Naomi L.C. Luban

High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin

In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 ± 50 pg/ml. In comparison, individuals with β-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 ± 9,600 pg/ml; range 4,800–248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.

Hydroxycarbamide versus chronic transfusion for maintenance of transcranial doppler flow velocities in children with sickle cell anaemia - TCD with Transfusions Changing to Hydroxyurea (TWiTCH): A multicentre, open-label, phase 3, non-inferiority trial

Background For children with sickle cell anaemia and elevated transcranial Doppler (TCD) flow velocities, regular blood transfusions effectively prevent primary stroke, but must be continued indefinitely. The efficacy of hydroxyurea in this setting is unknown. Methods TWiTCH was a multicentre Phase III randomised open label, non-inferiority trial comparing standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with abnormal TCD velocities but no severe vasculopathy. Iron overload was managed with chelation (Standard Arm) and serial phlebotomy (Alternative Arm). The primary study endpoint was the 24-month TCD velocity calculated from a general linear mixed model, with non-inferiority margin = 15 cm/sec. Findings Among 121 randomised participants (61 transfusions, 60 hydroxyurea), children on transfusions maintained <30% sickle haemoglobin, while those taking hydroxyurea (mean 27 mg/kg/day) averaged 25% fetal haemoglobin. The first scheduled interim analysis demonstrated non-inferiority, and the sponsor terminated the study. Final model-based TCD velocities (mean ± standard error) on Standard versus Alternative Arm were 143 ± 1.6 and 138 ± 1.6 cm/sec, respectively, with difference (95% CI) = 4.54 (0.10, 8.98), non-inferiority p=8.82 × 10−16 and post-hoc superiority p=0.023. Among 29 new neurological events adjudicated centrally by masked reviewers, no strokes occurred but there were 3 transient ischaemic attacks per arm. Exit brain MRI/MRA revealed no new cerebral infarcts in either arm, but worse vasculopathy in one participant (Standard Arm). Iron burden decreased more in the Alternative Arm, with ferritin difference −1047 ng/mL (−1524, −570), p<0.001 and liver iron difference −4.3 mg Fe/gm dry weight (−6.1, −2.5), p=0.001. Interpretation For high-risk children with sickle cell anaemia and abnormal TCD velocities, after four years of transfusions and without severe MRA vasculopathy, hydroxyurea therapy can substitute for chronic transfusions to maintain TCD velocities and help prevent primary stroke.BACKGROUND For children with sickle cell anaemia and high transcranial doppler (TCD) flow velocities, regular blood transfusions can effectively prevent primary stroke, but must be continued indefinitely. The efficacy of hydroxycarbamide (hydroxyurea) in this setting is unknown; we performed the TWiTCH trial to compare hydroxyurea with standard transfusions. METHODS TWiTCH was a multicentre, phase 3, randomised, open-label, non-inferiority trial done at 26 paediatric hospitals and health centres in the USA and Canada. We enrolled children with sickle cell anaemia who were aged 4-16 years and had abnormal TCD flow velocities (≥ 200 cm/s) but no severe vasculopathy. After screening, eligible participants were randomly assigned 1:1 to continue standard transfusions (standard group) or hydroxycarbamide (alternative group). Randomisation was done at a central site, stratified by site with a block size of four, and an adaptive randomisation scheme was used to balance the covariates of baseline age and TCD velocity. The study was open-label, but TCD examinations were read centrally by observers masked to treatment assignment and previous TCD results. Participants assigned to standard treatment continued to receive monthly transfusions to maintain 30% sickle haemoglobin or lower, while those assigned to the alternative treatment started oral hydroxycarbamide at 20 mg/kg per day, which was escalated to each participants maximum tolerated dose. The treatment period lasted 24 months from randomisation. The primary study endpoint was the 24 month TCD velocity calculated from a general linear mixed model, with the non-inferiority margin set at 15 cm/s. The primary analysis was done in the intention-to-treat population and safety was assessed in all patients who received at least one dose of assigned treatment. This study is registered with ClinicalTrials.gov, number NCT01425307. FINDINGS Between Sept 20, 2011, and April 17, 2013, 159 patients consented and enrolled in TWiTCH. 121 participants passed screening and were then randomly assigned to treatment (61 to transfusions and 60 to hydroxycarbamide). At the first scheduled interim analysis, non-inferiority was shown and the sponsor terminated the study. Final model-based TCD velocities were 143 cm/s (95% CI 140-146) in children who received standard transfusions and 138 cm/s (135-142) in those who received hydroxycarbamide, with a difference of 4·54 (0·10-8·98). Non-inferiority (p=8·82 × 10(-16)) and post-hoc superiority (p=0·023) were met. Of 29 new neurological events adjudicated centrally by masked reviewers, no strokes were identified, but three transient ischaemic attacks occurred in each group. Magnetic resonance brain imaging and angiography (MRI and MRA) at exit showed no new cerebral infarcts in either treatment group, but worsened vasculopathy in one participant who received standard transfusions. 23 severe adverse events in nine (15%) patients were reported for hydroxycarbamide and ten serious adverse events in six (10%) patients were reported for standard transfusions. The most common serious adverse event in both groups was vaso-occlusive pain (11 events in five [8%] patients with hydroxycarbamide and three events in one [2%] patient for transfusions). INTERPRETATION For high-risk children with sickle cell anaemia and abnormal TCD velocities who have received at least 1 year of transfusions, and have no MRA-defined severe vasculopathy, hydroxycarbamide treatment can substitute for chronic transfusions to maintain TCD velocities and help to prevent primary stroke. FUNDING National Heart, Lung, and Blood Institute, National Institutes of Health.

