Causes of iron overload in blood donors - a clinical study.

BACKGROUND AND OBJECTIVES: Despite the obligate iron loss from blood donation, some donors present with hyperferritinaemia that can result from a wide range of acute and chronic conditions including hereditary haemochromatosis (HH). The objective of our study was to investigate the causes of hyperferritinaemia in the blood donor population and explore the value of extensive HH mutational analyses. MATERIALS AND METHODS: Forty-nine consecutive donors (f = 6, m = 43) were included prospectively from the Capital Regional Blood Center. Inclusion criteria were a single ferritin value >1000 µg/l or repeated hyperferritinaemia with at least one value >500 µg/l. All donors were questioned about their medical history and underwent a physical examination, biochemical investigations and next-generation sequencing of HH-related genes, including the HFE gene, the haemojuvelin gene (HFE2/HJV), the hepcidin gene (HAMP), the ferroportin 1 gene (SLC40A1) and the transferrin receptor 2 gene (TFR2). RESULTS: Forty of 49 donors were mutation positive with a combined 69 mutations, 54 of which were located in the HFE gene. There were 11 mutations in the TFR2 gene, two mutations in the HFE2 gene and two mutations in the HAMP gene. Only four donors had apparent alternative causes of hyperferritinaemia. CONCLUSION: HH-related mutations were the most frequent cause of hyperferritinaemia in a Danish blood donor population, and it appears that several different HH-genotypes can contribute to hyperferritinaemia. HH screening in blood donors with high ferritin levels could be warranted. HH-related iron overload should not in itself result in donor ineligibility.

Safety and efficacy of the combination of pegylated interferon-α2b and dasatinib in newly diagnosed chronic-phase chronic myeloid leukemia patients.

Dasatinib (DAS) and interferon-α have antileukemic and immunostimulatory effects and induce deep responses in chronic myeloid leukemia (CML). We assigned 40 newly diagnosed chronic-phase CML patients to receive DAS 100 mg o.d. followed by addition of pegylated interferon-α2b (PegIFN) after 3 months (M3). The starting dose of PegIFN was 15 µg/week and it increased to 25 µg/week at M6 until M15. The combination was well tolerated with manageable toxicity. Of the patients, 84% remained on PegIFN at M12 and 91% (DAS) and 73% (PegIFN) of assigned dose was given. Only one patient had a pleural effusion during first year, and three more during the second year. After introduction of PegIFN we observed a steep increase in response rates. Major molecular response was achieved in 10%, 57%, 84% and 89% of patients at M3, M6, M12 and M18, respectively. At M12, MR(4) was achieved by 46% and MR(4.5) by 27% of patients. No patients progressed to advanced phase. In conclusion, the combination treatment appeared safe with very promising efficacy. A randomized comparison of DAS±PegIFN is warranted.

Prevalence and clinical significance of neutropenia discovered in routine complete blood cell counts: a longitudinal study.

BACKGROUND: Neutropenia, defined as an absolute blood neutrophil count (ANC) <1.5 G L(-1) , may accompany a variety of diseases. However, the clinical significance of neutropenia detected in a routine complete blood cell count is poorly understood. METHODS: Using a primary care resource, comprising more than 370 000 individuals, we assessed the association with a number of previously recognized conditions as well as all-cause mortality in the 4 years following the identification of neutropenia. By matching laboratory data with Danish nationwide health registers, risk estimates were assessed. RESULTS: Neutropenia was observed in approximately 1% of all individuals and was associated dose dependently with viral infections, haematological malignancies (but not autoimmune disorders or solid cancers) and mortality. Neutropenia was particularly associated with HIV, acute leukaemias and myelodysplastic syndromes. Odds ratios [95% confidence interval (CI)] for viral infections were 2.32 (1.84-2.91), 2.80 (2.20-3.57) and 4.77 (3.22-7.07) for subnormal (≥1.5-1.8 G L(-1) ), mild (≥1.0-1.5 G L(-1) ) and moderate-severe (≥0.0-1.0 G L(-1) ) neutropenic individuals, respectively (all P < 0.001). Likewise, odds ratios (95% CI) for haematological malignancies were 3.23 (2.35-4.45), 8.69 (6.58-11.47) and 46.03 (33.98-62.35 ), for the same neutropenia levels, respectively (all P < 0.001). Thus, the lower the ANC, the greater the likelihood of these diseases. The relative risk estimates observed for severe neutropenia corresponded to absolute risks of haematological malignancies and mortality from any cause of 40% and >50%, respectively. CONCLUSIONS: Neutropenia is an ominous sign necessitating careful follow-up. The risk estimates presented here support focusing attention to viral diseases and haematological malignancies when neutropenia is observed.

