Role of serum holotranscobalamin (holoTC) in the diagnosis of patients with low serum cobalamin. Comparison with methylmalonic acid and homocysteine.

Plasma holotranscobalamin (holoTC) transports active cobalamin. Decreased levels of holoTC have been considered to be the earliest marker of cobalamin (Cbl) deficiency. In this work, holoTC was evaluated in low or borderline serum Cbl (LB12) and a concordance analysis was carried out with methylmalonic acid (MMA) and homocysteine (Hcy). Levels of Cbl, holoTC, MMA, and Hcy were investigated in a reference group in 106 patients with LB12 (≤200 pmol/l) and in 27 with folate deficiency (FOL). HoloTC levels were evaluated by an automated immunoassay (Active B12, Abbott Lab, Abbott Park, IL, USA). Lower levels of holoTC were observed in both LB12 and FOL groups (reference group vs LB12; p < 0.0001. Reference group vs FOL; p = 0.002). HoloTC levels were lower in LB12 than in FOL (p = 0.001). In LB12, concordance between Hcy and MMA was 82.1 % (chi-square test, p < 0.001; Kappa Index, 0.64, p < 0.0001). Concordance between Hcy and holoTC was 62 % (chi-square test, p = 0.006; Kappa index, 0.245, p = 0.006). Concordance between holoTC and MMA was 55.6 % (p = 0.233). Some cases with LB12 and elevated MMA did not show decreased holoTC. By contrast, MMA and Hcy were not increased in some patients with low holoTC and LB12. In conclusion, levels of holoTC were decreased in LB12 and FOL. In LB12 patients, holoTC concordance with MMA was poor. MMA/Hcy levels were not increased in a significant number of subjects with LB12 and low holoTC. This profile was found in iron deficiency. The significance of these changes remains to be clarified.

Management of iron deficiency in various clinical conditions and the role of intravenous iron: Recommendations of the Spanish Erythropathology Group of the Spanish Society of Haematology and Haemotherapy. / Manejo del déficit de hierro en distintas situaciones clínicas y papel del hierro intravenoso: recomendaciones del Grupo Español de Eritropatología de la SEHH.

Iron deficiency due to blood loss, absorption disorders and dietary deficiencies causes iron-deficiency anaemia, whose treatment seeks to eliminate the underlying cause and restore haemoglobin and iron deposits. Typically, the latter 2 of these objectives can be achieved through oral iron therapy. Intravenous iron administration (IIA) should be limited to those patients refractory or intolerant to oral preparations or who require rapid repletion. The indiscriminate use of IIA can increase morbidity and mortality due to iatrogenic overload. This fact, coupled with the growing popularity of IIA and the lack of reference guidelines in Spanish, led the Spanish Erythropathology Group of the Spanish Society of Haematology and Haemotherapy to develop this study, which presents the main recommendations on the optimal use of IIA in iron deficiency and attempts to constitute reference guidelines on good practices for the clinical management of these conditions.

Vitamin B12 and folate levels increase during treatment of iron deficiency anaemia in young adult woman.

INTRODUCTION: The relationship between iron deficiency and vitamin B12 and folate was recognized several decades ago. Combined deficiency is important in clinical practice owing to its relationship with malabsorption syndromes. By contrast, iron deficiency and low levels of serum vitamin B12 with normal metabolic markers were often found mostly in young adults. In this work, vitamin B12/folate changes were investigated during treatment of iron deficiency anaemia (IDA) with pharmacological iron in young adult women. METHODS: A cohort of 35 young adult women with IDA was treated with oral iron. An haematological response was obtained in 97.2% at 4-month follow-up. Changes in serum vitamin B12, serum folate and other biochemical parameters were monitored. RESULTS: Treatment with iron increased significantly serum folate and vitamin B12 from baseline. This increase was also observed in vitamin B12 levels ≤200 pmol/L (six patients, 17.1%), in whom serum vitamin B12 was above 200 pmol/L at the end of the study in all cases. Other biochemical parameters also changed. Significant increases were seen for glucose (P = 0.012), uric acid (P < 0.001), total cholesterol (P = 0.023), HDL cholesterol (P = 0.026) and bilirubin (P < 0.001). Urea decreased significantly (P = 0.036). CONCLUSIONS: Data from our work suggest that iron deficiency could affect many metabolic pathways, including vitamin B12, folate and lipids. These changes normalize after iron therapy, even in women with baseline low levels of serum vitamin B12. Healthcare practitioners should be aware of these changes in IDA management. The mechanisms controlling these changes remain to be explained, but they are probably related to the control of iron homeostasis (iron deficiency mediated stimuli).

