP assay (titre of 0.1–0.18 U/ml).

The second patient, a 52-year-old woman with ESRF secondary to Goodpasture's syndrome, started haemodialysis in January 1999. S.c. epoetin-ß was begun with good response. In May 2002, her Hb dropped from 14.0 to 6.9 g/dl, requiring regular blood transfusions. Her reticulocyte count dropped to 6 x 109/l; bone marrow biopsy confirmed PRCA. Again, anti-erythropoietin antibodies were demonstrated by RIP assay (12 U/ml). Epoetin was stopped and she was treated with prednisolone, as above. After 3 months, the reticulocyte count recovered (96 x 109/l), with serum epoetin levels of 9.8 mU/ml (range 2–20) and, although still anaemic, she has been transfusion independent for >6 months. She remained antibody positive with a titre between 0.21 and 0.87 U/ml.

In light of these two recent cases of PRCA diagnosed within our unit, we decided to revisit ‘unexplained’ red cell aplasia in two previous cases that had been recognized as unusual cases of anaemia.

Patient 3 (a man of Pacific Island origin) was 55 years old with ESRF secondary to hypertension and started haemodialysis in November 1996. Prior to dialysis, epoetin had been provided through his general practitioner, for s.c. administration (probably epoetin-{alpha}).

On commencing haemodialysis, he was converted to s.c. epoetin-ß, initially responding very well. In November 1998 (30 months after beginning epoetin therapy; 24 months after converting to epoetin-ß), his Hb fell suddenly (by 1.2 g/dl/week), and he became transfusion dependent. His reticulocyte count was 3 x 109/l, with no other causes of anaemia to be found. Bone marrow biopsy (February 1999) demonstrated PRCA.

He was treated with oral prednisolone, at a dose of 60 mg daily, while treatment with epoetin was continued. Six weeks after starting steroid therapy, he was admitted acutely with Staphylococcus aureus meningitis, and the subsequent demonstration of mitral valve endocarditis. A prolonged antibiotic course brought about a complete microbiological response. Within 4 months of starting treatment, the dose of steroids had been reduced and withdrawn, in view of the staphylococcal sepsis and its presumed causative role in the red cell aplasia. He subsequently suffered a dense cerebrovascular accident and recurrent staphylococcal septicaemia, to which he succumbed.

Despite continuing treatment with epoetin and steroid withdrawal, he had become independent of transfusion, with Hb stable at ~9 g/dl and a reticulocyte count of 259 x 109/l.

Fortuitously, serum was obtained from the time of his illness for tissue typing and was available for examination for anti-erythropoietin antibodies. At the time of epoetin-resistant anaemia, his serum was positive for anti-erythropoietin antibodies, with a binding capacity of 18 U/ml.

Patient 4, a man of Indian origin, with ESRF secondary to hypertension, recommenced haemodialysis in 1996, following transplant failure (10 years after his first renal transplantation). Treatment was started with s.c. epoetin-ß in April 1997. In July 1998, an AV graft was removed from the forearm because of pseudo-aneurysm formation.

In November 1998, his Hb fell by 3.1 g/dl, to 8.5 g/dl over 1 month, and he became transfusion dependent (reticulocyte count reaching a nadir of 3 x 109/l). Bone marrow aspirate and trephine showed red cell aplasia with reduced megakaryocytes and reduced myeloid haemopoeisis (despite a normal peripheral white cell count and platelet count), i.e. not true PRCA. Treatment was commenced with oxymetholone (an androgenic anabolic steroid, licensed in the USA for treatment of aplastic anaemias) at a dose of 50 mg tds. Epoetin therapy was stopped in September 1999, due to a lack of efficacy.

In November 1999, a purulent AV graft was removed surgically from his arm, cultures from which yielded enterococci. Six weeks prior to this event, his reticulocyte count had been 6 x 109/l (having been transfused with 19 units of blood in a 3 month period); 6 weeks after surgery, it was 67 x 109/l (during the 3 months post-surgery he received only 5 units of blood). The resolution of absolute transfusion dependence, concurrent with the removal of a source of infection, led to a diagnosis of epoetin resistance due to infection (of note, his C-reactive protein had been normal when anaemia developed).

