Subarna Chakravorty

Sickle cell disease: a neglected chronic disease of increasing global health importance

Sickle cell disease (SCD) is a single gene disorder causing a debilitating systemic syndrome characterised by chronic anaemia, acute painful episodes, organ infarction and chronic organ damage and by a significant reduction in life expectancy. The origin of SCD lies in the malarial regions of the tropics where carriers are protected against death from malaria and hence enjoy an evolutionary advantage. More recently, population migration has meant that SCD now has a worldwide distribution and that a substantial number of children are born with the condition in higher-income areas, including large parts of Europe and North and South America. Newborn screening, systematic clinical follow-up and prevention of sepsis and organ damage have led to an increased life expectancy among people with SCD in many such countries; however, in resource-limited settings where the majority continue to be born, most affected children continue to die in early childhood, usually undiagnosed, due to the lack of effective programmes for its early detection and treatment. As new therapies emerge, potentially leading to disease amelioration or cure, it is of paramount importance that the significant burden of SCD in resource-poor countries is properly recognised.

How I manage neonatal thrombocytopenia

Although neonatal thrombocytopenia (platelet count < 150 × 109/l) is a common finding in hospital practice, a careful clinical history and examination of the blood film is often sufficient to establish the diagnosis and guide management without the need for further investigations. In preterm neonates, early‐onset thrombocytopenia (<72 h) is usually secondary to antenatal causes, has a characteristic pattern and resolves without complications or the need for treatment. By contrast, late‐onset thrombocytopenia in preterm neonates (>72 h) is nearly always due to post‐natally acquired bacterial infection and/or necrotizing enterocolitis, which rapidly leads to severe thrombocytopenia (platelet count < 50 × 109/l). Thrombocytopenia is much less common in term neonates and the most important cause is neonatal alloimmune thrombocytopenia (NAIT), which confers a high risk of perinatal intracranial haemorrhage and long‐term neurological disability. Prompt diagnosis and transfusion of human platelet antigen‐compatible platelets is key to the successful management of NAIT. Recent studies suggest that more than half of neonates with severe thrombocytopenia receive platelet transfusion(s) based on consensus national or local guidelines despite little evidence of benefit. The most pressing problem in management of neonatal thrombocytopenia is identification of safe, effective platelet transfusion therapy and controlled trials are urgently needed.

The risk of hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type 2.

Genetic disorders of lymphocyte cytotoxicity predispose patients to hemophagocytic lymphohistiocytosis (HLH). Reduced lymphocyte cytotoxicity has been demonstrated in Hermansky-Pudlak syndrome type 2 (HPS2), but only a single patient was reported who developed HLH. Because that patient also carried a potentially contributing heterozygous RAB27A mutation, the risk for HLH in HPS2 remains unclear. We analyzed susceptibility to HLH in the pearl mouse model of HPS2. After infection with lymphocytic choriomeningitis virus, pearl mice developed all key features of HLH, linked to impaired virus control caused by a moderate defect in CTL cytotoxicity in vivo. However, in contrast to perforin-deficient mice, the disease was transient, and all mice fully recovered and controlled the infection. An additional heterozygous Rab27a mutation did not aggravate the cytotoxicity defect or disease parameters. In the largest survey of 22 HPS2 patients covering 234 patient years, we identified only 1 additional patient with HLH and 2 with incomplete transient HLH-like episodes, although cytotoxicity or degranulation was impaired in all 16 patients tested. HPS2 confers a risk for HLH that is lower than in Griscelli or Chediak-Higashi syndrome, probably because of a milder defect in cytotoxicity. Preemptive hematopoietic stem cell transplantation does not appear justified in HPS2.

Disruption of AP3B1 by a chromosome 5 inversion: a new disease mechanism in Hermansky-Pudlak syndrome type 2.

