Kaveh Samii

Microangiopathic hemolytic anemia complicating FK506 (tacrolimus) therapy

We describe 3 episodes of microangiopathic hemolytic anemia (MAHA) in 2 solid organ recipients under FK506 (tacrolimus) therapy. In both cases, discontinuation of FK506 and treatment with plasma exchange, fresh frozen plasma replacement, corticosteroids, aspirin, and dipyridamole led to resolution of MAHA. In one patient, reintroduction of FK506 led to rapid recurrence of MAHA. FK506‐associated MAHA is probably rare but physicians must be aware of this severe complication. In our experience and according to the literature, FK506 does not seem to cross‐react with cyclosporin A (CyA), an immuno‐suppressive drug already known to induce MAHA.

Hemochromatosis gene mutations in chronic hepatitis C patients with and without liver siderosis.

Chronic hepatitis C is often associated with liver iron overload, which may affect the long‐term prognosis and the response to antiviral treatment. The occurrence of hemochromatosis (HFE) mutations were studied to determine whether may contribute to the liver iron overload of chronic hepatitis C patients. The prevalence of two HFE mutations (C282Y and H63D) in 120 chronic hepatitis C patients was determined and the findings were correlated with clinical, histological and virological features. Hepatic iron was determined semiquantitatively by a histochemical hepatic iron index, defined as the ratio of a histochemical staining score to the patients age, after correction for heterogeneous lobular iron distribution. Serum hepatitis C virus (HCV) RNA was measured by bDNA assay and typed by restriction fragment length polymorphism. Liver HCV RNA was measured by a semi‐quantitative strand‐specific reverse transcription‐polymerase chain reaction (RT‐PCR). Excess liver iron was stained in the liver of 36 patients (30%). Siderotic patients had the same geographic origin, serum and liver HCV RNA levels and H63D and C282Y mutations frequency as non‐siderotic patients. However, siderotic patients were older (P = 0.015), more frequently males (P = 0.02), less frequently infected with HCV genotype 3 (P = 0.037) and had a higher liver fibrosis score (P = 0.008). The liver iron content did not correlate with the serum or liver HCV RNA titers. Ten of the 36 patients with liver siderosis had neither a history of excess alcohol intake, multiple transfusions, or HFE mutations. In conclusion, the pathogenesis of the liver iron overload in chronic hepatitis C patients cannot be fully explained by the occurrence of HFE mutations. The exact mechanism of iron accumulation in these patients therefore remains unexplained. J. Med. Virol. 60:21–27, 2000.

Increased apoptosis in acquired sideroblastic anaemia

Idiopathic acquired sideroblastic anaemias (IASAs) form a subgroup of the myelodysplastic syndromes and are characterized by mitochondrial iron accumulation, bone marrow erythroid hyperplasia and decreased peripheral red blood cell counts. Increased intramedullary apoptosis of erythroid precursors is presumed to constitute the pathophysiological mechanism explaining this ineffective erythropoiesis, but if and how mitochondrial dysfunction is implicated in this process is currently unknown. We therefore studied bone marrow precursor cells obtained from nine patients with IASA for (i) caspase 3 activity, (ii) numbers of Annexin V‐ and 7‐amino‐actinomycin‐positive cells, (iii) numbers of cells with diminished mitochondrial membrane potential, ΔΨm, and (iv) numbers of cells producing reactive oxygen species (ROS), and we compared the results with those of five normal bone marrow samples. Compared with controls, we found increased caspase 3 activity in all IASA samples, which correlated with increased numbers of Annexin‐V‐positive cells (r = 0·7). Analysis of different subpopulations showed increased apoptosis in erythroid populations compared with myeloid and/or lymphoid populations in five out of nine cases, and increased apoptosis in the last two populations in four out of nine cases. As evidence of mitochondrial dysfunction, ΔΨm was found to be diminished in the erythroid subpopulations of all cases of IASA (66·6 ± 17% vs. 34·6 ± 12% in normals). ΔΨm decrease was correlated to Annexin V positivity (r = 0·7). Astonishingly, no difference was found between IASA and normal bone marrows with regard to the number of ROS‐producing cells. In fact, both groups exhibited a similar low proportion of ROS production (10·3 ± 7% in normals vs. 6·8 ± 5% in IASA). Taken together, our results show that mitochondria are clearly implicated in the apoptotic process in IASA patients. Whether this is a result of an intramitochondrial defect (e.g. Fe accumulation, secondary to mitochondrial or nuclear DNA mutations) or is secondary to an extracellular stimulus [e.g. tumour necrosis factor (TNF), Fas ligand (FasL)] remains to be determined.

