Laboratory predictors of sickle cell disease severity: a cross-sectional study.

Sickle cell disease (SCD) is the most common monogenic disorder, although the diversity and heterogenicity of clinical presentations render estimations of disease severity unpredictable. This cross-sectional study aimed to determine if laboratory markers could serve as indicators of SCD severity. We enrolled 90 adult patients with SCD with a mean age of 32.33 ± 11.84 years from the eastern province of Saudi Arabia, where SCD is more common than in other regions. Our study revealed a positive significant association between the number of hospitalizations and emergency visits with white blood cells (WBC) (R = 0.241, R = 0.207), respectively. Similarly, positive significant associations were found between the number of hospitalizations and emergency visits with platelets (R = 0.393, R = 0.276), respectively. Conversely, negative significant relationships were found between the number of hospitalizations and emergency visits (ER) with hemoglobin (Hb) F (R = -0.268, R = -0.263), respectively. Additionally, significant negative relationships were found between Hb F (R = -0.223) and the frequency of ICU admission. Only the number of hospitalizations and emergency visits annually were significantly predicted with P values of 0.021 and 0.038, respectively. Moreover, an increase in WBC was found to significantly increase the chance of undergoing splenectomy by 23.02%. SCD is a multisystemic disease with heterogeneous clinical presentations and disease severity. Inflammatory markers are valuable tools for better risk stratification and could be translated into developing new therapeutic strategies and modifying the treatment paradigm.

Lipid and hemolysis parameters predicting acute chest syndrome in adulthood with sickle cell disease.

Sickle cell disease (SCD) is a lifelong blood disorder affecting approximately 100,000 people in the United States and is one of the most common monogenic diseases. A serious complication of SCD is acute chest syndrome (ACS). ACS is a condition with a high rate of morbidity and mortality. The aim of the study was to assess hemolysis and lipid parameters in a cohort of confirmed SCD patients to predict ACS development in the following year.Standard lipid were performed (triglycerides, total cholesterol, high-density cholesterol, low-density cholesterol) panel to calculate of non-HDL-C, large buoyant LDL cholesterol (lbLDL-C) and small dense LDL cholesterol (sdLDL-C) with Sampson equation. Hemolysis and hematologic parameters were also evaluated.Among 91 patients included between September 2018 and June 2021, thirty-seven patients had history of ACS and 6 patients developed ACS during following year. In unadjusted logistic regression, total bilirubin was associated with ACS occurrence (RR: 1.2 [1.05-1.51] p = 0.013). Concerning lipid profile, non-HDL-C (RR: 0.87 [0.0.67-0.99] p = 0.04) and sdLDL-C (RR: 0.78 [0.49-0.96] p = 0.03) were associated with ACS occurrence decrease. C-reactive protein was associated with ACS occurrence (RR: 1.27 [1.065-1.85] p = 0.011).Based on these findings, this study demonstrated that several biomarker easily available can be used at steady state to predict ACS in the following year. The validation of these results are required to ensure the reproducibility of the findings.

Cardiac diastolic maladaptation is associated with the severity of exercise intolerance in sickle cell anemia patients.

This pilot study focusing on Sickle Cell Anemia (SCA) patients offers a comprehensive and integrative evaluation of respiratory, cardiovascular, hemodynamic, and metabolic variables during exercise. Knowing that diastolic dysfunction is frequent in this population, we hypothesize that a lack of cardiac adaptation through exercise might lead to premature increase in blood lactate concentrations in SCA patients, a potential trigger for acute disease complication. SCA patients were prospectively included in PHYSIO-EXDRE study and underwent a comprehensive stress test with a standardized incremental exercise protocol up to 4 mmol L-1 blood lactate concentration (BL4). Gas exchange, capillary lactate concentration and echocardiography were performed at baseline, during stress test (at ∼ 2 mmol L-1) and BL4. The population was divided into two groups and compared according to the median value of percentage of theoretical peak oxygen uptake (% V ˙ O 2 p e a k t h ) at BL4. Twenty-nine patients were included (42 ± 12 years old, 48% of women). Most patients reached BL4 at low-intensity exercise [median value of predicted power output (W) was 37%], which corresponds to daily life activities. The median value of % V ˙ O 2 p e a k t h at BL4 was 39%. Interestingly, diastolic maladaptation using echocardiography during stress test along with hemoglobin concentration were independently associated to early occurrence of BL4. As BL4 occurs for low-intensity exercises, SCA patients may be subject to acidosis-related complications even during their daily life activities. Beyond assessing physical capacities, our study underlines that diastolic maladaptation during exercise is associated with an early increase in blood lactate concentration.

