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Background In the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, variable phenotypic expression may delay diagnosis. Panleukopenia, malignancy, and chronic lung disease all affect morbidity and mortality risks. Routinely used treatments include immunoglobulins, granulocyte-colony stimulating factor (G-CSF), and antibiotics; recent trials with a target C-X-C chemokine receptor type 4 (CXCR4) antagonist show promising results. Objective We sought to characterize the largest cohort of patients with WHIM and evaluate their diagnostic and therapeutic management. Methods Data were collected from an international cohort of 18 patients with CXCR4 mutations. Results The clinical features manifested at 2.2 ± 2.6 years of age, whereas the disease diagnosis was delayed until 12.5 ± 10.4 years of age. Patients with WHIM commonly presented with a severe bacterial infection (78%). Pneumonia recurrence was observed in 61% of patients and was complicated with bronchiectasis in 27%. Skin warts were observed in 61% of patients at a mean age of 11 years, whereas human papilloma virus (HPV)-related malignancies manifested in 16% of patients. All the patients had severe neutropenia (195 ± 102 cells/mm3 at onset), whereas lymphopenia and hypogammaglobulinemia were detected in 88% and 58% of patients, respectively. Approximately 50% of patients received antibiotic prophylaxis, whereas G-CSF and immunoglobulin treatments were used in 72% and 55% of patients, respectively. Conclusions The WHIM syndrome onsets early in life and should be suspected in patients with chronic neutropenia. Patients with WHIM need careful monitoring and timely intervention for complications, mainly lung disease and HPV-related malignancies. We suggest that immunoglobulin therapy should be promptly considered to control the frequency of bacterial infections and prevent chronic lung damage.

The management of iron overload in thalassemia has changed dramatically since the implementation of magnetic resonance imaging, which allows detection of preclinical iron overload and prevention of clinical complications. This study evaluated the effect of deferasirox (DFX), the newest once-daily oral chelator, on cardiac function, iron overload and cardiovascular events over a longer follow up in a "real world" setting. Longitudinal changes in cardiac magnetic resonance T2*, cardiac function parameters and cardiovascular clinical events were assessed in a cohort of 98 TM patients exposed to DFX for a mean of 6.9 years (range 1.8-11.6 years). No cardiac death or incident heart failure occurred. Cardiac T2* significantly increased (+2.6 ± 11.9 msec; P = 0.035) in the whole population, with a significantly greater increase (+11.6 ± 15.5 msec, P = 0.019) in patients with cardiac iron overload (T2*20 ms). A significant improvement in left-ventricular ejection fraction (LVEF) (from 50.6 ± 6 to 60.2 ± 5; P = 0.001) was observed in 11 (84.6%) out of 13 patients who normalized cardiac function (LVEF56%). Arrhythmias were the most frequent cardiac adverse event noted but none led to DFX discontinuation. Our data indicate that DFX is effective in maintaining cardiac iron level in the normal range and in improving cardiac iron overload. No heart failure or cardiac death was reported over this longer observation up to 12 years. For the first time, a DFX-induced improvement in LVEF was observed in a subgroup of patients with abnormal cardiac function at baseline, a preliminary observation which deserves further evaluation.

Background Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with β-thalassemia given a bone marrow transplant. We investigated the relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for β-thalassemia.Design and Methods Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens.Results All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again.Conclusions Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with β-thalassemia given a cord blood transplant from a relative.

Objective: Effector phase of acute graft-versus-host disease (a-GVHD) is mainly mediated by donor-derived, anti-host cytotoxic T cells. T-cell homing into gut-associated lymphoid tissues is ascribed to the α4β7 integrin. We reasoned that development of intestinal a-GVHD might be triggered by recruitment in the intestinal mucosa of circulating, alloreactive, α4β7+ donor T cells. Therefore, we evaluated the correlation existing between circulating β7+ T-lymphocyte subsets early after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and occurrence of a-GVHD. Patients and Methods: Surface expression of β7 integrin on T cells was evaluated by means of direct immunofluorescence, in three-color analysis. Sixty-five patients given allo-HSCT were evaluated: 13 of them experienced intestinal a-GVHD, 14 developed a-GVHD without intestinal involvement, and 38 did not develop a-GVHD. Patients were studied early after initial signs of hematologic reconstitution and before occurrence of a-GVHD. Results: We found a significantly higher absolute number of CD8+ and a significantly lower percentage of CD8+CD45RA+β7+ T cells in patients with intestinal a-GVHD than in patients with a-GVHD without intestinal involvement (p = 0.003 and p = 0.003, respectively) or not experiencing a-GVHD (p = 0.02 and p = 0.002, respectively). In particular, we found that intestinal a-GVHD occurred in over 70% of patients showing an absolute number of CD8+ T cells ≥ 60 × 106/L and a percentage of circulating CD8+CD45RA+β7+ T cells < 35%. Conclusion: Measuring the absolute number of CD8+ T cells and percentage of CD8+CD45RA+β7+ T cells at time of hematologic reconstitution may help identify patients at risk of developing intestinal a-GVHD who could benefit from strategies aimed at hampering alloreactive T-cell homing to intestinal mucosa. © 2006 International Society for Experimental Hematology.