Cobalamin deficiency in patients infected with the human immunodeficiency virus.

Serum vitamin B12 levels are often low in human immunodeficiency virus (HIV)-infected patients. However, only a few patients appear to have actual vitamin B12 deficiency. A low red cell folate level accompanying the low vitamin B12 level makes the presence of vitamin B12 deficiency more likely. Our experience suggests that a low red cell folate level always indicates deficiency, but does not differentiate between vitamin B12 and folate deficiency. The deoxyuridine suppression test and the assay of serum or plasma total homocysteine and/or of methylmalonic acid levels can also be useful in the identification of patients with true vitamin B12 deficiency. HIV-positive patients frequently have absorption disorders, including vitamin B12 malabsorption. However, the correlation between vitamin B12 malabsorption and serum vitamin B12 and plasma homocysteine levels is poor. Abnormalities in vitamin B12-binding proteins, which are often found in HIV-positive patients, may explain many cases of low vitamin B12 levels. Current evidence suggests that low vitamin B12 levels are more common as the HIV disease progresses. The results of vitamin B12 treatment have been disappointing thus far, including the prevention of toxicity induced by azidothymidine. The possible role of vitamin B12 treatment in the long-term survival of HIV-infected patients is at present unknown. However, it is important to identify those patients who have real vitamin B12 deficiency to treat or prevent their hematologic and/or neurological symptoms.

Evaluation of immunoassays for the measurement of soluble transferrin receptor as an indirect biomarker of recombinant human erythropoietin misuse in sport.

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.

Iron overload might increase transplant-related mortality in haematopoietic stem cell transplantation.

Iron overload (IO) is associated with free radical generation and tissue damage. Our main objective was to ascertain if very high levels (VHL) of ferritin (>/=3000 microg/l) and transferrin saturation (TS) >/=100% during conditioning had an impact on overall survival (OS) and transplant-related mortality (TRM) after a haematopoietic stem cell transplantation (HSCT). Levels of ferritin and TS were measured at days -7 and -4, respectively, in 25 patients who underwent HSCT after CY/TBI. The group consisted of 20 men and five women with a median age of 40 years. Fifteen patients were autotransplanted and 10 allotransplanted. Nine of them had a diagnosis of AL, six of CML and 10 of lymphoma. Thirteen of them were in early and 12 in advanced status of disease. VHL of ferritin and TS >/=100% were associated with a decreased OS (P = 0.001 and P = 0.006, respectively) and an increased TRM (P = 0.003 and P = 0.004, respectively) in univariate survival analysis. Both variables remained significant at multivariate analysis for OS (P = 0.03 and 0.02, respectively) and TS was an independent factor for TRM (P = 0.01). Ferritin was very close to achieving statistical significance for TRM (P = 0.06) in multivariate analysis. In conclusion, VHL of ferritin and TS >/=100% at conditioning are associated with an increase in toxic deaths after transplant.

Frequent severe liver iron overload after stem cell transplantation and its possible association with invasive aspergillosis.

