Screening of subclinical functional hemoglobin and red blood cell abnormalities among blood donors of Fayoum University Hospital in Egypt: Are RET-He, and IRF useful screening tools?

BACKGROUND: The effectiveness of red cell transfusion in a given blood unit that relied on both quantity and quality of donated cells undoubtedly affects prognostic outcomes. OBJECTIVE: We aimed to determine the frequency of subclinical functional hemoglobin and red cell abnormalities in donated blood of Fayoum University Hospital in Egypt. Additionally, to assess the usefulness of reticulocyte mean hemoglobin content (RET-He) and immature reticulocyte fraction (IRF) as screening measures for such abnormalities. MATERIAL AND METHODS: This cross-sectional study enrolled 200 volunteer blood donors who met the national standard criterion of blood donation. Complete blood count with reticulocyte parameters, serum ferritin, sickling test, G6PD assay, Mentzer index, and naked-eye single tube red cell osmotic fragility test were carried out. RESULTS: Functional red cell abnormalities represented 44 % of this cohort. Out of them, 4.5 % had iron deficiency, 11 % had a positive sickling test, 19 % had G6PD deficiency, and 9.5 % had suspicious thalassemia. The sensitivity and specificity test for RET-He in selective identification of functional hemoglobin abnormalities in donated blood were 83.3 % and 61.2 %, respectively at a cutoff value of 26.9. Though there was no statistically significant effect of RET-He on the selective detection of G6PD deficiency, IRF had a statistically significant high level with a p-value of 0.04. CONCLUSION: Subclinical functional red cell abnormalities seem to be prevalent among blood donors. Reticulocyte/ erythrocyte indices could be useful screening tools for red cell abnormalities. Further studies are required for assessing the impact of transfusing such abnormalities to neonates and other critical recipients.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Infantile Pyknocytosis in a Premature Dichorionic Diamniotic Twin.

Infantile pyknocytosis is a rare and self-limiting cause of hemolytic anemia in neonates. It can result in severe anemia and hyperbilirubinemia. The pathogenesis is unknown: a genetic origin has been discussed; however, based on the current literature it is not clear which genetic mutations should be considered. We present a case of a premature twin, in whom genetic screening was performed. Genetic mutations in 46 genes associated with hereditary hemolytic anemia and dyserythropoietic anemia were tested. No mutations were found. In infantile pyknocytosis, a genetic defect in these genes is unlikely.

Autoimmune Liver Disease in Patients With Sickle Cell Disease.

OBJECTIVE: Hepatic and biliary tract diseases are common in sickle cell disease (SCD) patients, likely due to sickling, hemosiderosis, viral hepatitis, or cholelithiasis. Literature is lacking on associations between SCD, autoimmune hepatitis (AIH), and/or sclerosing cholangitis (SC)-together, autoimmune liver disease (AILD). We aimed to better understand the relationship of these diseases in pediatric patients. MATERIALS AND METHODS: A retrospective analysis of patients with SCD and AILD at the Children's Hospital of Philadelphia (January 2008 to August 2015). RESULTS: Seven patients, ages 8 to 23 years (3 males), were identified. Three had AIH, 2 SC, and 2 AIH/SC overlap, known as autoimmune SC. All patients with AIH treated with azathioprine significantly improved their liver enzymes. One patient with SC and inflammatory bowel disease underwent successful bone marrow transplant. Two SC patients died from SCD complications. CONCLUSIONS: In this cohort, there seems to be an association between SCD and AILD; SC in this population was severe. Physicians should be aware of this and evaluate patients with SCD and elevated liver enzymes for AILD.

The ADVIA2120i parameter Revised %MICRO is a surrogate marker of schistocyte formation after hematopoietic stem cell transplantation.

Objectives : Hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is an important early post-treatment condition. This study evaluated the Revised %MICRO, a parameter obtained from the ADVIA 2120i automated blood cell counter, as a surrogate marker of the schistocyte ratio. We hypothesized that individual differences between the %MICRO value and schistocyte ratio would remain constant. Design and Methods: EDTA-2K-treated peripheral blood samples were collected from 19 patients who underwent allogeneic HSCT from April 2014 to September 2018. First, the baseline difference, X, was calculated using a sample from the first day after HSCT as X = %MICRO (first day) - schistocyte ratio (first day). Next, the Revised %MICRO for each subsequent day was calculated as Revised %MICRO = %MICRO - X. We evaluated correlations of the schistocyte ratio with the calculated %MICRO and Revised %MICRO and the RBC fragment, RBC distribution width, %MICRO and Revised %MICRO data obtained from the ADVIA 2120i. Results : The mean schistocyte percentage and Revised %MICRO were both 0.4% ±â€…0.6. RBC fragments correlated weakly with the %MICRO and schistocyte ratio, respectively (r = 0.162 and r = 0.771, respectively), whereas the Revised %MICRO correlated strongly with the schistocyte ratio (r = 0.893). Conclusion : The Revised %MICRO appears to be a good surrogate of the schistocyte ratio in a clinical setting. J. Med. Invest. 67 : 250-254, August, 2020.

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene result in excessive extracellular Ca2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca2+ levels, promote major nuclear import of the Ca2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+ entry, highlighting that TAM and STRMK involve a common pathomechanism.

Hb Manitoba [α102(G9)Ser→Arg] in Pasifika: Tongan Case Report.

