Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

Impaired cellular and humoral immunity is a feature of Diamond-Blackfan anaemia; experience of 107 unselected cases in the United Kingdom.

Diamond-Blackfan anaemia (DBA) is a rare bone marrow failure syndrome characterised by anaemia, congenital anomalies and cancer predisposition. Although infections are the second leading cause of mortality in non-transplanted patients, immune function is largely unexplored. We identified quantitative deficits in serum immunoglobulins and/or circulating T, natural killer and B lymphocytes in 59 of 107 unselected patients (55·1%) attending our centre over a 7-year period. Immune abnormalities were independent of ribosomal protein genotype and arose in both steroid-treated and steroid-untreated patients. In summary, these data highlight the high prevalence and spectrum of infections and immune defects in DBA.

Hydroxyurea therapy in UK children with sickle cell anaemia: A single-centre experience.

INTRODUCTION: Despite the demonstrated efficacy of hydroxyurea therapy, children with sickle cell anaemia in the UK are preferentially managed with supportive care or transfusion. Hydroxyurea is reserved for children with severe disease phenotype. This is in contrast to North America and other countries where hydroxyurea is widely used for children of all clinical phenotypes. The conservative UK practice may in part be due to concerns about toxicity, in particular marrow suppression with high doses, and growth in children. METHODS AND RESULTS: We monitored 37 paediatric patients with sickle cell anaemia who were treated with hydroxyurea at a single UK treatment centre. Therapy was well tolerated and mild transient cytopenias were the only toxicity observed. Comparative analysis of patients receiving ≥26 mg/kg/day versus <26 mg/kg/day demonstrates increasing dose has a significant positive effect on foetal haemoglobin (Hb; 29.2% vs. 20.4%, P = 0.0151), mean cell volume (94.4 vs. 86.5, P = 0.0183) and reticulocyte count (99.66 × 109 /l vs. 164.3 × 109 /l, P = 0.0059). Marrow suppression was not a clinical problem with high-dose treatment, Hb 92.25 g/l versus 91.81 g/l (ns), neutrophil count 3.3 × 109 /l versus 4.8 × 109 /l (ns) and platelet count 232.4 × 109 /l versus 302.2 × 109 /l (ns). Normal growth rates were maintained in all children. Good adherence to therapy was a significant factor in reducing hospitalisations. CONCLUSION: This study demonstrates the effectiveness and safety in practice of high-dose hydroxyurea as a disease-modifying therapy, which we advocate for all children with sickle cell anaemia.

Band 3 nullVIENNA , a novel homozygous SLC4A1 p.Ser477X variant causing severe hemolytic anemia, dyserythropoiesis and complete distal renal tubular acidosis.

We describe the second patient with anionic exchanger 1/band 3 null phenotype (band 3 nullVIENNA ), which was caused by a novel nonsense mutation c.1430C>A (p.Ser477X) in exon 12 of SLC4A1. We also update on the previous band 3 nullCOIMBRA patient, thereby elucidating the physiological implications of total loss of AE1/band 3. Besides transfusion-dependent severe hemolytic anemia and complete distal renal tubular acidosis, dyserythropoiesis was identified in the band 3 nullVIENNA patient, suggesting a role for band 3 in erythropoiesis. Moreover, we also, for the first time, report that long-term survival is possible in band 3 null patients.

Two Novel Missense Mutations and a 5bp Deletion in the Erythroid-Specific Promoter of the PKLR Gene in Two Unrelated Patients With Pyruvate Kinase Deficient Transfusion-Dependent Chronic Nonspherocytic Hemolytic Anemia.

We report two children with severe chronic hemolytic anemia, the cause of which was difficult to establish because of transfusion dependency. Reduced erythrocyte pyruvate kinase activity in their asymptomatic parents provided the diagnostic clues for mutation screening of the PKLR gene and revealed that one child was a compound heterozygote of a novel paternally derived 5-bp deletion in the promoter region (c.-88_-84delTCTCT) and a maternally derived missense mutation in exon nine (c.1174G>A; p.Ala392Thr). The second child was a compound heterozygote of two novel missense mutations, namely a paternally derived exon ten c.1381G>A (p.Glu461Lys) and a maternally derived exon seven c.907-908delCC (p.Pro303GlyfsX12) variant.

The Prevalence of Sickle Cell Disease and Its Implication for Newborn Screening in Germany (Hamburg Metropolitan Area).

Sickle cell disease is among hereditary diseases with evidence that early diagnoses and treatment improves the clinical outcome. So far sickle cell disease has not been included in the German newborn screening program despite immigration from countries with populations at risk. To determine the birth prevalence we tested 17,018 newborns. High pressure liquid chromatography and subsequent molecular-genetic testing were used for the detection and confirmation of hemoglobin variants. The frequency of sickle cell disease-consistent genotypes was one in 2,385 newborns. Duffy-blood group typing showed evidence that affected children were likely of Sub-Saharan ancestry. An inclusion of sickle cell disease into the German newborn screening seems reasonable.

Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease.

Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice. In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet. Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD.

Point-of-care end-tidal carbon monoxide reflects severity of hemolysis in sickle cell anemia.

Carbon monoxide (CO) production from heme catabolism is increased with hemolysis. A portable end-tidal CO (ETCO) monitor was used to analyze breath samples in 16 children with sickle cell anemia (SCA, 5-14 years). Median (range) ETCO for SCA was 4.35 ppm (1.8-9.7) versus 0.80 ppm (0.2-2.3) for controls (P < 0.001). ETCOc >2.1 ppm provided sensitivity and specificity of 93.8% (69.8-99.8%) for detecting SCA. ETCO correlated with reticulocytosis (P = 0.015) and bilirubin (P = 0.009), and was 32% lower in children receiving hydroxyurea (P = 0.09). Point-of-care ETCO analysis may prove useful for non-invasive monitoring of hemolysis and as a screening test for SCA.

