The Impact of Ca2+ on Intracellular Distribution of Hemoglobin in Human Erythrocytes.

The membrane-bound hemoglobin (Hb) fraction impacts red blood cell (RBC) rheology and metabolism. Therefore, Hb-RBC membrane interactions are precisely controlled. For instance, the signaling function of membrane-bound deoxy-Hb and the structure of the docking sites in the cytosolic domain of the anion exchanger 1 (AE-1) protein are well documented; however, much less is known about the interaction of Hb variants with the erythrocyte's membrane. Here, we identified factors other than O2 availability that control Hb abundance in the membrane-bound fraction and the possible variant-specific binding selectivity of Hb to the membrane. We show that depletion of extracellular Ca2+ by chelators, or its omission from the extracellular medium, leads to membrane-bound Hb release into the cytosol. The removal of extracellular Ca2+ further triggers the redistribution of HbA0 and HbA2 variants between the membrane and the cytosol in favor of membrane-bound HbA2. Both effects are reversible and are no longer observed upon reintroduction of Ca2+ into the extracellular medium. Fluctuations of cytosolic Ca2+ also impact the pre-membrane Hb pool, resulting in the massive transfer of Hb to the cellular cytosol. We hypothesize that AE-1 is the specific membrane target and discuss the physiological outcomes and possible clinical implications of the Ca2+ regulation of the intracellular Hb distribution.

The continuing global challenges of treating patients with beta-thalassemia.

In this issue, Hokland et al. offer an interesting view of the different approaches on how to treat a beta-thalassemia patient. The principal concerns, that this report reveals, are the very wide differences in the facilities and economic resources available for the care of patients. Management of thalassemia should become a word wide health care priority and may include at last those two steps: national and international registries; national programs for screening couples at risk and providing preventive measures to prevent births of patients with thalassemia. Commentary on: Hokland et al. Thalassaemia-A global view. Br J Haematol. 2023;201:208-223.

The protective effect of the spleen in sickle cell patients. A comparative study between patients with asplenia/hyposplenism and hypersplenism.

Sickle cell disease (SCD) is caused by a point mutation in the beta-globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia, and progressive organ failure. Chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism can also occur with the loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The association of laboratory parameters with the spleen status including hyposplenism, asplenia, and splenomegaly/hypersplenism, and their implication in vaso-occlusive crisis and long-term complications in SCD remain to be determined. We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients: Group a) Patients with asplenia/hyposplenism (N = 22) (including auto-splenectomy and splenectomized patients) vs. Group b) patients with splenomegaly and or hypersplenism (N = 9). Laboratory studies included: Complete Blood Count, reticulocyte count, iron metabolism parameters, C Reactive Protein (CRP), Hb variant distribution, and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release, and K+ leakage, fetal RBC (F-Cells) and F-Reticulocytes, annexinV+, CD71+, oxidative stress measured by GSH presence in RBC and finally Howell Jolly Bodies count were all analyzed by Flow Cytometry. Scanning electron microscopy analysis of RBC was also performed. Patients with asplenia/hyposplenism showed significantly higher WBC, platelet, Hematocrit, hemoglobin S, CRP, D-dimer, Gamma Glutamyl Transferase (GGT), cholesterol, transferrin, annexin V+ RBCs, CD71+ RBCs, together with a markedly lower F Reticulocyte levels in comparison with splenomegaly/hypersplenism patients. In summary, important differences were also found between the groups in the studied RBCs parameters. Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia on laboratory parameters and in clinical manifestations, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach managing hypersplenism in SCD patients should be re-evaluated.

Associations between red blood cell indices and iron status and neurocognitive function in young adults: Evidence from memory and executive function tests and event-related potentials.

Cognitive impairment has been associated with anemia and iron deficiency; however, brain electrophysiological studies correlating red blood cell (RBC) indices and iron status to cognition in adulthood are scarce. We aimed to assess neurocognitive function in young adults of the general population and its correlation with RBC indices and iron status. Neurocognitive function was investigated using scalp-recorded event-related potentials (ERPs) within the context of a task-switching paradigm. ERPs and test performance were also compared across groups of "high"/"low" RBC and iron indices. Working memory was examined using the digit span test, in which mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and ferritin were found to be significant predictors of test performance, with higher MCH/MCV/ferritin being associated with better test scores. In the switching task, MCH, MCV, and ferritin were found to be significant predictors of task performance, with higher MCH/MCV/ferritin levels associated with a lower percentage of errors. Electrophysiological results showed that MCH and MCV were significant predictors of ERPs amplitude, with lower MCH/MCV levels associated with greater amplitude, which may reflect compensatory processes. P1, N1, P2, and P3 were greater for the low MCH/MCV groups. This is the first evidence of association between levels of MCH/MCV and brain function while engaged in an executive function task; possibly reflecting brain iron availability.