Hb Manitoba [α102(G9)Ser→Arg] results from an AGC>CGC or AGC>AGA substitution at codon 102 of the HBA1 or HBA2 genes. The variant is mildly unstable but carriers typically have normal clinical presentation and hematological profile. Hb Manitoba has not been reported in Pasifika of Tongan, Samoan or New Zealand (NZ) Maori descent before. The cases presented here support the findings from existing literature but include results from alternative methodology including capillary zone electrophoresis (CZE), which may slightly underestimate the true variant percentage. The subject of our case report, a Tongan male with microcytic indices, was shown to be heterozygous for Hb Manitoba III (HBA2: c.309C>A) coinherited with the -α3.7 (rightward) deletion.

Pathologic angiogenesis in the bone marrow of humanized sickle cell mice is reversed by blood transfusion.

Sickle cell disease (SCD) is a monogenic red blood cell (RBC) disorder with high morbidity and mortality. Here, we report, for the first time, the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vascular network of increased numbers of highly tortuous arterioles occupying the majority of the BM cavity, as well as fragmented sinusoidal vessels filled with aggregates of erythroid and myeloid cells. By in vivo imaging, sickle and control RBCs have significantly slow intravascular flow speeds in sickle cell BM but not in control BM. In sickle cell BM, we find increased reactive oxygen species production in expanded erythroblast populations and elevated levels of HIF-1α. The SCD BM exudate exhibits increased levels of proangiogenic growth factors and soluble vascular cell adhesion molecule-1. Transplantation of SCD mouse BM cells into wild-type mice recapitulates the SCD vascular phenotype. Our data provide a model of SCD BM, in which slow RBC flow and vaso-occlusions further diminish local oxygen availability in the physiologic hypoxic BM cavity. These events trigger a milieu that is conducive to aberrant vessel growth. The distorted neovascular network is completely reversed by a 6-week blood transfusion regimen targeting hemoglobin S to <30%, highlighting the plasticity of the vascular niche. A better insight into the BM microenvironments in SCD might provide opportunities to optimize approaches toward efficient and long-term hematopoietic engraftment in the context of curative therapies.

Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel ß-globin mutation.

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with ß-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the ß-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality.

Peripheral blood schistocytes in the acute phase after allogeneic or autologous stem cell transplantation assessed by digital microscopy.

INTRODUCTION: Early detection of endothelial graft-vs-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT) might help protect the patient from the late and severe complications of transplant-associated thrombotic microangiopathy (TA-TMA). Appearance of schistocytes in peripheral blood is one of the cardinal diagnostic features of TA-TMA. Our aim was to test the diagnostic accuracy and objectiveness of digital microscopy with respect to the recognition and quantification of schistocytes in the setting of endothelial GVHD. METHODS: Peripheral blood smears from 127 allogeneic and 63 autologous HSCT patients (January 2016-June 2017) were retrospectively examined before, 1 month and at 2-3 months after transplantation using digital microscopy. Peripheral blood smears from 31 healthy blood donors were also analyzed as the control group. RESULTS: Assessment by digital microscopy showed that schistocytes are significantly increased after 3 months from the allogeneic (P < .001) or the autologous HSCT (P < .001) compared to the control group. Significantly higher schistocyte counts were found in patients with acute GVHD (P = .05) and in patients with HLA mismatch (P = .005). Patients in the upper quartile of the schistocyte counts had significantly more frequently acute GVHD (P = .024) or HLA mismatch (P = .035). CONCLUSIONS: Schistocytes can be reliably counted by means of a digital microscopy system and are significantly higher in patients with acute GVHD and HLA mismatch. Inversely, patients with the highest numbers of schistocytes are more frequently affected by aGVHD, implying that high schistocyte counts after HSCT might be a surrogate marker for this complication.

Fetal hemoglobin and F-cell variance in mobilized CD34+ cell-transplanted rhesus monkeys.

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34+ cell transplantation. HbF protein expression peaked in the first 4-9 weeks posttransplant (0.99%-2.53%), and F-cells increased in the first 6-17 weeks posttransplant (8.7%-45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4-7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34+ cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development.

Metabolomic and molecular insights into sickle cell disease and innovative therapies.

Sickle cell disease (SCD) is an autosomal-recessive hemolytic disorder with high morbidity and mortality. The pathophysiology of SCD is characterized by the polymerization of deoxygenated intracellular sickle hemoglobin, which causes the sickling of erythrocytes. The recent development of metabolomics, the newest member of the "omics" family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic, and proteomic changes. Metabolomics changes respond faster to external stimuli than any other "ome" and are especially appropriate for surveilling the metabolic profile of erythrocytes. In this review, we summarize recent pioneering research that exploited cutting-edge metabolomics and state-of-the-art isotopically labeled nutrient flux analysis to monitor and trace intracellular metabolism in SCD mice and humans. Genetic, structural, biochemical, and molecular studies in mice and humans demonstrate unrecognized intracellular signaling pathways, including purinergic and sphingolipid signaling networks that promote hypoxic metabolic reprogramming by channeling glucose metabolism to glycolysis via the pentose phosphate pathway. In turn, this hypoxic metabolic reprogramming induces 2,3-bisphosphoglycerate production, deoxygenation of sickle hemoglobin, polymerization, and sickling. Additionally, we review the detrimental role of an impaired Lands' cycle, which contributes to sickling, inflammation, and disease progression. Thus, metabolomic profiling allows us to identify the pathological role of adenosine signaling and S1P-mediated erythrocyte hypoxic metabolic reprogramming and hypoxia-induced impaired Lands' cycle in SCD. These findings further reveal that the inhibition of adenosine and S1P signaling cascade and the restoration of an imbalanced Lands' cycle have potent preclinical efficacy in counteracting sickling, inflammation, and disease progression.

Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule.

Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar "stop-and-go" motion of sickle cell red blood cells on tumor factor-α-activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.