Influence of Human Immunodeficiency Virus—Infected Maternal Environment on Development of Infant Interleukin-12 Production

Monocyte-derived cytokine production by cord blood mononuclear cells (CBMC) from infants born to human immunodeficiency virus (HIV)-positive and -negative women was measured to determine whether monocyte dysfunction could contribute to the accelerated HIV disease of pediatric patients. Production of interleukin (IL)-12, but not that of tumor necrosis factor-alpha and IL-10, was reduced, compared with adult peripheral blood mononuclear cells (PBMC). This deficiency was more pronounced in infants of HIV-positive women, whose IL-12 production was also deficient. CBMC IL-12 levels were increased by interferon-gamma and CD40 ligand but remained deficient, compared with PBMC. IL-12 production was undetectable in 7 of 8 HIV-positive infants, in contrast to 21 of 26 uninfected infants. Uninfected infants of infected women exhibited an intermediate profile. These findings suggest that the maternal environment and/or exposure in utero to HIV products influence the newborns immune response and that the differences between infants born to HIV-positive and -negative women may persist.

Clinical heterogeneity in mitochondrial DNA deletion disorders: a diagnostic challenge of Pearson syndrome.

The clinical presentation of mitochondrial DNA (mtDNA) disorders is quite diverse. Very often, the initial symptoms do not fit a specific disease, and diagnosis is difficult to make. We describe a patient who presented with macrocytic anemia. Extensive biochemical and clinical work-up failed to provide an etiology for the macrocytic anemia. The patient over the course of 6 years developed gait problems, exercise intolerance, episodic vomiting, short stature, dermatological problems, and recurrent infection. At age 8 years she had encephalopathy with ataxia and dysphagia. The presence of elevated lactate, bilateral basal ganglia calcification, and ragged red fibers led to mtDNA mutational analysis. A novel 4.4-kb deletion from nucleotide position 10,560 to nucleotide position 14, 980 was identified in muscle biopsy. The same heteroplasmic mtDNA deletion was present in blood, buccal cells, and hair follicles, but not in mothers blood, consistent with sporadic mutation in the patient. This case emphasizes the importance of considering mtDNA disorder in patients with multisystemic symptoms that cannot be explained by a specific diagnosis.

Red blood cell transfusions in the newborn

The indications for red blood cell (RBC) transfusions in the newborn, particularly for the preterm neonate, remain controversial and variable, although the practice has been an integral part of neonatal care for decades. The hazards of RBC transfusions have been minimized over time, but residual risks remain and have led to re-evaluation of commonly accepted indications, generation of stringent transfusion guidelines, and development of alternate strategies to reduce donor exposure. Despite the increasing complexity of care, and the survival of smaller and sicker infants over the past few years, there has been a significant decline in the use of RBC transfusions.

Prevalence of conditions associated with human immunodeficiency and hepatits virus infections among persons with haemophilia, 2001-2003

