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Background: Iron deficiency anemia (IDA) is highly prevalent in women of child-bearing age. However, their nonhematological symptoms have been overlooked. This study aims to analyze the nonhematological features and symptoms of IDA in a group of women of reproductive age and the changes occurred during iron therapy. Materials and Methods: IDA women underwent dietary, physical activity, menstrual blood loss, and cognitive function assessment at baseline. Hematological and biochemical parameters were analyzed. Executive attention was tested by the flanker task and working memory by the 2-back task. Oral iron therapy (ferrous sulfate) was given to 35 women for 8 weeks and the changes in iron status, biochemical markers, cognitive function, and nonhematological symptoms were evaluated. Results: Patients presented nonhematological symptoms: pica, 32.4%; cheilitis, 20.6%; restless legs syndrome (RLS), 20.6%; diffuse hair loss, 55.9%; and ungual alterations, 38.2%. Two or more symptoms were present in 58.8% of women. Serum iron and working memory were correlated at baseline. Multivariate analyses show associations (odds ratio [OR], 95% confidence interval [CI]) between pica and reaction time in the working memory test (OR 2.14, 95% CI 1.19-3.87, p  = 0.012); RLS with total serum protein (OR 0.08, 95% CI 0.06-0.92, p  = 0.043); and cheilitis with mean corpuscular hemoglobin (OR 0.388, 95% CI 0.189-0.799, p  = 0.01). Pica, cheilitis, and RLS completely resolved with iron therapy, and ungual alterations and hair loss improved in 92.3% and 84.2% of women, respectively. Better performance in executive attention and working memory was observed after iron therapy. Conclusions: More attention should be given to the nonhematological manifestations of IDA to improve the quality of life of menstruating women., Competing Interests: No competing financial interests exist., (© Fernandez-Jimenez et al. 2020; Published by Mary Ann Liebert, Inc.)

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., (Copyright © 2019 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.)

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)., (© 2015 John Wiley & Sons Ltd.)

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.

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., (© 2013 John Wiley & Sons Ltd.)

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.

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.

This study aimed at assessing the relationship between thrombosis, hyperhomocysteinemia and vitamin B12 deficiency using a case-control study carried out in 326 patients with thrombosis (case group) and 351 patients from the same hospital (control group). Apart from the classic risk factors, a number of hematological variables were evaluated, including serum vitamin B12 (B12), red cell folate (RCF), and serum homocysteine (Hcy). An evaluation of serum methylmalonic acid (MMA) and a clinical study were carried out to investigate B12 pathology. Results of univariate analysis demonstrated decreased B12 levels in thrombosis (Student's t test, p < 0.0001). Vitamin B12 below 200 pmol/l (LB200) or below 150 pmol/l (LB150), and red cell folate below 600 nmol/l were found in 17.2, 8.6, and 2.2% of cases with thromboembolism, respectively. An increase in Hcy was detected in 86 cases with thrombosis (26.3%). An abnormality in vitamin B12 and/or renal function was found in 80% of cases with hyperHcy and thrombosis. The MMA increase demonstrated that vitamin B12 deficiency was present in these patients with low levels of vitamin B12 in serum, and the MMA levels were in concordance with Hcy levels. The clinical study revealed B12 malabsorption in most cases with LB200. Multivariate analysis showed that serum vitamin B12 (RR 0.998, CI 0.997-0.999) was moderately related to thromboembolism. The results indicated that vitamin B12 deficiency was common among patients with hyperhomocysteinemia and thrombosis. Moreover, HyperHcy was caused by vitamin B12 deficiency and/or chronic renal failure in most patients with thrombosis. As the main cause of vitamin B12 deficiency was vitamin malabsorption, parenteral vitamin B12 with or without folic acid should be administered for the treatment of this condition. However, it remains to be demonstrated whether this treatment approach prevents recurrent thromboses in patients with vitamin B12 deficiency and thrombosis, as suggested by some case reports.