In April 2000, following an unproductive investigation for a pyrexia of unknown origin and having become anaemic once more (with an acceptable degree of reticulocytosis, not suggestive of aplasia), he was treated empirically with anti-tuberculous triple therapy for a 6 month period (resulting in recovery of anaemia and resolution of pyrexia). Conversion to peritoneal dialysis (PD) due to loss of vascular access had occurred in May 2000, followed shortly after by an episode of fungal peritonitis. A follow-up bone marrow biopsy in May 2000 showed normal growth lines, and oxymetholone was discontinued in September 2000 (see Figure 1 and Table 1 for the series of events).



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Fig. 1. Sequential Hb, absolute and relative reticulocyte counts for patient 4 (see Table 1 for events).

 

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Table 1. Events in the history of patient 4, including the episode of red cell aplasia

 

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Table 2. Characteristics of PRCA patients

 
As he became anaemic again, s.c. epoetin-ß was re-introduced in September 2002, with the development of satisfactory Hb and reticulocyte indices (11.8–15.4 g/dl and 35–186 x 109/l, respectively) on a dose of 40 IU/kg/week. He subsequently died as a result of staphylococcal PD peritonitis.

Again, serum samples were taken for transplant work-up soon after the onset of anaemia, and were available for analysis. This patient proved to be anti-erythropoietin antibody positive by RIP assay (at a titre of >24 U/ml). Prior to his death, he remained weakly antibody positive by RIP assay (titres varying between 0.05 and 0.11 U/ml), while continuing to respond well to epoetin-ß therapy.

Reticulocyte counts in PRCA
We subsequently performed a retrospective review of our clinical database through the last 5 years, searching for paired Hb and reticulocytes (which had not been a routine component of haematological screening) in all dialysis patients. A total of 3252 paired, contemporaneous Hb and absolute reticulocyte results were analysed using Microsoft Excel (3735 paired Hb and relative reticulocytes results), revealing a median count of 63 x 109/l (2.2%). Mean reticulocyte count was 69±37 x 109/l (2.4±1.1%). A negative correlation existed between Hb and relative reticulocyte counts. Those who had an inappropriately low reticulocyte response to their anaemia (if adequately treated with epoetin) stood out clearly.

On examination of these paired results, the only patients in our records to show relative reticulocyte values falling below the 2.5th centile, with Hb <10 g/dl (with a history of epoetin exposure) are the four cases of PRCA we report here.

The identification (by discordant anaemia and reticulocyte response) of these four clinical cases serves to confirm the value of this approach.

Screening of the clinical databases of three of our regional units for discordant low Hb and reticulocyte counts has revealed one other epoetin-treated patient suitable for antibody screening [6] and may allow the biology of the epoetin immune response to be explored further.



   Discussion
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 Introduction
 Cases
 Discussion
 References
 
The use of epoetin recently has become associated with the induction of neutralizing antibody formation, causing PRCA. The presence of neutralizing anti-erythropoietin antibodies has also been reported in a case of PRCA with no prior exposure to epoetin [7].

The sole use of Eprex is believed to be associated with an incidence of 1.24 cases per 10 000 patient years in the period between 1998 and 2002 [8]. Cases of PRCA related to other brands of epoetin-{alpha}, epoetin-ß and darbepoetin remain extremely rare: a SwissMedic report from March 2003 estimated the incidence of PRCA associated with the sole use of epoetin-ß to be 0.14 per 10 000 patient years over the same time period [8], with a total of eight reported cases (including the three pure epoetin-ß cases reported here) globally. Theories regarding aetiology include route of administration (nearly all cases are related to s.c. administration), formulation (possibly related to removal of human serum albumin from Eprex in 1998, or silicon used within the same pre-filled syringes) and handling (warming to room temperature may allow denaturation of the glycoproteins, enhancing immunogenicity) although, to date, the causation is not established incontrovertibly.

Our patient 4 is one of the first documented cases of reinstitution of epoetin therapy after development of PRCA with anti-EPO antibodies, and the fact that he remained faintly positive by RIP assay is of interest. As with all of these cases, we do not know whether these are neutralizing antibodies, since bioassay to demonstrate neutralization of erythropoiesis in bone marrow cultures has not been performed. It has been shown, however, that there is a high degree of concordance between antibodies detected by the RIP assay used to diagnose these cases and neutralization of bone marrow culture erythropoiesis [2]. Such accidental re-exposure may be more common than has been suspected previously and carries obvious interest for the origin, persistence and biology of the antibody response.

It is also worthy of mention that while a case series (in preparation) suggests 27 reported cases of epoetin-associated PRCA in the UK between 1998 and 2003 (all related to s.c. therapy), the cases reported here are the only pure epoetin-ß cases.