Summary.  Before the mid‐1980s, haemophilia often was unknowingly treated with contaminated plasma products, resulting in high rates of human immunodeficiency virus (HIV‐1), hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. To estimate the impact of these infections, a new cohort was established. All HCV‐seropositive patients, age 13–88 years, at 52 comprehensive haemophilia treatment centres were eligible. Cross‐sectional data collected during April 2001 to January 2004 (median June 2002) were analysed. Plasma HIV‐1 and HCV RNA were quantified by polymerase chain reaction. Highly active antiretroviral therapy (HAART) was defined as use of at least three recommended medications. Among 2069 participants, 620 (30%) had HIV‐1. Of 1955 with known HBV status, 814 (42%) had resolved HBV and 90 (4.6%) were HBV carriers. Although 80% of the HIV‐1‐positive participants had ≥200 CD4+ cells μL−1, only 59% were on HAART. HIV‐1 RNA was undetectable in 23% of those not taking antiretroviral medications. Most (72%) participants had received no anti‐HCV therapy. HCV RNA was detected less frequently (59%) among participants treated with standard interferon plus ribavirin (P = 0.0001) and more frequently among HIV‐1‐positive than HIV‐1‐negative participants (85% vs. 70%, P < 0.0001). HIV‐1‐positive participants were more likely to have pancytopenia and subclinical hepatic abnormalities, as well as persistent jaundice, hepatomegaly, splenomegaly and ascites. HAART recipients did not differ from HIV‐negative participants in the prevalence of ascites. The clinical abnormalities were more prevalent with older age but were not confounded by HBV status or self‐reported alcohol consumption. Eleven participants presented with or previously had hepatocellular carcinoma or non‐Hodgkin lymphoma. Although prospective analysis is needed, our data reveal the scale of hepatic and haematological disease that is likely to manifest in the adult haemophilic population during the coming years unless most of them are successfully treated for HIV‐1, HCV or both.

Protease inhibitor and triple-drug therapy: cellular immune parameters are not restored in pediatric AIDS patients after 6 months of treatment.

Objective:To assess whether treatment of HIV-positive children by antiretroviral drugs for a 6-month period would improve immune function significantly. Design and methods:Immunological assessment of 89 HIV-positive children who received protease inhibitor monotherapy for 12–16 weeks as part of phase I/II studies, followed by triple antiretroviral therapy for an additional 12 weeks, was conducted. Immunological parameters were assessed in vitro at four time points (at enrollment, at weeks 2–4, at weeks 12–16, and at weeks 24–28). Assessments included: cytokine production by monocytes, T-cell proliferation to mitogen or recall antigens (including an HIV antigen) and apoptotic cell death. Plasma levels of tumor necrosis factor (TNF)-α and soluble TNF receptor (sTNF-R) were also measured, in addition to CD4+ T-lymphocyte counts and viral load. In addition, limited analyses were performed on samples from 17 children after 120 weeks of therapy, including 104 weeks of triple therapy. Results:At enrollment, the 89 children exhibited severe immune defects. Antiretroviral therapy raised CD4+ T-lymphocyte counts significantly and decreased viral loads. In contrast, the in vitro immune parameters studied were not improved, except for plasma levels of sTNF-RII which decreased in parallel with the decrease in viral load. In addition, there was a trend towards increased skin test reactivity for the ritonavir-treated children. No differences were seen in the immune parameters whether the patients were treated with mono- or triple therapy. Results obtained after 120 weeks of therapy demonstrated that defective interleukin-12 production was not restored by long-term therapy. Conclusions:After 6 months of therapy, with the exception of decreased sTNF-RII levels, and a trend towards increased skin test reactivity, restoration of several defective cellular immune responses did not occur despite significantly decreased viral loads and increased CD4+ T-lymphocyte counts.

Low prevalence of flower cells in U.S.A. blood donors infected with human T-lymphotrophic virus types I and II.

Large lymphocytes with basophilic cytoplasm and cleaved/cerebriform nuclei called flower cells have been described in human T‐lymphotrophic virus type I (HTLV‐I) seropositive individuals and may be precursors of adult T‐cell leukaemia (ATL). A cohort of 546 HTLV‐seropositive former blood donors, 32 HTLV‐positive sexual partners of these donors and 799 HTLV‐seronegative controls has been followed as part of the Retrovirus Epidemiology Donor Study. A novel methodology was developed to systematically review peripheral blood slides from these subjects for HTLV‐related lymphocyte abnormalities, using an algorithm based on morphologic features to objectively identify flower cells. The algorithm included: absence of azurophil granules; nuclear chromatin condensation; cell size >1.5 small lymphocytes; nuclear to cytoplasmic ratio >80%; and presence of nuclear folding/lobulation. Peripheral slides from subjects were screened by a medical technologist blinded to HTLV status. 6.8% of HTLV‐I subjects (P = 0.0001 versus seronegatives), 0.9% of HTLV‐II subjects and 1.1% of seronegatives were confirmed to have cells classified as flower cells by two haematologists using objective criteria, and blinded to serostatus. Despite the higher prevalence of flower cells in HTLV‐I positives, no clinical correlations were found. Longitudinal follow‐up may yield higher rates of cellular abnormalities as the sequelae of HTLV infection develop.

