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Background: Current guidelines support the use of erythropoiesis-stimulating agents for the treatment of anemia associated with low-risk myelodysplastic syndromes (MDS)., Design and Methods: Single-arm, open-label, multi-center, phase 2 trial that evaluated the efficacy and safety of darbepoetin alfa (DA) in patients with low or intermediate-risk MDS, hemoglobin <100 g/L, erythropoietin (EPO) levels <500 IU/L and transfusion requirements <2 units/month over the preceding 2 months. Erythroid response (major [MaR] or minor [MiR]) and fatigue (Functional Assessment of Cancer Therapy-Fatigue [FACT-F]) were evaluated at 8, 16 and 24 weeks. DA was initiated at 300 μg weekly. For patients who did not achieve MaR by 8 weeks, filgrastim 300 μg weekly was added., Clinical Trial Registration: clinicaltrials.gov identifier: NCT01039350., Results: Forty-four patients (72.7% transfusion independent) were included. Median age was 76.0 years (range 41.3-92.4), 54.5% were male, and 90.9% presented ECOG Status (0-1). Eighteen patients received filgrastim. An erythroid response was achieved by 31 of 44 patients (70.5%) at week 8 (47.7% MaR, 22.7% MiR), 31 of 44 patients (70.5%) at week 16 (61.4% MaR, 9.1% MiR), and 32 of 44 patients (72.7%) at week 24 (61.3% MaR, 11.4% MiR). Mean (95% CI) change in FACT-F at week 24 was 3.61 (0.72 to 6.51). Baseline EPO levels <100 IU/L were a predictive factor of response. DA was well tolerated. Four mild (two iron deficiencies, flu syndrome and headache) and one fatal (thromboembolic event) adverse events were considered related to darbepoetin alfa., Conclusions: A fixed dose of 300 μg of darbepoetin alfa weekly (with or without filgrastim) seems to be an effective and safe treatment for anemic patients with low or intermediate-risk MDS, low transfusion burden and EPO levels <500 IU/L. Results may not be extrapolable to unselected MDS patients.

The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU x l(-1) (s = 3.2) to 16.6 IU x l(-1) (s = 4.7)] and final exposures [from 4.6 IU x l(-1) (s = 1.4) to 24.8 IU x l(-1) (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.

Soluble transferrin receptor (sTfR) has been proposed as an indirect biomarker of the misuse of recombinant human erythropoietin in sport. An extended validation of four commercially available immunoassays for its measurement in serum is presented. Two ELISA techniques (ELISA1: Orion Diagnostica; ELISA2: R&D Systems), an immunoturbidimetric technique (Turbid: Roche Diagnostics), and a nephelometric technique (Nephel: Dade Behring) were investigated. Intra-laboratory precision better than 3% and correct accuracies were obtained for the Turbid and Nephel techniques using autoanalysers. Slightly worse precision (but always better than 11%) and correct accuracies were also obtained in almost all cases for the two ELISA techniques. Inter-laboratory results showed higher concordances for the ELISA procedures (intraclass correlation coefficients of 0.848 for ELISA1 and 0.973 for ELISA2 which was clearly better). Inter-technique correlations were good for the four techniques with lower dispersions found for the techniques using autoanalysers, i.e. Turbid and Nephel. While Turbid and ELISA1 results (expressed in mg/l) were comparable, results obtained with Nephel were approximately 2.7 times lower. The relationship between those three techniques was maintained when compared with ELISA2, which uses different units (nmol/l). We conclude that ELISA2 and Nephel in our hands were the most suitable techniques in terms of sensitivity, precision and accuracy, and adequacy of the calibration curve for the measurement of sTfR in real serum samples. Discrepancies observed in the results obtained with the different sTfR techniques showed that different reference standards were used and harmonization is recommended in order to obtain comparable results.

The measurement of serum erythropoietin (EPO) has been proposed as one of the indirect biomarkers for the detection of recombinant human EPO misuse in sport. An extended inter-laboratory validation of two commercial immunoassays for EPO measurement is described. A chemiluminescent immunoassay kit (CHEM) and an enzyme-linked immunosorbent assay kit (ELISA) were evaluated. The CHEM assay showed intra-laboratory precision better than 6% and correct accuracy values for all quality control samples tested. Precisions and accuracies better than 7 and 13%, respectively, were obtained for the ELISA assay for most of the quality control samples. The limit of quantification estimated for CHEM assay was lower than for the ELISA assay. Inter-laboratory concordance was good for both the assays, with lower dispersion shown by the CHEM assay. Results obtained with the ELISA assay were always lower than those of the CHEM assay. However, a good inter-technique correlation was obtained ([ELISA]=0.76 [CHEM]+0.06, r2=0.92). Quality control samples had a good stability after one and two freeze/thaw cycles and in simulated transportation conditions. In conclusion, CHEM and ELISA assays showed similar characteristics regarding intra-laboratory validation. Better inter-laboratory results were obtained with the CHEM assay and, hence, it is considered the recommended assay for anti-doping control analysis.