Temporal co-incidence of PRCA cases
The two pairs of cases (current and historical) show obvious temporal co-incidence: cases 1 and 2 in May 2002; cases 3 and 4 in November 1998. The two current cases were receiving haemodialysis at the same satellite unit at onset of disease (where s.c. epoetin is administered by nursing staff). In the two historical cases, there were no obvious geographical associations, although we cannot exclude their both receiving haemodialysis at our main unit at the same time (where, potentially, they could have received epoetin from the same batch). Personal communication with Professor Casadevall reveals that within her series [2], there were three episodes of PRCA from within the same unit over a 3 month period, which she attributed to the unforgiving handling characteristics of Eprex. Some evidence suggests that the formulation of epoetin-ß (in the form of Neorecormon) is more stable than Eprex and should not be as prone to the same instabilities. The temporal co-incidence of our cases does suggest an environmental factor, perhaps related to the patients rather than the epoetin preparation, such as a common viral infection provoking the immune system to develop anti-erythropoietin antibodies.

Screening of the prevalent population of the satellite dialysis unit where our recent cases occurred for the presence of less clinically significant anti-erythropoietin antibodies has been performed, with the identification (by RIP assay) of two patients displaying antibodies; neither of these has ever suffered an unexplained loss of sensitivity to epoetin products. The identification of PRCA may yet come to serve as a marker of other, subclinical, antibody responses in the local dialysis patient peer group and at least focuses screening for antibodies on a population that has demonstrated some potential hazard. In the historical cases, an episode of significant bacterial infection was co-incident, the treatment of which was temporally associated with resolution of the marrow aplasia. The relationship between anti-erythropoietin antibodies and PRCA is not simple apparently, with the relevance of other stimuli, such as infection, likely.

Summary
We describe two pairs of patients with PRCA associated with epoetin-ß. The two recent cases (2002) presented a typical profile, with complete or partial response to corticosteroids. Two historical cases identified by anecdote, and then by discordant low reticulocyte responses, had been diagnosed previously as due to sepsis-induced epoetin resistance. They showed a complete clinical picture, where both suffered a sudden fall in Hb consistent with aplasia (one with atypical bone marrow findings), and appear to have recovered under different treatment regimes. One patient was re-exposed to epoetin-ß without recurrent clinical disease. Remarkably, the cases were temporally related in 1998 and 2002, but physical association was not apparent in the historical pair. Mishandling of the epoetin product or a shared viral aetiology are possibilities that may be suspected.

Conflict of interest statement. Dr Tolman is in a research post funded by the Yorkshire Kidney Research Fund which receives support from a variety of commercial sources.



   References
 Top
 Introduction
 Cases
 Discussion
 References
 

  1. Bergrem H, Danielson BG, Eckhardt KU et al. An unusual case of anti-erythropoietin antibodies following recombinant human erythropoietin treatment. In: Erythropoietin: Molecular Physiology and Clinical Application. Marcel Dekker, New York; 1993: 266–275
  2. Casadevall N, Nataf J, Viron B et al. Pure red-cell aplasia and anti-erythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 2002; 346: 469–475[Abstract/Free Full Text]
  3. Press release from Johnson & Johnson. September 18 2002. http://www.jnj.com/news/jnj_news/20020917_142708.htm
  4. Breckenridge A. Eprex® (Epoetin Alfa) and Pure Red Cell Aplasia—Contraindication of Subcutaneous Administration to Patients with Chronic Renal Disease. Committee for the Safety of Medicines, Department of Health, London; 2002.
  5. Krüger A, Schröer W, Röhrs F, Vescio G. PRCA in a patient treated with epoetin beta. Nephrol Dial Transplant 2003 18: 1033–1034[Free Full Text]
  6. Tolman C, Will E, Bartlett C, Woodrow G. A discordant reticulocyte response to anaemia in EPO treated patients reveals PRCA in renal databases. Renal Association, Autumn Meeting; 2003 (A 36)
  7. Casadevall N, Dupuy E, Molho-Sabatier P et al. Brief report: autoantibodies against erythropoietin in a patient with pure red cell aplasia. N Eng J Med 1996; 334: 630–633[Free Full Text]
  8. Swissmedic report. March 26 2003. http://www.swissmedic.ch/de/fach/overall.asp?lang=1&theme=0.00086.0000100001_id=923&page=3
Received for publication: 8. 5.03
Accepted in revised form: 8. 3.04