Too much, too little, too soon, too late? Transfusion and long‐term survival in children

H ow blood transfusions impact patient outcome is of interest to health economists, insurers, transfusion medicine specialists, prescribing physicians, families, and most importantly, vulnerable patients. There are two different approaches to evaluating impact within the health care system: micro and macro. At the micro level, attention is focused on a single patient or group of patients, one provider at a time. Population-based clinical trials, clinical effectiveness studies, meta-analyses, and clinical experience are used to inform physician decision making on a micro level. Discussion of risks and benefits are implicit in the consent process and occur at the bedside with the physician, parent, and child (if age appropriate) reviewing how transfusion can alleviate symptoms and/or support a child’s hematopoietic system. In these cases, the physician’s allegiance to the patient is measured as an “n of 1” study, and there is no role for the discussion of cost implications, resource limitations, or societal burden. At the macro level, care is provided to populations of patients. In this setting, care that benefits the greatest number of patients while minimizing risks, complications, and costs in the aggregate is the focus. Measurement strategies like mortality and complication rates, proportions, incidence, and prevalence become important tools that help to inform health care policy decisions affecting access, cost, and quality. Those technologies and treatments, be they mechanical or biologic, that result in better outcomes at lower costs will have the greatest value to populations of patients. Admittedly, there is considerable interplay between the two care systems just defined. In reality, they are not discrete, but rather porous to effects in both directions and have important implications for transfusion medicine. In this issue of TRANSFUSION, Gauvin and colleagues describe a single institution’s experience with the long-term follow-up of patients who received blood transfusions between 1990 and 1992. Their demographic findings provide some insight regarding practice patterns in the early 1990s. From a diagnostic perspective, cardiac disease, malignancies, and operative procedures, specifically cardiac and orthopedic procedures, were the diagnoses most frequently requiring transfusion. Age was an important consideration for transfusion; nearly a third of patients were neonates and half were less than a year of age. The most frequently transfused product was red blood cells (RBCs) and 42.5 percent of patients received one to two transfusions. There are several important findings from this study when evaluated from the micro and macro level. At the micro level, the study provides an opportunity to contrast historical transfusion practices in children to those currently in use. The study also advances our understanding of some of the potential ill effects of transfusion and specifically focuses on the impact of transfusion on mortality. From a methodologic perspective, two specific concerns are raised on a macro level. A single-institution database to inform practices raises questions as to the generalizability of the findings at the intersection of the micro and macro levels. In addition, the ability of a small data set to reliably predict outcomes that are distinct in time or location raises analytical concerns. Historical perspectives are useful in providing context in caring for patients, which is why long-term follow-up studies are so valuable. The patient profiles of transfused pediatric patients have not changed dramatically. A recently published, multi-institutional analysis of transfusion practices in pediatric teaching hospitals found strikingly similar results to the study by Gauvin and colleagues. RBCs are the most frequent product transfused and age, race, disease severity score, and discharge disposition are all important demographic considerations for those who received transfusions. Are these similarities in findings in two studies of very different design and time periods good or bad news? At the micro level, progress in clinical transfusion medicine and provider prescribing behavior has not changed dramatically. To improve prescriber transfusion practices and outcomes, data regarding patterns and outcome need to be made available to that prescriber in “real time.” It is clear that transfusions provide benefit for specific subsets of patients with different disorders across a diverse range of conditions. No one would argue with the value of transfusion for acute bleeding, trauma, and specific disorders like sickle cell disease and symptomatic anemia. However, there is increasing attention in the literature to the potential adverse effects of transfusion, especially noninfectious risks, like transfusion-related acute lung injury (TRALI) and fatal or near-miss clerical and administration errors. Published data on transfusion complications in hospitalized children are rare, reflect a tendency to publish single-institution studies, use TRANSFUSION 2008;48:796-798.

Sickle Cell Disease Summit: From clinical and research disparity to action

The American Society of Pediatric Hematology/Oncology Sickle Cell Summit brought together a broad range of constituencies to identify a unified approach to healthcare and research disparities for sickle cell disease. Recommendations included the following: (1) speak with a unified voice representing all constituencies; (2) optimize access to care from knowledgeable health care providers and create a medical home for all individuals with the disease; (3) utilize population‐based surveillance to measure outcomes; (4) develop overall approaches to basic, translational, clinical, and health services research; (5) enhance the community role in advocacy, education, service, and fundraising. Taskforces were identified to effect implementation. Am. J. Hematol., 2009.