Back to the "Gold Standard": How Precise is Hematocrit Detection Today?

Introduction: The commonly used method for hematocrit detection, by visual examination of microcapillary tube, known as "micro-HCT", is subjective but remains one of the key sources for fast hematocrit evaluation. Analytical automation techniques have increased the standardization of RBC index detection; however, indirect hematocrit measurements by blood analyzer, the automated HCT, do not correlate well with "micro-HCT" results in patients with hematological pathologies. We aimed to overcome those disadvantages in "micro-HCT" analysis using "ImageJ" processing software. Methods: 223 blood samples from the "general population" and 19 from sickle cell disease patients were examined in parallel for hematocrit values using the automated HCT, standard "micro-HCT," and "ImageJ" micro-HCT methods. Results: For the "general population" samples, the "ImageJ" values were significantly higher than the corresponding values evaluated by standard "micro-HCT" and automated HCT, except for the 0 to 2 month old newborns, in which the automated HCT results were similar to the "ImageJ" evaluated HCT. Similar to the "general population" cohort, we found significantly higher values measured by "ImageJ" compared to either "micro-HCT" or the automated HCT in SCD patients. Correspondent differences for the MCV and MCHC were also found. Discussion: This study introduces the "micro-HCT" assessment technique using the image-analysis module of "ImageJ" software. This procedure allows overcoming most of the data errors associated with the standard "micro-HCT" evaluation and can replace the use of complicated and expensive automated equipment. The presented results may also be used to develop new standards for calculating hematocrit and associated parameters for routine clinical practice.

Characterization of acquired anemia in children by iron metabolism parameters.

Inflammatory states are associated with anemia of chronic disease and acute infection. Hepcidin, a regulator of iron metabolism, is involved in iron pathophysiology during inflammation. We investigated biochemical characteristics in children with anemia from different causes. Four patient groups (n = 38; mean age: 12.44 ± 4.35 years) were studied: (1) inflammatory bowel disease (IBD, 10 patients); (2) iron deficiency anemia (IDA, 12); (3) celiac disease (CD, 8); (4) acute infection (AI, 8). Laboratory measurements were evaluated at diagnosis: blood count, serum iron, transferrin, ferritin, vitamin B12, folic acid, CRP, erythropoietin, hepcidin and soluble transferrin receptor (sTfR). IDA patients had the lowest Hgb (6.9 ± 1.7 g/dL), MCV (63.2 ± 7.2 fL), iron (16.8 ± 13.5 µg/dL), ferritin (4.5 ± 4.5 ng/mL) and hepcidin (3.1 ± 0.8 ng/mL) values, and the highest transferrin and sTfR values. AI patients had the highest ferritin (156.2 ± 124.5 ng/mL), CRP (144.6 ± 94 mg/L) and hepcidin (74.67 ± 12.3 ng/ml) values. Overall, hepcidin levels correlated with CRP and with ferritin (r = 0.83 and 0.85, respectively). Elucidating specific etiology-related biochemical profiles in pediatric patients with anemia from different causes using a combination of laboratory biomarkers, including hepcidin, can help physicians treat the anemia.

A randomized, placebo-controlled, double-blind trial of canakinumab in children and young adults with sickle cell anemia.

Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in patients with sickle cell anemia (SCA) can activate the inflammasome, a multiprotein oligomer promoting maturation and secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). We hypothesized that IL-1ß blockade by canakinumab in patients with SCA would reduce markers of inflammation and clinical disease activity. In this randomized, double-blind, multicenter phase 2a study, patients aged 8 to 20 years with SCA (HbSS or HbSß0-thalassemia), history of acute pain episodes, and elevated high-sensitivity C-reactive protein >1.0 mg/L at screening were randomized 1:1 to received 6 monthly treatments with 300 mg subcutaneous canakinumab or placebo. Measured outcomes at baseline and weeks 4, 8, 12, 16, 20, and 24 included electronic patient-reported outcomes, hospitalization rate, and adverse events (AEs) and serious AEs (SAEs). All but 1 of the 49 enrolled patients were receiving stable background hydroxyurea therapy. Although the primary objective (prespecified reduction of pain) was not met, compared with patients in the placebo arm, patients treated with canakinumab had reductions in markers of inflammation, occurrence of SCA-related AEs and SAEs, and number and duration of hospitalizations as well as trends for improvement in pain intensity, fatigue, and absences from school or work. Post hoc analysis revealed treatment effects on weight, restricted to pediatric patients. Canakinumab was well tolerated with no treatment-related SAEs and no new safety signal. These findings demonstrate that the inflammation associated with SCA can be reduced by selective IL-1ß blockade by canakinumab with potential for therapeutic benefits. This trial was registered at www.clinicaltrials.gov as #NCT02961218.