Characterization of blood donors with high haemoglobin concentration.

BACKGROUND AND OBJECTIVES: The literature contains little on the prevalence and causes of high predonation haemoglobin levels among blood donors. This study aimed to characterize and develop an algorithm to manage would-be donors with polycythaemia. MATERIALS AND METHODS: Between November 2009 and November 2011, we offered haematology consultations to blood donors with repeated haemoglobin concentration (Hb) above the WHO limit for polycythaemia vera (PV) (10·2 and 11·5 mm/16·5 and 18·5 g/dl for women and men, respectively). Investigation of such donors included Hb, haematocrit, mean cell volume, erythropoietin, ferritin, platelet count and leucocyte count, JAK2 V617 and JAK2 exon12 analysis, as well as other routine measurements. RESULTS: Among 46 such donors, 39 had a history of smoking, which contributes to erythrocytosis. Two had PV, five had severe hypertension, one of them because of renal artery stenosis, and two had diabetes mellitus. Thus, we found a high morbidity among such donors. Of the 36 others, 30 donated again before May 2012, at which time the Hb was significantly lower. CONCLUSION: We recommend JAK2 V617 and JAK2 exon12 screening and clinical investigation for donors with concurrently high Hb, high haematocrit and iron deficiency. We also recommend that they stop or cut down on smoking to reduce the risk of thrombosis in general. We disqualified 10 of the donors.

Inheritance of the chronic myeloproliferative neoplasms. A systematic review.

This systematic review investigated the inheritance of the classical chronic myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and chronic myelogenous leukemia (CML). Sixty-one articles were included and provided 135 families with a total of 341 participants distributed to various subtypes of MPN: 50% PV, 23% ET, 14% PMF, 10% CML and 3% non-MPN hematological disorder. Women developed the disease earlier than men (43.1 years vs 47.3 years; p = 0.074), while the general average age of onset was 46 years, notably younger than sporadic cases. The clinical phenotype of the families showed a homogenous (67%) and a heterogeneous (33%) pattern, with the majority being PV-PV pairs (36%) and PV-PMF pairs (17%), respectively. This observation suggests that the susceptibility gene (or genes) is not restricted to one subtype supporting the hypothesis of a mutation in an early multipotent stem cell. Furthermore, a major subgroup of families provided evidence of an autosomal dominant (AD) inheritance with reduced penetrance. This study suggests that the origin of MPNs may occur in at least three different settings: (i) a sporadic, (ii) genetic heterogeneity with polygenetic and environmental impact and (iii) a familial phenotype following an AD inheritance.

Sustained major molecular response on interferon alpha-2b in two patients with polycythemia vera.

Quantitative assessment of the JAK2 V617F allele burden during disease evolution and ongoing myelosuppressive treatment is likely to be implemented in the future clinical setting. Interferon alpha has demonstrated efficacy in treatment of both chronic myeloid leukemia and the Philadelphia chromosome negative chronic myeloproliferative disorders. Reductions in the JAK2 V617F allele burden in patients treated with pegylated interferon alpha-2a (Peg-IFN-2a) have been demonstrated, although follow-up was relatively short. We report here the first profound and sustained molecular responses with a JAK2 V617F allele burden below 1.0% in two patients with polycythemia vera treated with interferon alpha-2b (IFN-2b). Discontinuation of IFN-2b in one of the patients was followed by a sustained long-lasting (12 months of follow-up) major molecular response.

[Primary cardiac lymphoma]. / Primaert, kardialt lymfom.

Primary cardiac non-Hodgkin lymphoma is very rare. Results recently published suggest that the prognosis is good, if the lymphoma is diagnosed early. The symptoms are nevertheless unspecific and a clinical investigation is often inconclusive. We report a case of a woman with symptoms of severe dyspnoea at rest, chest pain, and fatigue. The ECG showed a complete atrioventricular block. Magnetic resonance imaging (MRI) revealed a tumour in the right atrium and ventricle. A myocardial biopsy showed malignant non-Hodgkin lymphoma of the diffuse, large cell B-type. The patient was treated with chemotherapy and control MRI after four treatments showed complete regression of the tumour.

Combination chemotherapy with a five-drug regimen for invasive thymoma.