IRON FUNCTIONS: Iron is an essential micronutrient, as it is required for satisfactory erythropoietic function, oxidative metabolism and cellular immune response. IRON PHYSIOLOGY: Absorption of dietary iron (1-2 mg/day) is tightly regulated and just balanced against iron loss because there are no active iron excretory mechanisms. Dietary iron is found in haem (10%) and non-haem (ionic, 90%) forms, and their absorption occurs at the apical surface of duodenal enterocytes via different mechanisms. Iron is exported by ferroportin 1 (the only putative iron exporter) across the basolateral membrane of the enterocyte into the circulation (absorbed iron), where it binds to transferrin and is transported to sites of use and storage. Transferrin-bound iron enters target cells-mainly erythroid cells, but also immune and hepatic cells-via receptor-mediated endocytosis. Senescent erythrocytes are phagocytosed by reticuloendothelial system macrophages, haem is metabolised by haem oxygenase, and the released iron is stored as ferritin. Iron will be later exported from macrophages to transferrin. This internal turnover of iron is essential to meet the requirements of erythropoiesis (20-30 mg/day). As transferrin becomes saturated in iron-overload states, excess iron is transported to the liver, the other main storage organ for iron, carrying the risk of free radical formation and tissue damage. REGULATION OF IRON HOMOEOSTASIS: Hepcidin, synthesised by hepatocytes in response to iron concentrations, inflammation, hypoxia and erythropoiesis, is the main iron-regulatory hormone. It binds ferroportin on enterocytes, macrophages and hepatocytes triggering its internalisation and lysosomal degradation. Inappropriate hepcidin secretion may lead to either iron deficiency or iron overload.

The main objective of the study was to analyze the incidence of iron overload (IO) and its management in transfusion-dependent patients with low-risk myelodysplastic syndrome (MDS) before the license of deferasirox. This observational, cross-sectional, and multicenter study was conducted from January to May 2007 in 81 Spanish hospitals. Eligible patients had a low or intermediate-1 risk score and had to have received at least ten units of packed red blood cell (PRBC). Of the 549 patients analyzed, 75% had received more than 20 PRBC units since diagnosis; 14% had IO at diagnosis and 58% at last follow-up. Thirty-eight percent of patients received chelation therapy; of those, 92% were treated with desferrioxamine. Ferritin levels at the start of chelation therapy were higher than 1,000 microg/L in 76% and over 2,500 microg/L in 24% of patients. Of the 202 patients who received some form of chelation therapy, ferritin levels increased from a mean +/- SD of 1,986 +/- 1,398 to 2,480 +/- 1,648 microg/L at last follow-up in 86% (p < 0.001). In the remaining 29 patients treated with a minimally effective therapy, ferritin levels did not increase. Of these, only 11 patients received such therapy lasting more than 12 months. In conclusion, most low-risk transfusion-dependent MDS patients develop IO, but only a minority receives a minimally effective and timely iron chelation therapy.

Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y mutation of the HFE gene. Nevertheless, penetrance of the disease is very variable. In some patients, penetrance can be mediated by concomitant mutations in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP) and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in all patients by bidirectional direct cycle sequencing. Phenotype-genotype interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was found in only one out of 100 cases. Following, we performed a study of several members of that family, and we observed several members had a digenic inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the individuals who were homozygous for the C282Y mutation. One other patient presented a new polymorphism in the hemojuvelin gene, without consequences in iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin mutations are rare among Spanish HH patients, and their impact in this population is not significant.

Most hereditary haemochromatosis patients are homozygous for the C282Y mutation of the HFE gene. However, the phenotypic expression and clinical aggressiveness of the disease differs considerably from patient to patient. The main objective of this work was to study the role of variants in the SLC40A1 gene in the severity of iron overload and his clinical consequences in 100 Spanish probands homozygous for the C282Y mutation of the HFE gene. We performed automated sequencing of the coding regions, including intron-exon junctions of the SLC40A1 gene. We studied the association between polymorphisms in the SLC40A1 gene and median values of iron removed, taking into account statistical corrections for multiple comparisons. No pathogenic mutations in the SLC40A1 were detected. Five known single nucleotide polymorphisms (SNPs) were identified, and two of them were associated with phenotypic characteristics. IVS1-24 C>G was associated with the amount of iron removed and presence of liver disease: Of the 83 patients finally studied for this SNP, the amount of iron removed was above the median in 36 of 56 (64.3%) for C/C, in nine of 23(39.1%) for C/G and in zero of four (0%) for G/G patients (P=0.01). Liver damage was observed in 34 of 56 patients (60.7%) for C/C, in eight of 23 (34.8%) for C/G and in zero of four (0%) for G/G (P=0.01). Both associations remained significant at multivariate analysis (P=0.011 and P=0.023, respectively). IVS1-24 C>G on the ferroportin gene seems to be a genetic modifier for clinical aggressiveness of HFE1 haemochromatosis.

We studied the relationship between iron removed by venesection, sex, age, and clinical characteristics in a group of 100 Spanish probands with hereditary hemochromatosis (HH), all C282Y homozygous in the HFE gene. Iron overload was higher in men than in women (P < 0.0001) and increased with age (P = 0.02). Forty-four patients presented with liver disease (28 had fibrosis-cirrhosis of the liver), 24 with diabetes, 18 with arthropathy, and 13/73 men with impotence. No clinical consequences of hemochromatosis were observed in 43 patients. The number of clinical complications was higher in men (P = 0.01) and increased with age (P = 0.006) and with the amount of iron removed (P < 0.0001). The amount of iron removed was significantly higher by univariate analysis in patients with liver disease (P < 0.0001), diabetes (P = 0.007), arthropathy (P = 0.006), and impotence (P = 0.003) than in patients without these complications. In the multivariant analysis, only liver disease maintained a significant relationship with the amount of iron removed (P < 0.0001). Diabetes and arthropathy were closely related with previous liver disease, and impotence appeared mainly in hemochromatosic men with diabetes and alcoholism.

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.

Toxic-infectious complications may be related with iron toxicity after a stem cell transplant (SCT). Eighty one patients who underwent SCT were prospectively evaluated over 3 months for mucositis, bacteraemia and febrile days. Pre-SCT transferrin saturation (TS), ferritin level and the number of days with TS >or= 80% after transplant were determined. A ferritin level >1,500 microg/l predicted the appearance of severe mucositis, bacteraemia and days with fever in univariate (P = 0.03, P = 0.03 and P = 0.03) and multivariate analysis (P = 0.03, P = 0.006 and P = 0.002). Nevertheless, further statistical studies revealed that the predictive value of pre-SCT ferritin levels was restricted to AUTO-transplanted patients in both univariate (P = 0.05, P = 0.05 and P < 0.001) and multivariate (P = 0.03, P = 0.05 and P < 0.001) analysis, in contrast with the ALLO-transplanted group where this variable did not reach statistical significance. In conclusion, iron burden seems to influence the appearance of toxic-infectious complications during the first 3 months after transplant in AUTO-transplanted patients.

Background and Objective: Polycythemia vera (PV) and essential thrombocytemia (ET) are chronic myeloproliferative diseases (MPD) characterized by overactive hemopoiesis. A single point mutation of JAK2 (Val617Phe) has been detected in PV, ET and myelofibrosis (MF). The aim of this work was to investigate the JAK2 mutation in patients with MPD and to compare the results to those of the endogenous formation of BFU-E erythroid colonies (EEC). Finally, different sources of hematopoietic cells to obtain DNA were evaluated., Patients and Method: In this work 146 patents were studied (81 MPD: 27 PV, 28 ET, 11 MF and 15 with myeloid chronic leukemia). Moreover, 28 patients showed secondary polycythemias or reactive thrombocytosis, 8 MPD/myelodysplastic syndromes and 29 other disorders. In 54 patients, EEC were also evaluated. Peripheral blood cells were used as source of DNA in 122 patients, bone marrow in 33, cells from BFU-E in 14 and cells from EEC in 24 patients. Their DNA samples were analyzed using an allele-specific polimerase chain reaction methodology., Results: The JAK2 mutation was present in 96% of PV patients, 59% of ET and 63.6% of MF. None of the remaining patients showed this mutation. Diagnostic agreement was excellent between EEC and the mutation (kappa index = 0.93; 97% positive agreement and 95% negative agreement). DNA was obtained in 119 out of 122 samples from peripheral blood, in all patients with bone marrow, and in 50% of patients with BFU-E or EEC. In 7 cases, samples from different cell sources were studied. Their results were identical., Conclusions: The V617F mutation of JAK2 is present in most of PV patients and half of those with MF or ET. There is an excellent concordance with the EEC results.

Background and Objective: An essential step in the pathogenesis of hereditary hemochromatosis seems to be the increased expression of a duodenal divalent cation transporter (DMT1) responsible for absorption of non-heminic iron2+. The objective of the present study was to ascertain whether the competitive blockade of DMT1 by the administration of high doses of oral Mg2+ reduces iron absorption in patients homozygous for the C282Y mutation., Patients and Method: Iron absorption was evaluated by a low dose iron absorption test in 15 patients before and after treatment with oral magnesium (809.6 mg every 8 hours) for two weeks., Results: We did not observe secondary effects or significant differences in iron absorption before or after magnesium treatment (14.7 micromol/L; 95% confidence interval [CI], 9.8-19.6 vs 14.9 micromol/L; 95% CI, 8.5-21.4, P = 0.7)., Conclusions: Treatment with oral magnesium does not reduce iron absorption in homozygous C282Y patients. This treatment can not be used in these subjects.

Genetic control of soluble transferrin receptor (sTfR) levels was demonstrated using family-based studies (GAIT, Genetic Analysis of Idiopathic Thrombophilia project); moreover, a genetic relationship was observed between sTfR and the risk for thrombosis, suggesting that these phenotypes shared genetic determinants. We studied the regions that control sTfR. To assess such regions, a full genome scan was carried out using 604 highly polymorphic deoxyribonucleic acid markers (resolution 7.3 cM) in 21 extended pedigrees (358 individuals). Then, a quantitative trait linkage analysis was performed using variance components methods. The genomewide scan linkage analysis showed two regions (quantitative trait locus or QTL) with significant limit of detection (LOD) scores (2q23.14, LOD score = 2.64, nominal p = 0.00024; 3q21.2, LOD score = 1.94, nominal p = 0.0014). There were no obvious candidate genes in these regions. In conclusion, this linkage analysis suggested the existence of a QTL in 2q23.14 that probably harbored a gene (or genes) controlling sTfR levels. Moreover, a second linkage signal was observed in 3q21.2; albeit the evidence for this second locus was lower. The next step will be to identify the gene(s) and its possible involvement in thrombosis and iron homeostasis.

Background: Cobalamin attached to transcobalamin II (TC II), known as holo-TC II, is the active cobalamin fraction taken up by tissues. Holo-TC II is also the form in which absorbed cobalamin enters the circulation from the ileum. Therefore, holo-TC II has been proposed variously as a marker of cobalamin adequacy, cobalamin absorption, or both, including even its advocacy as a surrogate Schilling test. Such claims carry conflicting diagnostic implications because metabolic adequacy and absorption are not identical., Objective: The objective was to examine metabolic and absorptive influences on holo-TC II., Design: Treated patients with pernicious anemia (PA), who have abnormal absorption but a normal metabolic status, were chosen as the model to differentiate between the effects of the 2 cobalamin-related characteristics. Serum holo-TC II and indexes of cobalamin metabolism in 23 treated patients were compared with those of 6 untreated PA patients (abnormal absorption and metabolic status) and 33 control subjects (normal absorption and metabolic status)., Results: Holo-TC II, which correlated directly with cobalamin and inversely with homocysteine, was significantly higher in treated PA patients in metabolic remission than in untreated PA patients (74 +/- 59 compared with 9 +/- 6 pmol/L) and was significantly lower than in control subjects (105 +/- 58 pmol/L), although the latter difference was small and the values overlapped greatly., Conclusions: Metabolic cobalamin status is a major determinant of serum holo-TC II. Absorption status may have mild influence as well, although other explanations remain possible. Serum holo-TC II cannot be used clinically to diagnose cobalamin malabsorption because of overlap with normal values. The influences on holo-TC II are complex and require careful analysis.

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.

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.

Aim: To determine the efficacy of flow cytometry (FC) in the assessment of bone marrow (BM) in B-cell non-Hodgkin lymphoma (B-NHL). FC is a common practice, but is far from being validated., Methods and Results: Morphological analysis and FC immunophenotyping were performed on 421 samples. T-cell lymphomas, Hodgkin's disease, chronic lymphocytic leukaemia and hairy cell leukaemia were not included in the study. Clonality was assessed by the standard kappa/lambda/CD19 test. Aberrant immunophenotypes present in the B-cell subpopulation were also investigated. A double-step procedure was employed in all cases to increase the sensitivity of the FC procedure. Of 380 evaluable samples, 188 corresponded to follicular lymphoma (FL), 58 to diffuse large B-cell lymphoma (DLBCL), 57 to mantle cell lymphoma (MCL), seven to Burkitt's lymphoma and the remaining 70 samples to other low-grade lymphomas. Morphological marrow infiltration was found in 148 cases, and flow immunophenotyping identified 138 cases with BM involvement. A concordance between the two methods was detected in 298 cases (79%). There was a discordance in 82 cases (21%): morphology positive/FC negative in 46 cases and morphology negative/FC positive in 36 (61% of all cases with discordance were from FL). There was no difference in outcome when patients with discordances were compared with patients without discordances., Conclusions: Most samples showed concordance between morphological and FC results. FC identified BM involvement in the absence of morphological infiltration. Morphology/FC discordance seems to have no influence on the outcome of FL patients., (Copyright 2004 Blackwell Publishing Limited)

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.

Biochemical iron overload (IO) is a frequent metabolic abnormality. It may be caused by several diseases, and data regarding the relative frequency of these are scant. A single diagnostic protocol including clinical, biochemical, and genetic data was used to diagnose the cause of biochemical IO in a group of 150 patients referred by general practitioners. Severe alterations of the HFE gene (42 patients, 28%), hepatitis C virus infection (33 patients, 22%), and dysmetabolic syndrome with iron overload (DSIO) (22 patients, 15%) emerged as the main causes, and other single causes were found in 20 patients (13%). In 19 patients (13%), multiple causes of IO were found, and in 14 patients no cause was found, 5 of whom had classical criteria of genetic hemochromatosis (GH) without HFE mutations. Transferrin saturation index (TSI) had a very low positive predictive value (0.16) for GH among patients with biochemical IO in this setting. In conclusion, 90% of patients with biochemical IO were diagnosed with a specific disorder. GH, hepatitis C infection, and DSIO were the major causes, and a large group of patients had multiple causes of IO. TSI is not a useful indicator of GH in patients referred by general practitioners.

Background: Before the advent of highly active antiretroviral therapy (HAART), 20% and 10% of HIV-infected patients had low vitamin B-12 and red blood cell folate (RBCF) concentrations, respectively. However, few patients had real vitamin B-12 deficiency., Objective: We evaluated the prevalence of low vitamin B-12 and RBCF concentrations in HIV-infected patients receiving HAART and the usefulness of serum homocysteine (sHcy) for differentiating patients with deficiency from those with harmlessly low vitamin B-12., Design: The prevalence of low vitamin B-12 and RBCF was evaluated in 126 HIV-infected patients receiving HAART. Moreover, sHcy concentrations were evaluated in 40 HIV-infected patients with low vitamin B-12 and in 37 HIV-infected patients with low RBCF and were compared with those in 128 HIV-infected patients with normal vitamin B-12 and RBCF. sHcy was used to monitor treatment with vitamin B-12 and folic acid in 28 patients (24 with low vitamin B-12 and RBCF and 4 with hyperhomocysteinemia but normal vitamin B-12 and RBCF)., Results: The prevalence of low vitamin B-12 was significantly lower in patients receiving HAART than in previously studied patients who did not receive HAART (8.7% compared with 27%). Nine of the 40 patients (22.5%) with low vitamin B-12 (< or = 200 pmol/L) had hyperhomocysteinemia (> 17.5 micromol homocysteine/L). Nineteen (51.4%) of the 37 patients with low RBCF (< or = 580 nmol/L, percentile 10) had hyperhomocysteinemia. Among the 9 patients with an RBCF concentration < or = 450 nmol/L (percentile 2.5), all had hyperhomocysteinemia. The treatment with vitamin B-12 and folic acid normalized sHcy concentrations., Conclusions: The prevalence of low vitamin B-12 decreased after the introduction of HAART. The study of sHcy is useful for detecting HIV-infected patients with low vitamin B-12 and real deficiency.

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.

Hydrops fetalis is rarely caused by congenital dyserythropoietic anemia (CDA). We report a patient with hydrops fetalis as a result of severe anemia. This patient needed intrauterine transfusions from 21 weeks of gestation until birth. The hematologic study showed an atypical CDA (hydrops fetalis-associated CDA) characterized by features resembling CDA type II, but negative acidified serum lysis test (HEMPAS negative). The patient was regularly transfused for a year, after which an allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling was successfully carried out. His actual hemoglobin is 127 g/L, and he has not received transfusions for more than a year. In conclusion, intrauterine transfusions and BMT could cure an otherwise lethal atypical CDA.

Cytochrome b(5) reductase (b5R) deficiency manifests itself in 2 distinct ways. In methemoglobinemia type I, the patients only suffer from cyanosis, whereas in type II, the patients suffer in addition from severe mental retardation and neurologic impairment. Biochemical data indicate that this may be due to a difference in mutations, causing enzyme instability in type I and complete enzyme deficiency or enzyme inactivation in type II. We have investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations predicted amino acid substitutions at sites not involved in reduced nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) binding, as deduced from a 3-dimensional model of human b5R. This model was constructed from comparison with the known 3-dimensional structure of pig b5R. The seventh mutation was a splice site mutation leading to skipping of exon 5 in messenger RNA, present in heterozygous form in a patient together with a missense mutation on the other allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in nonessential regions of the enzyme. In contrast, 2 other substitutions, known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH binding. Thus, these data support the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form.

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.