Clinical features and viral kinetics in a rapidly cured patient with Ebola virus disease: a case report

BACKGROUND A detailed description of viral kinetics, duration of virus shedding, and intraviral evolution in different body sites is warranted to understand Ebola virus pathogenesis. Patients with Ebola virus infections admitted to university hospitals provide a unique opportunity to do such in-depth virological investigations. We describe the clinical, biological, and virological follow-up of a case of Ebola virus disease. METHODS A 43-year-old medical doctor who contracted an Ebola virus infection in Sierra Leone on Nov 16, 2014 (day 1), was airlifted to Geneva University Hospitals, Geneva, Switzerland, on day 5 after disease onset. The patient received an experimental antiviral treatment of monoclonal antibodies (ZMAb) and favipiravir. We monitored daily viral load kinetics, estimated viral clearance, calculated the half-life of the virus in plasma, and analysed the viral genome via high-throughput sequencing, in addition to clinical and biological signs. FINDINGS The patient recovered rapidly, despite an initial high viral load (about 1 × 10(7) RNA copies per mL 24 h after onset of fever). We noted a two-phase viral decay. The virus half-life decreased from about 26 h to 9·5 h after the experimental antiviral treatment. Compared with a consensus sequence of June 18, 2014, the isolate that infected this patient displayed only five synonymous nucleotide substitutions on the full genome (4901A→C, 7837C→T, 8712A→G, 9947T→C, 16201T→C) despite 5 months of human-to-human transmission. INTERPRETATION This study emphasises the importance of virological investigations to fully understand the course of Ebola virus disease and adaptation of the virus. Whether the viral decay was caused by the effects of the immune response alone, an additional benefit from the antiviral treatment, or a combination of both is unclear. In-depth virological analysis and randomised controlled trials are needed before any conclusion on the potential effect of antiviral treatment can be drawn. FUNDING Geneva University Hospitals, Swiss Office of Public Health, Swiss Agency for Development and Cooperation, and Swiss National Science Foundation.

Electron Transfer Dissociation Mass Spectrometry of Hemoglobin on Clinical Samples

A mass spectrometry-based assay combining the specificity of selected reaction monitoring and the protein ion activation capabilities of electron transfer dissociation was developed and employed for the rapid identification of hemoglobin variants from whole blood without previous proteolytic cleavage. The analysis was performed in a robust ion trap mass spectrometer operating at nominal mass accuracy and resolution. Subtle differences in globin sequences, resulting with mass shifts of about one Da, can be unambiguously identified. These results suggest that mass spectrometry analysis of entire proteins using electron transfer dissociation can be employed on clinical samples in a workflow compatible with diagnostic applications.

Hematologic modifications in natalizumab-treated multiple sclerosis patients An 18-month longitudinal study

Objective: To monitor the hematologic modifications in the peripheral blood of patients with relapsing-remitting multiple sclerosis treated with natalizumab. Methods: The cohort included 44 patients with relapsing-remitting multiple sclerosis treated monthly with natalizumab for 18 months. Peripheral blood was collected before treatment initiation and on a monthly basis during the treatment course. Complete blood cell count was performed using automated hematology systems. Blood smears were prepared and analyzed when abnormal values were detected. Results: Mean total white blood cell, lymphocyte, and eosinophil counts were significantly higher 1 month after treatment initiation and remained stable during the 18 months of follow-up. Monocyte counts increased progressively during the 18-month treatment with natalizumab. Erythroblasts and neutrophil precursors were absent before treatment initiation but were present in 16% and 6.8% of patients, respectively, 1 month after the first natalizumab infusion. The proportion of patients with erythroblasts and neutrophil precursors remained stable throughout the 18-month follow-up period. On an individual patient basis, a fluctuating level of erythroblasts and neutrophil precursors was observed. No difference in mean erythrocyte, hemoglobin, hematocrit, thrombocyte, and neutrophil levels was observed before and after 18 months of natalizumab treatment. No cases of myelodysplastic syndrome or acute leukemia were observed. Conclusion: Chronic treatment with natalizumab is associated with significant modifications in complete blood cell count, including emergence of hematopoietic precursors that are not present in peripheral blood under normal conditions. None of these modifications were associated with malignancy.

Quantitative mass spectrometry analysis of intact hemoglobin A2 by precursor ion isolation and detection.

Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a β-thalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R(2) = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories.