Nine patients with stage III and stage IV thymoma were treated with cisplatin, vincristine, lomustine, cyclophosphamide and prednisolone. Two patients (22%) obtained remission, and four patients (44%) showed no change for 11 to 31 months. It was found that this five-drug regimen did not improve the results obtained by other chemotherapy modalities for thymoma.

Type III procollagen N-peptide correlates with beta-2-microglobulin in myelomatosis.

The serum concentration of the N-terminal peptide of type III procollagen (PIIINP) was determined in 32 patients with myelomatosis (MM). Four subjects were studied at the time of diagnosis and the remaining patients at variable intervals from diagnosis. Serum concentration of beta-2-microglobulin (B2m) was measured in 31 patients. Serial measurements of both substances were performed in 20 patients. Serum PIIINP was increased in MM as compared with healthy control subjects (P < 0.001). Patients with active disease had significantly higher propeptide values (median 7.4; range 3.8-11.2) as compared to those with stable disease (median 4.3; range 2.2-9.6) (P < 0.009). A highly significant correlation existed between circulating PIIINP and B2m (P < 0.001). It is concluded that MM elicits a stromal reaction as reflected by parallel increases in serum PIIINP and serum B2m. In subsets of patients, e.g. those with non-secretory myeloma and in patients with smouldering disease, serum PIIINP may even be superior to B2m as an indicator of disease activity.

Multiple myeloma treated with mitoxantrone in combination with vincristine and prednisolone (NOP regimen) versus melphalan and prednisolone: a phase III study. Nordic Myeloma Study Group (NMSG).

One-hundred-and-fifty-one patients with previously untreated multiple myeloma were allocated to treatment with either NOP regimen (mitoxantrone 16 mg/m2 and vincristine 2 mg day 1 and prednisolone 250 mg day 1-4 and 17-20) or M+P regimen (melphalan 0.25 mg/kg and prednisolone 100-200 mg/day day 1-4). Both regimens were repeated every 4 weeks and were scheduled for 1 year. Seventy-seven patients were treated with NOP and 74 patients with M+P. No major clinical differences were recorded between the groups before treatment. Sixty percent of the patients responded (CR+PR) to NOP versus 64% to M+P (NS). The time to progression was 16 months (95% C.L. 14-51) in the NOP group versus 21 months (95% C.L. 15-27) in the M+P group (NS). The median survival was 14 months (7-21) in the NOP group and 31 months (21-43) in the M+P group (p = 0.02). NOP was significantly more toxic than M+P. Seven patients treated with NOP died due to infection and neutropenia and 1 patient died of cardiac toxicity, in contrast to 1 death due to infection and neutropenia in the M+P group. Gastrointestinal toxicity was acceptable in both groups. In conclusion, NOP was inferior to M+P as primary treatment of multiple myeloma.

Human neutrophil structure and function with special reference to cytochrome b559 and beta 2-microglobulin.

Neutrophil granulocytes are the most important white blood cells in the combat of non-viral infections. Circumstantial evidence indicates that neutrophils in addition modulate the inflammatory process. Production of neutrophils takes place in the bone marrow, and mature cells egress to the circulation. Neutrophils emigrate following activation from the vessels into the tissues (chemotaxis). During this process neutrophils generate reactive oxygen species (respiratory burst) and mobilize intracellular compartments (degranulation). By degranulation, neutrophils exercise influence on nearby cells or bacteria by extracellular release of intragranular proteins (exocytosis), and intensify plasma membrane-related processes, such as chemotaxis and respiratory burst, by translocation of membrane-bound proteins to the surface (upregulation). Ultimately, microorganisms may be killed intracellularly following engulfment (phagocytosis). The thesis presents results of protein-chemical analysis of human neutrophils, based on studies of intact cells and subcellular structures (subcellular fractionation). By fractionation, azurophil granules and specific granules can be disunited from each other and from plasma membrane and secretory vesicles. Only partial separation of plasma membrane and secretory vesicles can be obtained. Subcellular structures are identified by markers, e.g. vitamin B12 binding protein for specific granules, and latent alkaline phosphatase for secretory vesicles. The studies demonstrated tetranectin in neutrophils, localized exclusively in the secretory vesicles. Tetranectin was released by incubation of neutrophils in the presence of weak, inflammatory stimuli and paralleled the upregulation of alkaline phosphatase, but preceded degranulation of specific granules. Alkaline phosphatase has previously been employed as a plasma membrane marker. A novel ELISA for HLA class I antigen was introduced as a new plasma membrane marker. Results obtained by this assay showed upregulation of alkaline phosphatase occurring without a concurrent redistribution of HLA antigen. This indicates that the two proteins are localized in separate compartments. Upregulation of alkaline phosphatase induced by weak stimuli, however, paralleled the translocation of cytochrome b559, anticipated to be the terminal component in the respiratory burst, and known to be localized primarily in the specific granules. The present studies indicate that 15% of cytochrome b is localized in the secretory vesicles. An ELISA was established for quantitation of beta 2-microglobulin, the light chain of HLA class I antigens. The concentration of beta 2-microglobulin in plasma from patients with chronic myeloid leukaemia was found to correlate with the concentration of vitamin B12 binding protein.4+ Measurements in neutrophils demonstrated 65% of the total content of beta 2-microglobulin to be localized in the specific granules, and 20% to be present in secretory vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Human neutrophil gelatinase: a marker for circulating blood neutrophils. Purification and quantitation by enzyme linked immunosorbent assay.