Deviating from safety guidelines during deferiprone therapy in clinical practice may not be associated with higher risk of agranulocytosis.

BACKGROUND: A risk associated with the iron chelator deferiprone is the development of neutropenia or agranulocytosis. Accordingly, the product label recommends weekly blood monitoring and immediate interruption of treatment upon detection of an absolute neutrophil count (ANC) <1.5 × 10(9)/L, out of concern that continued therapy might lead to a more severe drop. However, it is uncertain how these recommendations are followed under real-life conditions and, if they are not followed, whether continuation of therapy results in increased incidence of agranulocytosis. PROCEDURE: This non-interventional surveillance program assessed the monitoring of deferiprone therapy in clinical practice. A total of 294 patients with transfusion-dependent anemias received deferiprone, as monotherapy or with another chelator, for up to 1 year. The participating physicians were not given any instructions about treatment and monitoring beyond being referred to the information in the package insert. RESULTS: ANC monitoring was conducted at an average interval of 5 ± 4 weeks, and deferiprone was not always interrupted upon detection of neutropenia. One patient (0.3%) experienced agranulocytosis, and nine others (3%) experienced a total of 11 episodes of neutropenia. All neutropenia episodes resolved; median time to resolution was similar whether or not treatment was interrupted; and no case of neutropenia progressed to agranulocytosis. CONCLUSIONS: These data indicate that less frequent ANC monitoring and continuation of deferiprone therapy during neutropenia are not associated with prolonged neutropenia or with progression to agranulocytosis.

Does glucose-6-phosphate dehydrogenase deficiency worsen the clinical features of sickle cell disease? A multi-hospital-based cross-sectional study.

BACKGROUND: The impact of glucose-6-phosphate dehydrogenase deficiency(G-6-PD) on the clinical course of sickle cell disease(SCD) is still controversial. The objectives of this study were to determine the prevalence of G-6-PD deficiency in patients with SCD and its effect on their clinical course. METHODS: A cross-sectional study of 122 SCD patients and 211 healthy blood donors was conducted in Kisangani city. Data were collected through clinical examination supplemented by patient medical records, and laboratory tests based on a survey form. G-6-PD activity was measured by spectrophotometry and the screening for SCD by the HemoTypeSC® rapid test. Statistical analysis was done using SPSS ver. 20.0. RESULTS: The prevalence of G-6-PD deficiency did not differ between SCD and non-SCD subjects, 35.2% vs. 33.6% respectively(p = .767). When comparing the hemoglobin level between SCD patients with and without G-6-PD deficiency, no significant difference was observed. However, in the 6 months prior to the study, SCD patients with G-6-PD deficiency had on average more transfusions than non-deficient SCD patients, 0.64 ± 0.897 vs. 0.24 ± 0.486(p = .004). Similarly, considering the clinical events of the last 12 months prior to the study, there were more hospitalizations, major vaso-occlusive crises and anemia requiring blood transfusion among G-6-PD deficient SCD patients compared to no-deficient, respectively 1.42 ± 1.451vs. 0.76 ± 1.112(p = .007); 1.37 ± 1.092 vs. 0.85 ± 1.014(p = .005); 0.74 ± 0.902 vs. 0.38 ± 0.739 (p = .007). CONCLUSION: The prevalence of G-6-PD deficiency in SCD patients was high but did not differ from that observed in controls. In addition, G-6-PD deficiency appeared to worsen the clinical features of SCD. Nevertheless, prospective studies further clarifying this observation are needed.

Synergistic Integration of Laboratory and Numerical Approaches in Studies of the Biomechanics of Diseased Red Blood Cells.

In red blood cell (RBC) disorders, such as sickle cell disease, hereditary spherocytosis, and diabetes, alterations to the size and shape of RBCs due to either mutations of RBC proteins or changes to the extracellular environment, lead to compromised cell deformability, impaired cell stability, and increased propensity to aggregate. Numerous laboratory approaches have been implemented to elucidate the pathogenesis of RBC disorders. Concurrently, computational RBC models have been developed to simulate the dynamics of RBCs under physiological and pathological conditions. In this work, we review recent laboratory and computational studies of disordered RBCs. Distinguished from previous reviews, we emphasize how experimental techniques and computational modeling can be synergically integrated to improve the understanding of the pathophysiology of hematological disorders.

Evaluation of OSMOCELLS, a new semi-automatic device for osmotic fragility assessment.

INTRODUCTION: The osmotic fragility (OF) test was a central test for the diagnosis of hereditary red blood cell (RBC) disorders (mostly hereditary spherocytosis (HS), but thalassaemia as well). Nowadays although the traditional multitubes method has lost a prominent place, many laboratories still perform such a laboured test, despite the lack of standardization. In fact, the evaluation of OF may offer an inexpensive screening for RBC disorders. We present a new semi-automatic device, allowing the continuous recording of OF, by an updated dialysis method. METHODS: Repeatability, stability over time, influence of the anticoagulant were evaluated among a population of healthy blood donors. The test was then performed among patients presenting inherited RBC disorders (HS or haemoglobinopathies) where OF is typically altered. RESULTS: Repeatability was excellent; the parameters were greatly influenced by the nature of the anticoagulant and interestingly appeared stable for 48 h. Patients with RBC disorders displayed the expected profile in regard with their disease: patients with HS all presented an increased OF while patients with haemoglobinopathy displayed resistant profiles. CONCLUSION: The device offers a substantial improvement in terms of standardization and consistency of the results and may offer a considerable gain for general laboratories.

A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review.

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue.