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.

Combined cobalamin and iron deficiency anemia: a diagnostic approach using a model based on age and homocysteine assessment.

Macrocytosis, the hallmark of cobalamin/folate deficiency anemia, is frequently absent. Clinicians have to be aware of coexisting conditions that can mask the macrocytosis expression of megaloblastic anemia, especially iron deficiency. The objective of this work was to investigate the degree of overlap between iron deficiency anemia (IDA) and cobalamin deficiency and to develop a predictive model for differentiating IDA from combined deficiency. A prospective case and control study was carried out to investigate vitamin B12 and folate status in iron deficiency anemia. A total of 658 patients were recruited, 41 of whom (6.2 %) were excluded. The remaining 617 subjects consisted of 130 controls and 487 with IDA. Low vitamin B12 (LB12) was considered when serum vitamin B12 was ≤200 pmol/L. High serum homocysteine (Hcy) was defined by Hcy >17 µM/L. A multivariate analysis (including a logistic regression) was performed to develop a diagnostic model. Low vitamin B12 levels were found in 17.8 % of IDA subjects. Ten out of 11 subjects (91 %) with IDA and serum vitamin B12 (B12) ≤100 pmol/L showed vitamin B12 deficiency. Moreover, vitamin B12 deficiency was demonstrated in 48 % of cases with IDA and B12 between 101 and 150 pmol/L and in 40 % with IDA and B12 between 151 and 200 pmol/, respectively. As a result of multivariate logistic analysis, neutrophil counts and age predicted subjects with vitamin B12 ≤200 and Hcy >17 µmol/L, [Formula: see text]. Using the age of 60 as a cutoff, sensitivity was 91 % (39 out of the 43 patients with vitamin B12 deficiency and IDA were identified). In summary, low vitamin B12 was found in 18 % of patients with IDA. Vitamin B12 deficiency was demonstrated in many patients with LB12 and IDA. Age over 60 years was used to separate patients with combined deficiency (sensitivity 91 %). Therefore, for a diagnostic purpose, serum vitamin B12 should be evaluated in IDA patients over 60 years. This diagnostic model needs to be validated in a different population.

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.

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.

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.

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.

Enhanced risk of thrombotic disease in patients with acquired vitamin B12 and/or folate deficiency: role of hyperhomocysteinemia.

A number of studies have identified elevated levels of homocysteine (Hcy) as a risk factor for thrombosis. Given the relationship between Hcy and thrombosis, a high prevalence of thrombosis would be expected in patients with megaloblastic anemia. The aim of our study was to determine whether an acquired vitamin B12/folate deficiency is a risk factor for thrombosis. A retrospective case and control study was performed that included 193 cases with reduced levels of vitamin B12/folate. The cases were divided initially into two groups (105 with serum vitamin B12 < or =150 pmol/l and/or low red cell folate < or = 450 nmol/l and 88 with serum vitamin B12 between 150 and 200 pmol/l and/or red cell folate between 450 and 590 nmol/l). The control group consisted of 87 additional patients who had normal levels of serum vitamin B12, red cell folate, and normal renal function. Serum Hcy, thrombotic events, and risk factors were evaluated in all participants. Eight patients (9%) in the control group had had previous vascular events although only three of these events (37.5%) were observed between the vitamin study and 2 years prior to the study. In the case group, 20% of the patients had a history of thrombosis. In contrast with controls, 85% of cases suffered thrombosis between the time they were diagnosed and 2 years prior to the time they were diagnosed as showing a vitamin deficiency. Multivariate analysis demonstrated that vitamin deficiency was a significant risk factor for arterial thrombosis [adjusted odds ratio (OR) 3.3, confidence interval (CI) 1.1-10.2]. However, when hyperhomocysteinemia was included in the analysis, vitamin deficiency was no longer a risk factor, suggesting that hyperhomocysteinemia was responsible for arterial thrombotic risk in these patients (adjusted OR 2.5, CI 1.1-5.8). As a consequence of hyperhomocysteinemia, patients with acquired vitamin deficiency of vitamin B12/folate had a high risk of thrombosis. However, a more extensive study that controls risk variables and genetic factors is needed to sort out the various contributing factors.

The S65C mutation in Spain. Implications for iron overload screening.

Hereditary hemochromatosis is related to mutations of the HFE gene. The role of the S65C mutation of this gene was evaluated in a Spanish population, consisting of 100 controls and 41 patients who had resulted positive to screening for iron overload. Only one patient was heterozygous for the S65C mutation, so the S65C mutation is infrequent in our area. Nevertheless, it is advisable to search for this mutation in cases with iron overload and heterozygosity for the C282Y or H63D mutations of the HFE gene.