Comparing Two Types of Rabbit ATG prior to Reduced Intensity Conditioning Allogeneic Hematopoietic SCT for Hematologic Malignancies

Different rabbit polyclonal antilymphocyte globulins (ATGs) are used in allogeneic hematopoietic stem cell transplantation (alloHSCT) to prevent graft-versus-host disease (GvHD). We compared 2 different ATGs in alloHSCT after reduced intensity conditioning (RIC) for hematological malignancies. We reviewed 30 alloHSCT for hematologic malignancies performed between 2007 and 2010 with fludarabine and i.v. busulfan as conditioning regimen. Patients alternatingly received Thymoglobulin or ATG-F. Median followup was 3.3 (2.5–4.5) years. Adverse events appeared to occur more frequently during Thymoglobulin infusion than during ATG-F infusion but without statistical significance (P = 0.14). There were also no differences in 3-year overall survival (OS), disease-free survival (DFS), relapse incidence, and transplant related mortality (TRM) in the Thymoglobulin versus ATG-F group: 45.7% versus 46.7%, 40% versus 33.7%, 40% versus 33.3%, and 20% versus 33.3%. The same held for graft failure, rejection, infectious complications, immune reconstitution, and acute or chronic GvHD. In patients transplanted for hematologic malignancies after RIC, the use of Thymoglobulin is comparable to that of ATG-F in all the aspects evaluated in the study. However due to the small number of patients in each group we cannot exclude a possible difference that may exist.

Restoration of hematopoiesis in a case of myelodysplastic syndrome associated with systemic lupus erythematosus treated with rituximab

Dear Editor, Experimental and clinical evidence indicates that autoimmune mechanisms contribute to the pathogenesis of cytopenias in some cases of myelodysplastic syndrome (MDS). Dysregulation in inflammatory cytokine production, skewing of the T-cell repertoire, and specific T-cell responses directed to antigens overexpressed by dysplastic clones have been reported [1]. Furthermore, MDS are frequently associated with systemic autoimmune disorders [2, 3], and immunosuppressive regimens have been successfully employed to restore hematopoiesis in selected MDS patients [4]. A 61-year-old Caucasian woman known for a long standing SLE was admitted for shortness of breath and dizziness worsening over 2 weeks. Before admission, SLE was stable for many years in the absence of any ongoing immunosuppressive treatment although medication history included previous treatments with chlorambucil and cyclophosphamide. Clinical examination revealed pallor without jaundice, lymphadenopathy, or hepatosplenomegaly. Laboratory investigations showed severe non-regenerative macrocytic anemia (hemoglobin (Hb) 40 g/l, MCV 100.9 fl, reticulocytes 4.6 10/l) with normal leukocytes (WBC 4.3 10/l), and platelets counts (Plt 294 10/l). Lactate dehydrogenase was 380 U/l, haptoglobin 294 mg/l, and unconjugated bilirubinemia 7 mmol/l. Serological testing failed to detect any acute or chronic viral infection. Direct Coombs’s test was strongly positive for immunoglobulin G and complement C3d. Bone marrow examination showed severe dysplasia affecting all three lineages without blasts excess. Interestingly, we observed a severe erythroid hypoplasia associated with specific maturation block in erythroid-lineage cells which were constituted by proerythroblasts and basophilic erythroblasts in the virtual absence of polychromatophilic and orthochromatophilic erythroblasts (Fig. 1a, left panel). Cytogenetic analysis showed normal female karyotype. Molecular analysis revealed a missense mutation in the exon 6 of the U2AF1 gene (c.470A>C, p.Q157P). A diagnosis of MDS of the refractory cytopenia with multilineage dysplasia (RCMD) subtype was established, and massive transfusional support was initiated with only slight increase in Hb levels (Fig. 1b). No alloimmunization was detected. We hypothesized a pathogenic role of warm autoantibodies in red blood cell transfusion refractoriness, and corticosteroid treatment was initiated with no improvement. According to previous reports [5], we initiated rituximab (RTX) 375mg/m/week for 4 weeks. A prompt increase in Hb levels was observed, and by the end of RTX treatment, transfusion independence was obtained (Fig. 1b). Bone marrow examination performed 3 months after RTX treatment still showed persistent signs of three lineages dysplasia but revealed restoration of erythroid maturation with reappearance of significant numbers of polychromatophilic and orthochromatophilic erythroblasts (Fig. 1a, right panel). During follow-up, we observed stable Hb levels, a normalized MCV, and sustained reticulocyte counts indicating persistence of an effective erythropoiesis. Unfortunately, 3 years after initial MDS diagnosis, the patient presented with pancytopenia due to acute myeloid leukemia (AML) transformation. * Federico Simonetta federico.simonetta@hcuge.ch

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an Aγ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.