This retrospective study was conducted to determine whether the response to splenectomy is related to the response to previous treatments. We examined the records of 90 children splenectomized for chronic ITP. Platelet counts were constantly>50x10(9)/L in 68 patients (75%). An improvement in the quality of life was observed in 79 (85%). The success of splenectomy was strongly correlated with a good response to previous treatment. A negative response to any of the prior treatments had no predictive value. This finding is relevant when elective splenectomy is considered as a treatment option.

Summary The risk of developing hepatocellular carcinoma (HCC) in patients with thalassaemia is increased by transfusion-transmitted infections and haemosiderosis. All Italian Thalassaemia Centres use an ad hoc form to report all diagnoses of HCC to the Italian Registry. Since our last report, in 2002, up to December 2012, 62 new cases were identified, 52% of whom were affected by thalassaemia major (TM) and 45% by thalassaemia intermedia (TI). Two had sickle-thalassaemia (ST). The incidence of the tumour is increasing, possibly because of the longer survival of patients and consequent longer exposure to the noxious effects of the hepatotropic viruses and iron. Three patients were hepatitis B surface antigen-positive, 36 patients showed evidence of past infection with hepatitis B virus (HBV). Fifty-four patients had antibodies against hepatitis C virus (HCV), 43 of whom were HCV RNA positive. Only 4 had no evidence of exposure either to HCV or HBV. The mean liver iron concentration was 8 mg/g dry weight. Therapy included chemoembolization, thermoablation with radiofrequency and surgical excision. Three patients underwent liver transplant, 21 received palliative therapy. As of December 2012, 41 patients had died. The average survival time from HCC detection to death was 11·5 months (1·4–107·2 months). Ultrasonography is recommended every 6 months to enable early diagnosis of HCC, which is crucial to decrease mortality.

This report represents an attempt to define the rate of beta-spectrin de novo mutations affecting mRNA accumulation in patients with hereditary spherocytosis (HS). 19 HS children with haematologically normal parents and varying degrees of spectrin deficiency were studied. 13 of the 19 cases who were heterozygous at the genomic level for polymorphisms in the beta-spectrin coding region were further studied. However, in an analysis of reverse-transcripted amplified cDNA from the regions of the polymorphisms, seven patients appeared to be homozygous, suggesting the occurrence of de novo mutational events affecting expression of one beta-spectrin allele. We conclude that in HS patients with isolated spectrin reduction and normal parents the apparently recessive pattern of inheritance may frequently be associated with de novo monoallelic expression of beta-spectrin.

Objective: To determine the efficacy of high-dose dexamethasone in chronic idiopathic thrombocytopenic purpura of childhood. Methods: Seventeen patients entered the protocol. Dexamethasone was to be given orally in two divided doses at a dosage of 20 mg/m 2 for 4 consecutive days every 28 days for six courses. Results: One month after the end of the sixth course, six patients (35%) had platelet values within the normal range. One year later, five patients (29%) still have normal platelet values. Five patients discontinued treatment before completion because of lack of response and in one case for important side effects. Duration of the disease before treatment was inversely correlated with response to dexamethasone: 5 of 10 patients who had had thrombocytopenia for 30 months or less went into remission, as opposed to none of the seven who had been sick for a longer period ( p = 0.04). Side effects included fatigue or irritability, anxiety, abdominal pain, striae, hirsutism, acne, and weight gain. Conclusions: Contrary to what is observed in adults, in our patients pulsed dexamethasone therapy did not prove to be uniformly effective. However, in view of its effectiveness in a third of the patients, acceptable side effects, and low cost, we believe that this treatment could be considered in patients with chronic idiopathic thrombocytopenic purpura who do not tolerate the disease well, especially if no more than 3 years have elapsed since diagnosis. Larger studies will be necessary to define which patients will respond to this type of therapy.

Nucleated red blood cells (NRBCs) are present in the peripheral blood of several hematological and non-hematological conditions, usually associated with bad prognosis. The lack of an easy, rapid and reliable NRBCs count method did no’t allow one to know the incidence of NRBCs and to quantify them: the count was usually done during the microscopic revision of a blood smear; this is the reason we found few studies on NRBCs automated count in the literature. The aim of this study was the evaluation of the presence and the quantification of NRBCs in some onco-hematological disorders.This study analyzed 478 patients with the automated hematology analyzer Sysmex XE2100. The range of NRBCs were calculated in the peripheral blood at diagnosis, at hematological remission and during therapy.NRBCs are present in the peripheral blood of a high number of hematological diseases and are related to ineffective erythropoiesis or stress erythropoiesis or primary alterations of hematopoiesis. NRBCs were found in nearly all onco-hematological diseases at diagnosis, but not in all patients. NRBCs were frequently found during chemotherapy and absent at remission.To the authors’ knowledge, this is the first study that gives a range for NRBCs count in the peripheral blood of these diseases.

We report the long-term follow-up (median 39.5 months) of 49 paediatric patients (33 females and 16 males) with refractory symptomatic immune thrombocytopenic purpura (ITP) treated with rituximab. The overall response rate was 69% (34/49 patients). Twenty-one responders had a platelet count >50 x 10(9)/l at a median 20.2 months from treatment. Kaplan-Meier analysis showed a probability of relapse-free survival (RFS) of 60% at 36 months from the first rituximab infusion. The number of infusions and a previous splenectomy did not influence overall response rate. Patients who achieved complete response were significantly older at diagnosis and first rituximab infusion than partial responders (P = 0.027). Older children displayed a significantly greater probability of sustained response (RFS) at 36 months than younger children (88.9% vs. 56.7%, P = 0.037). Earlier responses (within 20 d from treatment) were significantly associated with both complete (P = 0.004) and sustained response (P = 0.002). Only mild and transient side-effects were observed in 9/49 children; no major infections nor delayed toxicities were recorded during the follow-up.

Persistent production of fetal hemoglobin (HbF) in adult has ameliorative effects on hemoglobinopathies and great efforts are currently made to achieve an exhaustive understanding of the molecular mechanisms of the switching in globin gene expression. One of the factors reported to be associated with the expression of fetal globin genes is the Xmn I Ggamma-158 polymorphism, although it is still unclear if it is involved in this mechanism either by itself or in strong linkage disequilibrium with other loci. Here, we report a novel effect of the Xmn I Ggamma-158 site that was found associated with a significant delayed decline of HbF production in infant age. The prolonged decay trend was enhanced when the Ggamma-158 C-->T substitution was co-inherited with a beta-thalassemic trait. Our observations reinforce the hypothesis that this region plays an important role in the expression of the gamma-globin genes and give new insights on the intriguing and still poorly understood mechanisms of globin gene expression switching.

Warm-type idiopathic autoimmune hemolytic anemia (AIHA) is a relatively common hematologic disorder resulting from autoantibody production against red blood cells. Steroids represent the first-line therapeutic option, and immunosuppressive agents as well as splenectomy are used for refractory cases. Recently, the anti-CD20 monoclonal antibody rituximab has been shown to control autoimmune hemolysis in patients with refractory chronic disease. We report results from a retrospective analysis of 11 adult patients receiving rituximab for steroid-refractory AIHA of the warm type at a mean age of 55 yr (range 23-81 yr). All patients were given methyl-prednisolone as first-line treatment and some of them also received azathioprine and intravenous high-dose immunoglobulins. One patient underwent splenectomy. All patients were considered refractory to steroids and/or immunosuppressive drugs and all were then given weekly rituximab (375 mg/m 2 ) for four consecutive weeks. An increase in hemoglobin (Hgb) levels in response to rituximab, with a mean increment of 3.3 g/dL (95% Cl 2.1-4.4), was observed in all cases. Four patients required packed red cell transfusions before starting rituximab and all became transfusion-free. At a mean follow-up of 604 d (range 30-2884 d) since the treatment of AIHA with rituximab, all patients are alive, eight (73%) of them in complete remission (CR) and three (27%) in partial remission (PR). A moderate hemolysis still persisted in six (54%) patients. In conclusion, our experience clearly demonstrates that anti-CD20 monoclonal antibody rituximab is an effective and safe alternative treatment option for idiopathic AIHA, in particular, for steroid-refractory disease.