Background and Objective: Anemia leading to transfusion is probably the most important problem in patients with myelodysplastic syndromes (MDS). Human recombinant erythropoietin (rHuEpo) and granulocyte colony-stimulating factor (G-CSF) have been used to treat patients with anemia of MDS, but fewer than 50% respond. The aim of this work was to evaluate the benefit of rHuEpo +/- G-CSF treatment and to isolate the response predictive variables in a group of selected patients with MDS., Design and Methods: A non-randomized multicenter trial was carried out in 32 patients with MDS. The inclusion criteria were age >= 18 years, refractory anemia (RA) or refractory anemia with ringed sideroblasts, Hb <= 100 g/L or receiving transfusions and serum erythropoietin <= 250 U/L. These patients were treated with subcutaneous rHuEpo (300 U/kg) three times a week for 8 weeks. In the case of partial response (PR) or no response (NR) subcutaneosly administered G-CSF (1 microg/kg) three times a week was added to the rHuEpo for 8 more weeks. If the patient achieved complete response (CR) or PR in the second phase, he was included in a follow-up phase of 24 weeks in which the dose of growth factors was tapered down. Several variables, including the score published by the Scandinavian-American group, were used as possible predictive variables., Results: An erythroid response was observed in 16 patients (50%); in 12 it was a CR and in 4 it was a PR. During the period of rHuEpo administration, 7 CR and 4 PR (34.4%) were documented. Of the 14 patients in whom G-CSF was added to rHuEpo, 7 (50%) responded (3 CR and 4 PR). No major side-effects associated with growth factors were observed. The multivariate analysis showed that of the different variables evaluated only the Scandinavian-American response score was significant with a relative probability of response of 11.8 (95% confident intervals: 2.5-53) when this score was > +1 (77% of cases responded). In contrast, when this score was <= 1 only 15 % of the cases responded., Interpretation and Conclusions: Use of the Scandinavian-American response score is to be recommended in a patient-oriented approach to treating MDS cases with the Epo and G-CSF. Treatment with rHuEpo and G-CSF is safe, its main drawback being its cost. However, a long-term study evaluating the regimen's cost-benefit ratio is warranted.

Background and Objective: It has been suggested that the determination of serum erythropoietin (sEpo) may be useful in distinguishing between polycythemia vera (PV), relative polycythemia and secondary polycythemia (SP), but no conclusive evidence has yet been provided for this. In the present work, we evaluated the role of sEpo in the differential diagnosis of polycythemia vera and its usefulness in the follow-up of PV patients., Methods: sEpo was assessed in 190 patients with polycythemia of different etiologies. A follow-up study was carried out in some of these patients (27 with secondary polycythemia and 17 with polycythemia vera)., Results: sEpo levels were higher in SP than in PV and relative polycythemia. There were, however, differences with regard to the various etiologies of SP. Polycythemia related to congenital heart disorders showed the highest levels of sEpo of the SP. When a study was conducted, sEpo alone as a diagnostic parameter displayed an efficiency of more than 90% and the most discriminating value was 5 U/L. Using lower levels (below 2 U/L) and higher levels (above 12 U/L), it was possible to distinguish between SP and PV, although an important overlap was detected between these limits (approximately 50% of cases). The follow-up study showed that in half the patients with SP the levels of sEpo were at times < 12 U/L and at other times greater than this value. At least three determinations were necessary to detect an elevated reading. In PV after venesection there was an increase in sEpo in some cases, although most of the time there was no change., Interpretation and Conclusions: Using sEpo, it was possible to differentiate between PV and SP, despite an important overlap. A follow-up study demonstrated that the increase in sEpo was intermittent in SP and that in many of these cases more than one determination could be helpful.

We studied erythropoiesis in 31 patients with vitamin B12 deficiency by measuring serum erythropoietin (s-Epo), serum transferrin receptor (s-TfR, taken as an index of total erythroid activity), reticulocyte count, and the reticulocyte maturation index (RMI). s-Epo and s-TfR were measured with commercial immunoassays, whereas reticulocyte count and RMI were determined by flow cytometry. s-Epo (123 +/- 196 U/L) and s-TfR (4.1 +/- 2 mg/L) levels were increased in patients with vitamin B12 deficiency. The absolute reticulocyte counts were decreased (29 +/- 18 x 10(9)/L) with a relative increase in the most immature fractions (RMI: 29.6 +/- 18%). A significant negative relationship was found between s-Epo and Hb level (r = -0.65, p < 0.0001). On the average, however, s-Epo was inappropriately low for the degree of anemia, since the observed/predicted (O/P) s-Epo ratio was 0.80 +/- 0.28 in vitamin B12 deficiency vs 1.00 +/- 0.16 in a group of patients with iron deficiency anemia. It is concluded that at least a portion of patients with vitamin B12 deficiency have serum erythropoietin levels that are inappropriately low for the degree of anemia.

To evaluate the role of erythropoietin (Epo) in the erythroid abnormalities often found in athletes, Epo was evaluated by radioimmunoassay in endurance runners (ER). In a first study, 46 experienced ER, 11 with iron deficiency (ID group), were studied during a training period. In ID and non-ID runners, serum Epo (SEpo) levels were similar to sedentary controls (ID = 19.1 +/- 4.9 U/L, non-ID = 19.7 +/- 5.5 U/L and controls 19.7 +/- 9.2 U/L). In a second study, serum and urine erythropoietin (UEpo) levels were evaluated in 17 ER during a 6-hour race. Samples were taken before the race (pre-race), immediately following (6-hour) and 4 days after (post-race). No differences were observed in SEpo levels (pre-race = 19.8 +/- 4.1 U/L, 6-hour = 21.2 +/- 4.9 U/L and post-race = 21 +/- 4 U/L), but UEpo increased following the race (pre-race = 15.4 +/- 9.6 U/L, 6-hour = 26.1 +/- 6.2 U/L and post-race = 14.1 +/- 6.5 U/L) (p < 0.0001) and this UEpo increase was related to urine creatinine changes (rs = 0.79, p < 0.00001). In conclusion, SEpo in ER does not differ from sedentary values and does not vary with competition; however, UEpo increases during a long-distance race. These data may be important for a correct evaluation of Epo abusers and sports anemia.

True erythrocytosis is a relatively common complication of successful renal transplantation. From a group of 17 patients with post-transplant erythrocytosis (PTE), four underwent selective venous catheterization of the native and transplanted kidneys because of arterial hypertension. In three who presented with active PTE at the time the procedure was performed the peripheral blood (PB) concentration of EPO was elevated, and the level of erythropoietin (EPO) in native kidney veins was significantly higher than the PB and allograft EPO levels. Additionally, only one of the three cases had high levels of plasma renin activity (PRA). The fourth patient showed normal levels of EPO and PRA in PB and in venous blood from the native and transplanted kidneys. However, the PTE had subsided 4 months before the performance of the catheterization after he redeveloped terminal renal failure; the loss of a functioning allograft might have blunted the overproduction of EPO by the native kidneys. In conclusion, in patients with PTE, inappropriate EPO production seems to originate from the diseased native kidneys, and there seems to be no correlation between the production of EPO and the PRA.

Erythroid alterations were studied in 136 patients with rheumatoid arthritis (RA). Anemia was present in 75 cases. A definite diagnosis was determined in 65. The most frequent anemia was that of chronic disease (ACD) (43 cases); 14 patients with ACD presented with moderate to severe anemia. Prevalence of deficiencies were also high (15 cases had iron deficiency anemia, IDA). Serum erythropoietin levels were different in patients with RA compared with a healthy control group (p < 0.00001). Serum erythropoietin was increased in ACD (49 +/- 28.8 U/l) with respect to both RA (38.6 +/- 12.7 U/l, p = 0.0036) and controls (18.2 +/- 7.6 U/l, p < 0.00001). Although hemoglobin (Hb) was similar in ACD and IDA, serum erythropoietin in ACD was lower than in IDA (p = 0.01). There was a negative relationship between Hb and serum erythropoietin in ACD (r = -0.42, p = 0.005). In conclusion, almost 50% of patients with RA have anemia and ACD is the most frequent. As serum erythropoietin in ACD is blunted, patients with moderate to severe ACD are possible candidates for erythropoietin treatment.

Background: In order to know the influence of dialysis treatment in erythropoietin production, serum erythropoietin (Ep) has been studied in patients with anemia due to chronic renal failure (CRF)., Methods: Thirty six of them in hemodialysis (HD), 10 in continuous ambulatory peritoneal dialysis (CAPD) and 18 in predialysis stage (PD) and their results were compared to two control groups, including 72 healthy controls (HC) and the second one 89 iron deficiency anemia patients (ID)., Results: Patients had lower Hb and Ep levels than the other groups. Although Ep was higher in CAPD and PD. Ep levels were similar to HC values, and lower than ID levels. It could be demonstrated any correlation between Hb and Ep in CRF patients, however a negative exponential correlation was demonstrated in ID patients between Hb and Ep (r = -0.83; p less than 0.00001). In summary, Ep is higher in CAPD and in PD than in HD, but the levels are lower than they should be expected., Conclusions: These data confirm an Ep production failure in most of IRC patients and it seems likely that Ep treatment could be effective to treat the anemia of CRF.