Iron overload is associated with free radical generation and tissue damage. Our main objective was to ascertain the frequency and severity of iron overload in a group of 59 patients who died after conventional-intensity autologous (n=24) or allogeneic (n=35) haematopoietic stem cell transplantation (HSCT). A second objective was to investigate associations between liver-iron concentration and causes of transplant-related mortality. The median age was 41 years (range, 19-66), 41 were males and 18 females. In total, 26 patients had acute leukaemia or MDS, 10 CML, 17 lymphoma, four myeloma and two aplastic anaemia. The median hepatic iron concentration (HIC) was 138 micromol/g dry weight (7.7 mg/g; range 31-631 micromol/g). In total, 4/32 (12%) patients with HIC <150 micromol/g and 10/27 (37%) with hepatic iron > or =150 micromol/g showed invasive aspergillosis at autopsy (P=0.035). This was significant in multivariate analysis (RR 9.0; 95% CI 1.6-50.3, P=0.012). In conclusion, severe iron overload is frequent in patients who die following HSCT and is associated with invasive aspergillosis.

Changes in reticulocyte fractions during peripheral stem cell harvesting: role in monitoring stem cell collection.

We prospectively evaluated the changes in immature reticulocyte fractions during peripheral blood stem cell mobilization to determine any possible relationship with mobilization of stem cells into the peripheral blood. Circulating neutrophils, immature reticulocyte fractions (% of HFR + MFR) (HMFR) (measured by flow cytometry), circulating CD34+ cells (measured by flow cytometry) and CFU-GM (measured by semisolid media assay in the apheresis fluid) were closely monitored following priming with chemotherapy and colony-stimulating factors in 15 patients with hematological or solid tumors (group I). Day 0 was defined as the day on which the neutrophil count fell below 0.5 x 10(9)/l. In a second group of nine patients (group II) reticulocyte fractions and CD34+ cells were measured directly on the days on which they were predicted to increase using the data from group I. Reticulocyte counts and HMFR were also monitored in 18 patients who were mobilized with G-CSF alone. In group I, a significant rise in HMFR and CD34+ cells occurred on days 2, 4 and 6, and a linear correlation between HMFR on day 2 and CD34+ cells on day 4 was demonstrated (P = 0.0068, r = 0.74). In group II similar patterns of recovery were found. During mobilization with G-CSF alone HMFR significantly increased on days 2, 4 and 6 of treatment with respect to baseline values, and a multiplicative relationship between the increase of HMFR and neutrophils was observed (r = 0.707, P < 0.00001). Unfortunately, patients who did not mobilize CD34+ cells (one in groups I and II and three in the G-CSF group) showed similar HMFR kinetics to those who mobilized CD34+ cells. An increase in immature reticulocyte fractions precedes the presence of circulating CD34+ cells by about 2 days in patients mobilized with chemotherapy and growth factors, and it could thus serve as an indirect surrogate marker for monitoring the timing of stem cell harvesting. An unexpected increase of HMFR during treatment with G-CSF alone was found, indicating an effect of this factor on erythropoiesis in vivo.

[Atypical familial microcytosis: a familial and genetic study]. / Microcitosis familiares atípicas: estudio familiar y genético.

The characteristics of familiar atypical microcytosis studied during one year were evaluated. Out of 149 patients with microcytosis in whom iron deficiency was ruled out, a heterogenous beta-thalassemia was diagnosed in 72 cases, a heterozygous delta beta-thalassemia in 16 cases and a hemoglobinopathy in 3 cases. The microcytosis was related to an inflammatory anemia in 12 cases and to an hemopathy in 9 cases. An atypical microcytosis was detected in 37 patients. A familiar and molecular analysis was carried out to detect alpha-thalassemia in cases with atypical microcytosis. It was possible to complete the familiar and molecular analysis in 35 out of 37 cases, and an alpha-thalassemia was observed in 31 patients. Most cases proved to be heterozygous or homozygous-alpha 3.7-thalassemia. No patient with heterozygous alpha zero-thalassemia was found. Most cases of familiar atypical microcytosis in our country are due to -alpha 3.7-thalassemia. Bearing these findings in mind, this analysis should only be used in situations where a problem of prenatal diagnosis is present. Moreover, systematic molecular analysis of familiar atypical microcytosis could be justified if the MCV is lower than 75 fl.

[Influence of chronic renal failure and of its treatment on serum erythropoietin]. / Influencia de la insuficiencia renal crónica y de su tratamiento en la eritropoyetina sérica.

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.

Immune complexes and persistent high levels of serum vitamin B12.

INTRODUCTION: Patients with persistent high levels of serum vitamin B12 were often referred to Hematology departments. In this study, characteristic of patients with serum vitamin B12 levels higher than 2500 pmol/L (high B12) were studied. METHODS: Prevalence of high B12 was evaluated during a 10-month period. Samples with high B12 were incubated with polyethylene glycol (PEG) and a new measurement of vitamin B12 was carried out using the supernatant. As a pilot study, 26 frozen samples with high B12 were evaluated for changes in vitamin B12 after PEG. Moreover, a prospective study was carried out during three consecutive months. Size exclusion chromatography was employed to demonstrate the presence of immune complexes (ICs) with plasma vitamin B12-binding proteins in some serum samples with high B12. RESULTS: Prevalence of high B12 was 1.3%. Results from 26 frozen samples and from a prospective study (28 cases) showed that undergoing vitamin B12 treatment was the main cause of high B12. However, ICs were detected in 10 frozen samples and in seven cases (25%) of the prospective study, respectively. Serum vitamin B12 decreased to normal values after precipitation with PEG, and size exclusion chromatography confirmed ICs. An association with autoimmune or hematological disorders was observed. CONCLUSIONS: In patients with repeatedly high B12 levels, ICs were detected in approximately 25% of samples. Precipitation with PEG is an easy method to confirm the presence of ICs and to evaluate serum vitamin B12 levels in these patients.

Darbepoetin alfa for anemia in patients with low or intermediate-1 risk myelodysplastic syndromes and positive predictive factors of response.

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.

Vitamin B-12 abnormalities in HIV-infected patients.

A prospective study of 60 consecutively admitted patients with HIV infection was performed to document the prevalence, etiology and manifestations of low serum vitamin B-12 in such patients. Low serum B-12 levels were found in 10 patients (16.7%). In 6, vitamin B-12 absorption was impaired and hog intrinsic factor addition did not improve it. Patients with low vitamin B-12 levels showed lower hemoglobin, leukocytes, lymphocytes, CD4 lymphocytes and CD4/CD8 lymphocyte ratio than HIV patients with physiological serum vitamin B-12 levels. However, bone marrow megaloblastosis was found in only 3 low vitamin B-12 patients and the deoxyuridine suppression test was pathological in only 1 case. In 7 patients, parenteral treatment was begun with variable response despite serum vitamin B-12 correction. In conclusion, low serum vitamin B-12 is often found in HIV-infected patients and it could be related to malabsorption, but clear megaloblastic abnormalities and treatment response could not be demonstrated. A decreased concentration of the serum binders due to disturbances in the leukocytes and related immunocompetent cell may play an additional role.

[Hematologic changes induced by exertion during a long-distance race]. / Cambios hematológicos inducidos por el ejercicio durante una carrera de largas distancias.

PURPOSE: To evaluate the haematological changes induced by a long-distance race in well trained runners. PATIENTS AND METHODS: The haematological changes presented by 17 runners (15 men and 2 women) were assessed in a 6-hour race. For this purpose samples were examined before the race, 2 hours later, 4 hours later, at the end of the race, and 4 days after the trial. RESULTS: Haemoglobin rates, as well as leucocyte, neutrophil, monocyte and platelet counts were increased, probably due to decreased plasma volume. Haemolytic traits were seen as well, namely, increased reticulocyte count and decreased haptoglobin. High transferrin-transport capability and serum B12 levels were also present. Mild haematuria was found without any haemosiderinuria, siderinuria or haemoglobinuria. All these changes recovered in the post-race study. CONCLUSION: Changes in the major haematologic values are present during long-distance races, some of them due to haemoconcentration. Leucocytosis, haemolysis and haematuria are noteworthy, and these findings must be borne in mind when evaluating the health of runners.

Postrenal transplant erythrocytosis: further evidence implicating erythropoietin production by the native kidneys.

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.

Serum transferrin receptor in polycythemia.

We measured serum transferrin receptor (sTfR) levels in 22 patients with polycythemia vera and in 26 cases of secondary polycythemia. In our study, raised sTfR levels in both polycythemia groups were related to iron deficiency.

The role of serum transferrin receptor in the diagnosis of iron deficiency.

BACKGROUND AND OBJECTIVE: Iron deficiency anemia (IDA) is often associated with inflammatory disorders. The most conventional parameters of iron metabolism are therefore affected, making the evaluation of iron status difficult. Serum transferrin receptor (sTfR) levels are raised in iron deficiency but are not influenced by inflammatory changes. The aim of this study was to investigate the role of sTfR in differentiating IDA with inflammatory features. DESIGN AND METHODS: A diagnostic study of sTfR measured by immunoassay was carried out in IDA and anemia of chronic disorders (ACD). The cut-off points of sTfR and the ratio of sTfR/serum ferritin, which were obtained after comparing IDA and ACD, were applied to a group of 64 patients with mixed iron patterns (MIX) (16 with ACD and 48 with IDA). RESULTS: The best cut-off point of sTfR between IDA and ACD was 4.7 mg/L. Applying this cut-off to the MIX group, an efficiency of 87% was obtained (sensitivity 92% and specificity 81%). This level of sTfR correctly classified 53 out of 64 cases of the MIX group (83%). Using the ratio of sTfRx 100/serum ferritin, the best cut-off point was 8 (efficiency 100%), which correctly classified 62 out of 64 cases of the MIX group (97%). INTERPRETATION AND CONCLUSIONS: This study demonstrates that sTfR in conjunction with other iron parameters is very useful in iron deficiency evaluation, especially in hospital practice. Iron treatment should be considered in patients with mixed patterns of iron status, in which the diagnosis of IDA versus ACD is difficult, when the levels of sTfR exceed the cut-off point.

Vitamin B12 transport proteins in patients with HIV-1 infection and AIDS.

BACKGROUND: Low vitamin B12 levels (B12) are often observed in patients infected with human immunodeficiency virus type 1 (HIV-1). The causes underlying this finding are thought to be intestinal malabsorption and/or abnormalities in the vitamin plasma binding proteins (BP). MATERIAL AND METHODS: Serum levels of B12 and BP were studied in eighty HIV-1-positive patients, 55 of whom met the diagnostic criteria for AIDS. Subjects were divided into various subgroups: non-AIDS HIV-1 positive versus AIDS; low serum B12 levels (DB12, < 150 pmol/L) versus normal serum B12 levels (NB12); and the results obtained were compared both between groups and with respect to a reference population (RF) of normal volunteers. RESULTS: Low levels of serum B12 were found in 14 patients (17.5%), without differences between the AIDS and non-AIDS subgroups. The levels of holohaptocorrin (holoHP) were lower in the DB12 group than in the NB12 and RF groups (p < 0.01), and no differences were found between the AIDS and non-AIDS groups. The levels of apotranscobalamin (apoTC) were higher in the AIDS group than in the non-AIDs and RF subjects (p < 0.01), but no differences were found between the DB12 and NB12 groups. Likewise, no differences were noted in the levels of holoTC between the DB12 and NB12 groups. A positive correlation between neutrophil counts and free serum haptocorrin levels (apoHP) (rs = 0.36; p = 0.002), and a negative one between the former and the levels of apoTC (rs = -0.3; p = 0.009) were observed. Furthermore, a positive correlation was detected between the erythrocyte sedimentation rate and the levels of apoHP and TC. CONCLUSIONS: Low serum levels of HP in HIV-1 positive patients could lead to the low levels of serum vitamin B12 frequently observed in this patient population, while the high levels of TC could merely represent a non-specific marker of inflammation (acute phase, reactant).

Serum erythropoietin and erythroid activity in vitamin B12 deficiency.

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.