Objective Functional recovery of B lymphocytes after hematopoietic stem cell transplantation (HSCT) can take up to 2 years. HSCT recipients may obtain protective titers of pathogen-specific antibody through vaccination, but optimal timing of reimmunization remains to be defined. Patients and Methods In this study, we evaluated the reconstitution of B-cell number and activity in 139 children given HSCT, by B-cell subset phenotyping and in vitro immunoglobulin (Ig) production. Results Patients were longitudinally studied at 3, 6, 12, and 18 to 24 months after transplantation. At all time points, recipients displayed a significantly higher percentage of naive (IgD + CD27 − ) B cells and showed significantly lower production of stimulated in vitro Ig as compared to healthy controls. Moreover, during follow-up, we observed an increase in the proportion of patients who had CD27 + B subsets and who were able to mount in vitro Ig production greater than the 5th percentile. Conclusion Similar to what has been described in adults, most children lack memory B cells and produce low amounts of Ig. However, the number of B cells, as well as their function, gradually recovered over time and the spread of data we observed suggests that the reimmunization schedule should be individualized for each patient. It remains to be defined in a prospective clinical study the time point at which a patient should start reimmunization. A reasonable hypothesis to be explored is the time point at which a percentage of memory B cells greater than the 5th percentile of normal controls is reached.

We report on 14 children (seven boys, seven girls) with chronic idiopathic thrombocytopenic purpura (ITP) refractory to multiple treatments, who were given a short-term therapy (range between 6 and 10 weeks) with high doses of cyclosporin A (CyA) (median, 10 mg/kg/d). Six patients experienced adverse events and one developed severe systemic mycosis during therapy. A complete response (CR) was observed in four patients and a partial response (PR) in three patients. Only the four CR patients, who were all girls, had a sustained response. These data suggest that CyA may be effective in some children with chronic symptomatic ITP.

SummaryThe increasing number of diagnosed cases of inherited thrombocytopenias, owing to the routine practice of including platelet counts in blood tests, suggests that this condition is not so rare as expected. In the majority of cases, the molecular basis of the disease is unknown, although the defect is likely to affect thrombocytopoiesis and regulation of the normal platelet count. Here we report a genomewide search in a large Italian family affected by autosomal dominant thrombocytopenia. Patients showed a moderate thrombocytopenia with minimal symptoms characterized by normocellular bone marrow, normal medium platelet volume, and positive aggregation tests. Microsatellite analysis demonstrated that the disease locus (THC2) is linked to chromosome 10p11.1-12, within a candidate region of 6 cM between markers D10S586 and D19S1639. A maximum LOD score of 8.12 at recombination fraction .00 was obtained with the microsatellite D10S588. These data localized the first locus of an autosomal dominant thrombocytopenia, and the subsequent identification of the gene will provide new insight into the basic mechanism of megakaryocytopoiesis disorders.

BackgroundGain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined.Methodology/principal findingsWe describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G-->T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34(+) cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway.Conclusions/significanceOur data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.

Summary Lipid abnormalities, including low levels of all fractions of serum lipids, have been repeatedly reported in all phenotypes of β-thalassemia. Unexpectedly, in more recent studies, the concentration of total cholesterol (TC) and high- and/or low-density lipoprotein cholesterol (HDL-C and LDL-C) has been found in β-thalassemia intermedia (TI) patients even lower than in thalassemia major, without a clear explanation of pathophysiology of these findings. This lack of information prompted us to evaluate the plasma lipids and lipoproteins pattern in the TI patients followed in our department; the data were compared with those found in hereditary spherocytosis patients. Furthermore, in both groups of patients, the erythroid bone marrow activity was evaluated, utilizing the level of soluble transferrin receptors (sTfR) in the plasma. Both groups of patients showed similar lipid abnormalities (low-TC, HDL-C and LDL-C) and the same increase of sTfR, with significantly lower hemoglobin levels in TI patients. Data analysis of our study shows that the lipid profile in TI patients is not influenced by age, sex, liver injury, hemoglobin or ferritin levels; the higher erythroid bone marrow activity with the enhanced cholesterol consumption could be the dominant mechanism implicated in the lipid abnormalities of TI patients.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder. We report three patients with WHIM syndrome who are affected by Tetralogy of Fallot (TOF). This observation suggests a possible increased risk of TOF in WHIM syndrome and that birth presentation of TOF and neutropenia should lead to suspect WHIM syndrome.