Renal function in ß-thalassemia major patients treated with two different iron-chelation regimes.

BACKGROUND: Renal injury in transfusion dependent ß thalassemia patients (TDT) has been attributed to iron overload, chronic anemia and iron-chelation therapy (ICT) toxicity. We studied renal function in TDT patients treated with two different ICT regimes. PATIENTS AND METHODS: We studied 36 TDT patients: 26 received deferasirox (DFX) and 10 were treated with deferoxamine (DFO) +/- deferiprone (DFP). RESULTS: Increased uNAG was found in 30% of the DFX group vs. 10% of the DFO+/-DFP group, the mean uNAG level in the DFX group was significantly higher than in the DFO+/-DFP group, (P < 0.05). A moderate negative correlation was found between uNAG levels and mean serum ferritin for the prior 10 years (P = 0.03), more pronounced for the DFO+/-DFP group. Twenty nine patients had had their renal function evaluated 10 years earlier; eGFR significantly declined in patients switched to DFX (P = 0.0093) but not in patients who continued DFO+/-DFP. CONCLUSIONS: A high prevalence of renal tubular damage was observed in our TDT patients, particularly those treated with DFX; uNAG was negatively associated with mean 10-year serum ferritin, suggesting ICT's involvement in tubular injury. A significant decline in eGFR compared to a decade earlier was observed only in patients currently treated with DFX. Strict follow-up of renal function in TDT patients is warranted.

Extracellular Vesicle MicroRNA That Are Involved in ß-Thalassemia Complications.

Beta thalassemia major (ßT) is a hereditary anemia characterized by transfusion-dependency, lifelong requirement of chelation, and organ dysfunction. MicroRNA (miRNA) can be packed into extracellular vesicles (EVs) that carry them to target cells. We explored EV-miRNA in ßT and their pathophysiologic role. Circulating EVs were isolated from 35 ßT-patients and 15 controls. EV miRNA was evaluated by nano-string technology and real-time quantitative polymerase chain reaction (RT-qPCR). We explored effects of EVs on cell culture proliferation, apoptosis, and signal transduction. Higher amounts of small EV (exosomes) were found in patients than in controls. The expression of 21 miRNA was > two-fold higher, and of 17 miRNA < three-fold lower in ßT-EVs than control-EVs. RT-qPCR confirmed differential expression of six miRNAs in ßT, particularly miR-144-3p, a regulator of erythropoiesis. Exposure of endothelial, liver Huh7, and pancreatic 1.1B4 cells to ßT-EVs significantly reduced cell viability and increased cell apoptosis. ßT-EV-induced endothelial cell apoptosis involved the MAPK/JNK signal-transduction pathway. In contrast, splenectomized ßT-EVs induced proliferation of bone marrow mesenchymal stem cells (BM-MSC). In summary, the miR-144-3p was strongly increased; ßT-EVs induced apoptosis and decreased endothelial, pancreatic, and liver cell survival while supporting BM-MSC proliferation. These mechanisms may contribute to ßT organ dysfunction and complications.

Pediatric Otogenic Cerebral Sinus Vein Thrombosis and Thrombophilia.

BACKGROUND: Otogenic cerebral sinus vein thrombosis (CSVT) is a rare but severe complication of otitis media in children. To date, the role of prothrombotic evaluation is still controversial. OBJECTIVES: To report the clinical manifestations, prothrombotic evaluation, and current management of CSVT. METHODS: We performed a retrospective study of nine pediatric patients with otogenic CSVT who underwent prothrombotic evaluation between 2008 and 2018. RESULTS: Prominent clinical features included persistent otorrhea (88.8%), signs of mastoiditis (88.8%), high fever ≥ 38.3°C (100%), a classic spiking fever pattern (55.5%), and neurological signs (55.5%). A subperiosteal abscess (66.6%) was the most common otitis media complication associated with mastoiditis and CSVT. No microorganism was identified in 55.5% of patients. Cultures collected from ear secretions had a low yield (6.25%). However, PCR assays had a high detection rate (100%; n=3). The prothrombotic evaluation demonstrated an abnormal LAC-dRVVT ratio (6/9), elevated Factor VIII (5/8) (and a combination of both in four patients), antiphospholipid antibodies (2/8), and high homocysteine levels (1/5).The surgical intervention of choice included one-sided mastoidectomy with myringotomy and ventilation-tube placement on the affected side (77.7%). There were no mortalities and no long-term sequela except chronic otitis media (22.2%). CONCLUSIONS: Our findings demonstrate good outcomes for otogenic CSVT treatment with intravenous antibiotics, anticoagulation, and conservative surgical intervention, which supports the current trend in management. The prothrombotic evaluation revealed transient inflammation-related risk factors but did not alter management. Further prospective multicenter studies are needed to determine its relevance.

Severe Protein C Deficiency due to Novel Biallelic Variants in PROC and Their Phenotype Correlation.

Severe protein C deficiency due to biallelic PROC mutations is an extremely rare thrombophilia, most commonly presenting during the neonatal period as purpura fulminans. Despite treatment, severe morbidity and mortality are frequent. The current study reports 3 unrelated patients harboring novel homozygous PROC mutations and their clinical phenotypes. We discuss how the cytoprotective activity of protein C and its role in the stabilization of endothelial barriers may account for the unique symptoms of this thrombophilia.

Campylobacter gastroenteritis in children in north-eastern Israel comparison with other common pathogens.

Gastroenteritis is common among children. Campylobacter jejuni is one of the main causative bacterial pathogens, together with Shigella, Salmonella and invasive Escherichia coli. Campylobacteriosis is a zoonotic, usually self-limited disease that does not always require antibiotic treatment. In cases of protracted diarrhoea in healthy children or immunocompromised patients, antibiotic treatment is recommended, and the drug of choice is still macrolides, with very low resistance rates in Campylobacter species. However, it is crucial to isolate the causative organism, because some cases, such as Shigella encephalitis, call for initiation of empiric antibiotic treatment. In this study, we compared the incidence, epidemiology, clinical findings and laboratory results of gastroenteritis with dysentery caused by these organisms in children in our area. C. jejuni was found to be the leading pathogen in children hospitalized with bacterial gastroenteritis, followed by Shigella and Salmonella. Macrolides were the drug of choice for Campylobacter, and ceftriaxone and ciprofloxacin were the best empiric treatments for Shigella and Salmonella, respectively.

Immune Thrombocytopenia Secondary to Hodgkin's Lymphoma in Children

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disorder of variable origin that results in bleeding and decreased platelet count. Autoimmune abnormalities have been described in patients with malignancies including non-Hodgkin's lymphoma but are rarely described in patients with Hodgkin's lymphoma. OBJECTIVES: To describe an unusual presentation of Hodgkin's lymphoma in an unusual age and alarm pediatricians of the challenging diagnosis. METHODS: We present two cases that highlight an unusual clinical presentation of childhood Hodgkin's lymphoma occurring at an atypical age. RESULTS: Over a 4-year period, two children aged 5 and 6 years were admitted for suspected ITP, both had cervical lymphadenopathy. Bone marrow examination showed no evidence of tumor or fibrosis. Biopsy of the lymph node was possible only after administration of intravenous immunoglobulins and normalization of the platelet count. Platelet counts increased after initiation of chemotherapy. CONCLUSIONS: The identification of the clinical presentation of ITP as a possible presentation of Hodgkin's lymphoma is important to facilitate timely diagnosis and management.

Coagulation tests or standardized questionnaire, which is better as a predictor of bleeding? A prospective study among children before tonsillectomy and/or adenoidectomy.

OBJECTIVE: The value of pre-operative coagulation testing for adenotonsillar surgery is controversial. The purpose of this study was to evaluate the role of routine coagulation tests and a standardized questionnaire in children before tonsillectomy and/or adenoidectomy. RESULTS: A total of 143 children were prospectively enrolled in the study between 2013 and 2017, 81 males (56.6%) and 62 females (43.4%), age range 1 to 18 years (median age 5 years). Eighteen bleeding events were documented, three of them required treatment in the operating room. Abnormal coagulation tests were not associated with higher odds of bleeding after surgery. Higher risk of bleeding (p = 0.01) was associated with an abnormal standardized medical questionnaire.

Characterization and genotype-phenotype correlation of patients with Fanconi anemia in a multi-ethnic population.

Fanconi anemia (FA), an inherited bone marrow failure (BMF) syndrome, caused by mutations in DNA repair genes, is characterized by congenital anomalies, aplastic anemia, high risk of malignancies and extreme sensitivity to alkylating agents. We aimed to study the clinical presentation, molecular diagnosis and genotype-phenotype correlation among patients with FA from the Israeli inherited BMF registry. Overall, 111 patients of Arab (57%) and Jewish (43%) descent were followed for a median of 15 years (range: 0.1-49); 63% were offspring of consanguineous parents. One-hundred patients (90%) had at least one congenital anomaly; over 80% of the patients developed bone marrow failure; 53% underwent hematopoietic stem-cell transplantation; 33% of the patients developed cancer; no significant association was found between hematopoietic stem-cell transplant and solid tumor development. Nearly 95% of the patients tested had confirmed mutations in the Fanconi genes FANCA (67%), FANCC (13%), FANCG (14%), FANCJ (3%) and FANCD1 (2%), including twenty novel mutations. Patients with FANCA mutations developed cancer at a significantly older age compared to patients with mutations in other Fanconi genes (mean 18.5 and 5.2 years, respectively, P=0.001); however, the overall survival did not depend on the causative gene. We hereby describe a large national cohort of patients with FA, the vast majority genetically diagnosed. Our results suggest an older age for cancer development in patients with FANCA mutations and no increased incidence of solid tumors following hematopoietic stem-cell transplant. Further studies are needed to guide individual treatment and follow-up programs.

Attention, response inhibition and brain event-related potential alterations in adults with beta-thalassaemia major.

We investigated neural correlates of cognitive function in adults with beta thalassaemia major (ß-TM) compared to healthy controls using scalp-recorded event-related potentials (ERPs). Event-related potential studies in the field of ß-TM are scarce and mostly limited to children. A stop-signal task was used to evaluate indices of attention and response inhibition function, considered to be the hallmark of executive control. Correlations between task performance, ERPs and haemoglobin were also examined. Results showed impaired cognitive performance in ß-TM patients, as indicated by longer response times than controls. Haemoglobin was negatively correlated with response times to Go stimuli. Electrophysiological results indicated significant ß-TM-related alterations in neuronal activity, reflected in greater peak amplitudes of several task-related ERP components. A possible interpretation of these ERP results is that ß-TM patients need to recruit additional brain resources when dealing with cognitive challenge. Significant correlations were found between levels of haemoglobin and amplitude of all ERP components; the lower the haemoglobin, the more pronounced the ERPs amplitude. The present study represents a novel investigation of cognitive function and related brain dynamics in ß-TM in adult. Integrating neuropsychological assessment and interventions into traditional disease management, may be imperative in achieving a better quality of life for these patients.

Growth hormone deficiency in children with antenatal Bartter syndrome.

Background Bartter syndrome is a group of rare autosomal-recessive renal disorders characterized by hypokalemic hypochloremic metabolic alkalosis associated with severe growth failure; the exact causes for growth retardation are unclear. GH deficiency (GHD) has been reported in a few cases of Bartter syndrome. The aim of our study was to determine the prevalence of GHD in children with antenatal Bartter syndrome and to assess their response to GH therapy. Methods Ten patients aged 1.5-14.5 years and diagnosed with antenatal Bartter syndrome were enrolled. Seven children with short stature underwent GH stimulation tests. Results Common presenting symptoms were failure to thrive and polyuria. The mean patient height at study entry was -2.7 standard deviation (SD) (range 0.89 to -5.95) and mean weight (SD) was -1.7 (range 1.89 to -4.11). A decline in height and weight (SD) was observed over the years. GHD was diagnosed in four children and GH therapy was started in all of them. Two patients responded very well and gained >1 SD in height, one patient stopped therapy due to non-adherence and one had a poor response. Conclusions In addition to other important causes for poor growth in antenatal Bartter syndrome, our findings suggest that GHD should also be considered as a cause of growth retardation and therefore, clinical assessment of the GH axis is recommended. GH therapy has a role in the treatment of growth failure in some individuals with Bartter syndrome.

Extracellular Vesicle Characteristics in ß-thalassemia as Potential Biomarkers for Spleen Functional Status and Ineffective Erythropoiesis.