Human neutrophil gelatinase was purified to apparent homogeneity. The N-terminal amino-acid sequence of the purified enzyme could be aligned to an internal part of the cDNA-derived amino-acid sequence of 92-kDa type IV collagenase from SV 40-transfected human lung fibroblasts and from a TPA differentiated monocytic cell line, U937. Total amino-acid composition of U937 and neutrophil gelatinases was identical. Gelatinase was susceptible to treatment with o- and n-glycanase, indicating that posttranslational addition of oligosaccharide side chains occurs. An enzyme-linked immunosorbent assay for gelatinase was developed using specific polyclonal rabbit antibodies. The assay was specific, sensitive, accurate, and reproducible. Ninety percent range for plasma gelatinase from normal subjects was 17.3 to 102.9 ng/ml. In patients treated with cytostatic agents for non-Hodgkin's lymphoma, there was a parallel drop in plasma gelatinase and peripheral granulocyte count. This indicates that plasma gelatinase is a marker for circulating neutrophils. Plasma gelatinase does not seem to reflect bone marrow cellularity.

Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.

Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin).

Subcellular fractionation studies were performed on human neutrophils stimulated with ionomycin (a Ca(2+)-specific ionophore). The results of these studies revealed NADPH-oxidase activity, without any additive, both in the plasma membrane and in the specific granule fractions. After comparing these results with the NADPH oxidase activity induced by the ionophore in intact neutrophils, in differentiated HL-60 cells and in neutrophil cytoplasts, we conclude that ionomycin preferentially activates the NADPH oxidase pool located in the membrane of specific granules. Furthermore, we suggest that incorporation of granule membrane into the plasma membrane makes the associated NADPH oxidase less sensitive to activation induced by a rise in [Ca(2+)]i.

Stimulus-dependent secretion of plasma proteins from human neutrophils.

In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli.

Chronic granulomatous disease with partial deficiency of cytochrome b558 and incomplete respiratory burst: variants of the X-linked, cytochrome b558-negative form of the disease.

Five male patients from four different families presented with a clinical record of chronic granulomatous disease (CGD): recurrent infections of the skin and/or respiratory tract with catalase-positive microorganisms, sometimes in combination with granulomata and/or abscesses in various organs. These patients differed from "classical" forms of the disease in that their neutrophils, although deficient in killing in vitro of Staphylococcus aureus, contained a decreased but measurable amount of cytochrome b558 (10-60% of normal on a heme basis), causing weak staining in the nitroblue tetrazolium dye test and a depressed respiratory burst after contact of the cells with fluid or particulate activators of the NADPH:O2 oxidoreductase. In the cell-free activation system, the defect in the patients' cells was localized in the membrane fraction. In each of the four families, the cellular abnormalities showed an X-linked inheritance. Fusion experiments performed with the monocytes from these patients and those from patients with classical X-linked, cytochrome b558-negative (Xb(0)) or autosomal, cytochrome b558-positive (Ab+) CGD showed complementation of NADPH:O2 oxidoreductase activity in the latter but not in the former combination. Thus, the unusual CGD patients represent variant forms of Xb(0) CGD, with mutations in the gene coding for the beta subunit of cytochrome b558 that do not cause complete loss of this protein.

Refractory myelomatosis treated with mitoxantrone in combination with vincristine and prednisone (NOP-regimen): a phase II study. The Nordic Myeloma Study Group (NMSG)

In a phase II study, patients with refractory myelomatosis were treated with a combination chemotherapy (NOP regimen): mitoxantrone (bolus injection of 4 mg/m2 on days 1-4), vincristine (continuous infusion of 0.4 mg/24 h on days 1-4) and prednisone (250 mg/d on days 1-4 and 17-20). The treatment was repeated every 4 weeks. Ninety-two patients were treated after they were found refractory to treatment with melphalan and prednisone (and occasionally vincristine) (n = 50) or more intensive treatment regimens (n = 42) including anthracyclines (n = 18). Response (greater than or equal to 50% reduction of M protein) was obtained in 23 patients and minor response (clinical improvement but less than 50% reduction in M protein) in 22 patients. The median duration of the response was 7.5 months. Equal response rates were observed irrespective of the type of previous treatment. The major toxicity was myelosuppression with severe granulocytopenia and infections. However, the frequency decreased throughout the cycles. The NOP treatment is recommended in refractory myelomatosis, especially in patients refractory to other intensive regimens. Patients in a poor clinical condition or with thrombocytopenia before treatment should have a reduced mitoxantrone dose in the first treatment cycles.

Neutrophil beta-2 microglobulin: an inflammatory mediator.

Beta-2 microglobulin (beta 2m) constitutes the light invariant chain of HLA class I antigen, and is a constituent of mobilizable compartments of neutrophils. Two forms of beta 2m exist: native beta 2m and proteolytically modified beta 2m (Des-Lys58-beta 2m), which shows alpha mobility in crossed radioimmuno-electrophoresis. The modification of native beta 2m can be executed by membrane-associated activity of mononuclear cells, and Des-Lys58-beta 2m augments the production of interleukin 2. In this study we present evidence that human neutrophils contain native beta 2m in specific granules, secretory vesicles, and plasma membrane. Beta 2m was released in the native form from neutrophils in response to stimulation with chemotactic stimuli and phorbol ester. The results of experiments designed to study the modification of native beta 2m by neutrophils indicated that neutrophils do not participate in the proteolysis of beta 2m. However, we demonstrated that native beta 2m following degranulation may be transformed to Des-Lys58-beta 2m by lymphocytes. We suggest that neutrophil beta 2m following exocytosis may be transformed to Des-Lys58-beta 2m, acting as an extracellular messenger between granulocytes and lymphocytes in the inflammatory focus.

Mixed enzyme-linked immunosorbent assay (MELISA) for HLA class I antigen: a plasma membrane marker.

This study introduces a simple, reproducible assay for HLA class I antigen using antibodies against beta 2-microglobulin and the heavy chain on HLA. The sandwich technique was named mixed enzyme-linked immunosorbent assay (MELISA), and was designed for identification of plasma membranes in neutrophil subcellular fractions. The subcellular localization of HLA was identical to that of other plasma membrane markers, [3H]concanavalin A and detergent-independent alkaline phosphatase, and was unchanged by stimulation of cells by weak and strong secretagogues. In addition to the presence as part of the HLA complex in the plasma membrane uncomplexed beta 2-microglobulin is present in the specific granules of neutrophils. However, the release of beta 2-microglobulin from intact neutrophils stimulated with formyl-methionylleucylphenylalanine was much higher than could be explained by exocytosis of specific granules. Subcellular fractionation studies demonstrated that beta 2-microglobulin is localized in fractions characterized by latent alkaline phosphatase and released from this novel secretory compartment in response to stimulation with formyl-methionylleucylphenylalanine.

Quantitative analysis of phospholipids and demonstration of plasmalogen in human neutrophil subcellular fractions by high-performance liquid chromatography.

The subcellular distribution of phospholipids in unstimulated neutrophils was investigated by high-performance liquid chromatography of lipid extracts of granule and plasma membranes obtained from Percoll density gradients. The mobile phase contained acetonitrile:methanol:85% phosphoric acid (131:3:0.8 v/v/v). Post-nuclear supernatants contained 2.3 micrograms lipid phosphorus/mg protein. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine were demonstrated in azurophil granules, specific granules and in a combined fraction of secretory granules and plasma membranes. Separate estimates of each phospholipid class by peak areas obtained by high-performance liquid chromatography showed that secretory granules and plasma membranes contained most of the phosphatidylinositol and phosphatidylcholine (p less than 0.005 vs peaks areas obtained in azurophil and specific granules), whereas a major part of the phosphatidylethanolamine was located in the specific granules (p less than 0.005 vs peak areas obtained in azurophil granules, and plasma membrane and secretory granules). High-performance liquid chromatography proved to be a useful principle for the demonstration of plasmalogen because the acidic solvent caused hydrolysis of phosphatidalethanolamine, which was recovered as lysophosphatidylethanolamine. Additionally, sphingomyelin was demonstrated in all subfractions by thin-layer chromatography.