BackgroundHermansky-Pudlak syndrome 2 (HPS2; OMIM #608233) is a rare, autosomal recessive disorder caused by loss-of-function genetic variations affecting AP3B1, which encodes the β3A subunit of the adaptor-related protein complex 3 (AP3). Phenotypic characteristics include reduced pigmentation, absent platelet dense granule secretion, neutropenia and reduced cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function. To date HPS2 has been associated with non-synonymous, stop-gain or deletion-insertion nucleotide variations within the coding region of AP3B1.Case presentationWe describe a consanguineous female infant with reduced pigmentation, neutropenia and recurrent infections. Platelets displayed reduced aggregation and absent ATP secretion in response to collagen and ADP, indicating a platelet dense granule defect. There was increased basal surface expression of CD107a (lysosome-associated membrane protein 1(LAMP-1)) on NK cells and CTLs from the study subject and a smaller increase in the percentage of CD107a positive cells after stimulation compared to most healthy controls. Immunoblotting of protein extracts from EBV-transformed lymphoblasts from the index case showed absent expression of full-length AP-3 β3A subunit protein, confirming a phenotypic diagnosis of HPS2.The index case displayed a homozygous pericentric inv(5)(p15.1q14.1), which was also detected as a heterozygous defect in both parents of the index case. No loss of genetic material was demonstrated by microarray comparative genome hybridisation at 60kb resolution. Fluorescence in-situ hybridisation using the 189.6kb probe RP11-422I12, which maps to 5q14.1, demonstrated dual hybridisation to both 5q14.1 and 5p15.1 regions of the inverted Chr5. The RP11-422I12 probe maps from intron 1 to intron 16 of AP3B1, thus localising the 5q inversion breakpoint to within AP3B1. The probe RP11-211K15, which corresponds to an intergenic region on 5p also showed dual hybridisation, enabling localisation of the 5p inversion breakpoint.ConclusionThis case report extends the phenotypic description of the very rare disorder HPS2. Our demonstration of a homozygous Chr5 inversion predicted to disrupt AP3B1 gene provides a novel pathogenic mechanism for this disorder.

Impairment of neutrophil oxidative burst in children with sickle cell disease is associated with heme oxygenase-1

Sickle cell disease is a risk factor for invasive bacterial infections, and splenic dysfunction is believed to be the main underlying cause. We have previously shown that the liberation of heme in acute hemolysis can induce heme oxygenase-1 during granulopoiesis, impairing the ability of developing neutrophils to mount a bactericidal oxidative burst, and increasing susceptibility to bacterial infection. We hypothesized that this may also occur with the chronic hemolysis of sickle cell disease, potentially contributing to susceptibility to infections. We found that neutrophil oxidative burst activity was significantly lower in treatment-naïve children with sickle cell disease compared to age-, gender- and ethnicity-matched controls, whilst degranulation was similar. The defect in neutrophil oxidative burst was quantitatively related to both systemic heme oxygenase-1 activity (assessed by carboxyhemoglobin concentration) and neutrophil mobilization. A distinct population of heme oxygenase-1-expressing cells was present in the bone marrow of children with sickle cell disease, but not in healthy children, with a surface marker profile consistent with neutrophil progenitors (CD49dHi CD24Lo CD15Int CD16Int CD11b+/−). Incubation of promyelocytic HL-60 cells with the heme oxygenase-1 substrate and inducer, hemin, demonstrated that heme oxygenase-1 induction during neutrophilic differentiation could reduce oxidative burst capacity. These findings indicate that impairment of neutrophil oxidative burst activity in sickle cell disease is associated with hemolysis and heme oxygenase-1 expression. Neutrophil dysfunction might contribute to risk of infection in sickle cell disease, and measurement of neutrophil oxidative burst might be used to identify patients at greatest risk of infection, who might benefit from enhanced prophylaxis.

Associations between environmental factors and hospital admissions for sickle cell disease

Sickle cell disease is an increasing global health burden. This inherited disease is characterized by a remarkable phenotypic heterogeneity, which can only partly be explained by genetic factors. Environmental factors are likely to play an important role but studies of their impact on disease severity are limited and their results are often inconsistent. This study investigated associations between a range of environmental factors and hospital admissions of young patients with sickle cell disease in London and in Paris between 2008 and 2012. Specific analyses were conducted for subgroups of patients with different genotypes and for the main reasons for admissions. Generalized additive models and distributed lag non-linear models were used to assess the magnitude of the associations and to calculate relative risks. Some environmental factors significantly influence the numbers of hospital admissions of children with sickle cell disease, although the associations identified are complicated. Our study suggests that meteorological factors are more likely to be associated with hospital admissions for sickle cell disease than air pollutants. It confirms previous reports of risks associated with wind speed (risk ratio: 1.06/standard deviation; 95% confidence interval: 1.00–1.12) and also with rainfall (1.06/standard deviation; 95% confidence interval: 1.01–1.12). Maximum atmospheric pressure was found to be a protective factor (0.93/standard deviation; 95% confidence interval: 0.88–0.99). Weak or no associations were found with temperature. Divergent associations were identified for different genotypes or reasons for admissions, which could partly explain the lack of consistency in earlier studies. Advice to patients with sickle cell disease usually includes avoiding a range of environmental conditions that are believed to trigger acute complications, including extreme temperatures and high altitudes. Scientific evidence to support such advice is limited and sometimes confusing. This study shows that environmental factors do explain some of the variations in rates of admission to hospital with acute symptoms in sickle cell disease, but the associations are complex, and likely to be specific to different environments and the individual’s exposure to them. Furthermore, this study highlights the need for prospective studies with large numbers of patients and standardized protocols across Europe.

Are the risks of treatment to cure a child with severe sickle cell disease too high

Haematopoietic stem cell transplantation from alternative donors is curative for children with sickle cell disease, but risk of death is high. David Rees and colleagues call for greater caution in its use

G235 Prevalence Of Nocturnal Enuresis and Proteinuria In Children With Sickle Cell Disease and Its Relation To Severity Of Painful Crises

Nocturnal enuresis and albuminuria or proteinuria are markers of renal damage in sickle cell disease (SCD) and commonly develop early on in life. Proteinuria progresses with age, leading to chronic kidney disease in adulthood. The aims of this study were to identify the prevalence of enuresis and albuminuria/proteinuria in paediatric patients with SCD and to determine the relationship between these and various demographic and clinical variables. Methods A cross sectional study with qestionnaire-based interviews themed on nocturnal enuresis was undertaken for children with SCD. Results: Fifty-six patients were recruited to the study (27 females). Twenty patients (35.7%) met the DSM IV (Diagnostic and Statistical Manual of Mental Disorders, fourth edition) criteria for nocturnal enuresis compared to 5% prevalence in children in the general population. There was a significant association between enuresis and age, overactive bladder (OAB) symptoms, sleep-disordered breathing (SDB), painful crises and regular transfusions. Fourteen out of 29 patients (48.3%) with OAB symptoms reported nocturnal enuresis compared to six out of 27 patients (22.2%) who did not (p < 0.05). Of the patients reporting SDB, 48.6% were enuretic compared to 14.3% who did not (p < 0.01). Incidence of painful crises per month was higher for the enuretic group (2.29 vs. 0.88, p < 0.05). There were significantly more enuretic children in the 6–9 year age group (6/15) than the 1–15 and 16–17 year age groups (8/26 and 1/15 respectively, p < 0.05). Abstract G235 Table 1 Prevalence of albuminuria or proteinuria across patient characteristics (n = 52) Patient Albuminuria or proteinuria % P value Gender Male 2/28 10.7 Female 5/24 37.5 0.149 Age group 6–9 1/15 6.7 10–15 3/26 11.5 16–17 3/11 27.3 0.146 Hydroxycarbamide Yes 1/9 11.1 No 3/31 9.7 0.900 Regular blood transfusion Yes 3/12 25.0 No 3/31 9.7 0.193 Haematuria – or trace 2/45 4.4 +, ++, +++ 5/7 71.4 <0.001 BP Normotensive 5/41 12.2 Hypertensive 2/9 22.2 0.432 Current enuresis Yes 0/15 0.0 No 7/37 18.9 0.070 Conclusions Nocturnal enuresis and proteinuria are prevalent at an early age in many children with SCD. Early identification and initiation of treatment may delay onset of complications. Questioning parents on enuresis, OAB and SDB symptoms and undertaking regular urinalysis on younger age groups is a practical and cost-effective surveillance method.

The cause of sudden anemia revealed by the blood film

A three and a half-year-old girl of Indian ethnic origin presented with a 2-day history of fever and sore throat. She had also been passing red urine for the previous 24 hours. Her blood count showed: white cell count 27.5 x 10/l, red cell count 2.76 x 10/l, hemoglobin concentration 81 g/l, hematocrit 0.22 l/l, mean cell volume 80 fl, mean cell hemoglobin 29.6 pg, mean cell hemoglobin concentration (MCHC) 370 g/l. The increased MCHC suggested the presence of spherocytes, irregularly contracted cells or some other hyper-dense cells. A blood film confirmed the presence of numerous spherocytes and in addition was leucoerythroblastic and showed red cell agglutination and erythrophagocytosis by neutrophils (Figures). Other neutrophils had large vacuoles, similar in size to an erythrocyte, containing amorphous debris. There were also atypical lymphocytes, which appeared reactive. Lactate dehydrogenase and bilirubin were increased. The reticulocyte count was initially normal (46 x 10/l) but subsequently rose. The combination of marked spherocytosis, red cell agglutinates and erythrophagocytosis was considered strongly suggestive of paroxysmal cold haemoglobinuria (PCH) and confirmatory tests were performed. A direct antiglobulin test was positive for complement (111) and negative for immunoglobulin (Ig) G. A Donath – Landsteiner test was positive. Red cell transfusion was required and full recovery had occurred by 3 weeks. Although the Donath-Landsteiner antibody is an IgG antibody it is usual for the direct antiglobulin test in PCH to show complement only since the antibody that binds to the cell and fixes complement in the cold detaches from the red cell membrane on warming. Confirmation of the diagnosis is by demonstration of the presence of an anti-P antibody and biphasic haemolysis. However the blood film appearances are highly characteristic and permit a rapid presumptive diagnosis.

Congenital acute megakaryoblastic leukemia

A newborn baby girl, born to non-consanguineous Northern European parents, presented with an epistaxis at 10 days of age. She was found to have hepatosplenomegaly. There were no clinical features of Down syndrome. Her full blood count showed: white blood cell count 36 3 10/l, hemoglobin concentration 133 g/l, and platelet count 74 3 10/l. Her peripheral blood film (images) showed numerous blast cells with distinctive features. They had a high nucleocytoplasmic ratio and small indistinct nucleoli. The scanty cytoplasm varied from weakly to moderately basophilic and extended into irregular basophilic blebs and fronds. There were also some giant platelets. The cytological features were those of acute megakaryoblastic leukemia. Bone marrow aspiration was difficult. The hemodilute, aparticulate aspirate showed 18% blast cells. Cytogenetic analysis of aspirated bone marrow showed t(1;22)(p13;q13). This subtype of acute myeloid leukemia is associated with an RBM15-MKL1 fusion gene. It usually presents within the first 6 months of life and at least four congenital cases have been reported previously, in addition to a previous brief report of this case [1–3]. Occurrence in identical twins at the age of two months provides further evidence of an intrauterine origin [4]. The bone marrow blast cell percentage may be below 20%. Following several courses of combination chemotherapy, remission was achieved but the bone marrow remained hypocellular with low peripheral blood counts. Following sibling hematopoietic stem cell transplantation, she achieved long-term relapse-free survival.