ß-thalassemia major (ß-TM) is a therapeutically challenging chronic disease in which ineffective erythropoiesis is a main pathophysiological factor. Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells into biological fluids; they are involved in intercellular communication and in multiple physiological and pathological processes. The chaperone heat-shock protein 70 (HSP70), which is released from cells via EVs, aggravates ineffective erythropoiesis in ß-TM. We propose that ß-TM EVs may show specific signatures, reflecting disease mechanisms, stages and severity. Our study aims were to define EV profiles in ß-TM patients, investigate the influence of hypersplenism and splenectomy on EV features, and explore the association of circulating EVs with ineffective erythropoiesis and iron-overload parameters. We characterized circulating EVs in 35 transfusion-dependent ß-thalassemia patients and 35 controls using several techniques. Nanoparticle-tracking analysis revealed increased EV concentration in patients vs. controls (P = 0.0036), with smaller EV counts and sizes in patients with hypersplenism. Flow cytometry analysis showed lower levels of RBC and monocyte EVs in patients vs. controls. RBC-EV levels correlated with patient hematocrit, reflecting degree of anemia. The procoagulant potential of the EVs evaluated by flow cytometry revealed lower levels of endothelial protein C receptor-labeled EVs in patients vs. controls, and increased tissue factor-to-tissue factor pathway inhibitor-labeled EV ratio in splenectomized patients, suggesting a hypercoagulable state. Protein content, evaluated in EV pellets, showed increased levels of HSP70 in patients (P = 0.0018), inversely correlated with transfusion requirement and hemoglobin levels, and positively correlated with reticulocyte, erythropoietin and lactate dehydrogenase levels. This first description of EVs in patients with hypersplenism reveals the spleen's importance in EV physiology and clearance. Circulating EV-HSP70 levels were associated with markers of ineffective erythropoiesis, hemolysis and hematological disease severity. EV analysis in ß-TM-reflecting spleen status, hypercoagulability state and ineffective erythropoiesis-may serve as a biomarker of disease dynamics, supporting both anticipation of the risk of complications and optimizing treatment.

Detection of ß-Thalassemia Carriers by Red Cell Parameters Obtained from Automatic Counters using Mathematical Formulas.

BACKGROUND: ß-thalassemia major is a severe disease with high morbidity. The world prevalence of carriers is around 1.5-7%. The present study aimed to find a reliable formula for detecting ß-thalassemia carriers using an extensive database of more than 22,000 samples obtained from a homogeneous population of childbearing age women with 3161 (13.6%) of ß-thalassemia carriers and to check previously published formulas. METHODS: We applied a mathematical method based on the support vector machine (SVM) algorithm in the search for a reliable formula that can differentiate between thalassemia carriers and non-carriers, including normal counts or counts suspected to belong to iron-deficient women. RESULTS: Shine's formula and our SVM formula showed >98% sensitivity and >99.77% negative predictive value (NPV). All other published formulas gave inferior results. CONCLUSIONS: We found a reliable formula that can be incorporated into any automatic blood counter to alert health providers to the possibility of a woman being a ß-thalassemia carrier. A further simple hemoglobin characterization by HPLC analysis should be performed to confirm the diagnosis, and subsequent family studies should be carried out. Our SVM formula is currently limited to women of fertility age until further analysis in other groups can be performed.

STAT3 gain-of-function mutations associated with autoimmune lymphoproliferative syndrome like disease deregulate lymphocyte apoptosis and can be targeted by BH3 mimetic compounds.

Autoimmune lymphoproliferative syndrome (ALPS) is typically caused by mutations in genes of the extrinsic FAS mediated apoptotic pathway, but for about 30% of ALPS-like patients the genetic diagnosis is lacking. We analyzed 30 children with ALPS-like disease of unknown cause and identified two dominant gain-of-function mutations of the Signal Transducer And Activator Of Transcription 3 (STAT3, p.R278H, p.M394T) leading to increased transcriptional activity. Hyperactivity of STAT3, a known repressor of FAS, was associated with decreased FAS-mediated apoptosis, mimicking ALPS caused by FAS mutations. Expression of BCL2 family proteins, further targets of STAT3 and regulators of the intrinsic apoptotic pathway, was disturbed. Cells with hyperactive STAT3 were consequently more resistant to intrinsic apoptotic stimuli and STAT3 inhibition alleviated this effect. Importantly, STAT3-mutant cells were more sensitive to death induced by the BCL2-inhibitor ABT-737 indicating a dependence on anti-apoptotic BCL2 proteins and potential novel therapeutic options.