Hydroxyurea dose optimisation for children with sickle cell anaemia in sub-Saharan Africa (REACH): extended follow-up of a multicentre, open-label, phase 1/2 trial.

BACKGROUND: Realizing Effectiveness Across Continents with Hydroxyurea (REACH) is an open-label non-randomised trial of hydroxyurea (hydroxycarbamide) in children with sickle cell anaemia in sub-Saharan Africa. The short-term results of REACH on safety, feasibility, and effectiveness of hydroxyurea were published previously. In this paper we report results from extended hydroxyurea treatment in the REACH cohort up to 8 years. METHODS: In this open-label, non-randomised, phase 1/2 trial, participants were recruited from four clinical sites in Kilifi, Kenya; Mbale, Uganda; Luanda, Angola; and Kinshasa, Democratic Republic of Congo. Eligible children were 1-10 years old with documented haemoglobin SS or haemoglobin Sß zero thalassaemia, weighing at least 10 kg. Participants received fixed-dose hydroxyurea of 17.5 (±2.5) mg/kg per day for 6 months (fixed-dose phase), followed by 6 months of dose escalation (2·5-5·0 mg/kg increments every 8 weeks) as tolerated, up to 20-35 mg/kg per day (maximum tolerated dose; MTD), defined as mild myelosuppression. After the MTD was reached, hydroxyurea dosing was optimised for each participant on the basis of changes in bodyweight and laboratory values over time (MTD with optimisation phase). After completion of the first 12 months, children with an acceptable toxicity profile and favourable responses were given the opportunity to continue hydroxyurea until the age of 18 years. The safety and feasibility results after 3 years has been reported previously. Here, haematological responses, clinical events, and toxicity rates were compared across the dosing phases (fixed-dose hydroxyurea vs MTD with optimisation phase) as protocol-specified outcomes. REACH is registered on ClinicalTrials.gov (NCT01966731) and is ongoing. FINDINGS: We enrolled 635 children between July 4, 2014, and Nov 11, 2016. 606 children were given hydroxyurea and 522 (86%; 266 [51%] boys and 256 [49%] girls) received treatment for a median of 93 months (IQR 84-97) with 4340 patient-years of treatment. The current (Oct 5, 2023) mean dose is 28·2 (SD 5·2) mg/kg per day with an increased mean haemoglobin concentration (7·3 [SD 1·1] g/dL at baseline to 8·5 [1·5] g/dL) and mean fetal haemoglobin level (10·9% [SD 6·8] to 23·3% [9·5]) and decreased absolute neutrophil count (6·8 [3·0] × 109 cells per L to 3·6 [2·2] × 109 cells per L). Incidence rate ratios (IRR) comparing MTD with fixed-dose hydroxyurea indicate decreased vaso-occlusive episodes (0·60; 95% CI 0·52-0·70; p<0·0001), acute chest syndrome events (0·21; 0·13-0·33; p<0·0001), recurrent stroke events (0·27; 0·07-1·06; p=0·061), malaria infections (0·58; 0·46-0·72; p<0·0001), non-malarial infections (0·52; 0·46-0·58; p<0·0001), serious adverse events (0·42; 0·27-0·67; p<0·0001), and death (0·70; 0·25-1·97; p=0·50). Dose-limiting toxicity rates were similar between the fixed-dose (24·1 per 100 patient-years) and MTD phases (23·2 per 100 patient-years; 0·97; 0·70-1·35; p=0·86). Grade 3 and 4 adverse events were infrequent (18·5 per 100 patient-years) and included malaria infection, non-malarial infections, vaso-occlusive pain, and acute chest syndrome. Serious adverse events were uncommon (3·6 per 100 patient-years) and included malaria infections, parvovirus-associated anaemia, sepsis, and stroke, with no treatment-related deaths. INTERPRETATION: Hydroxyurea dose escalation to MTD with dose optimisation significantly improved clinical responses and treatment outcomes, without increasing toxicities in children with sickle cell anaemia in sub-Saharan Africa. FUNDING: US National Heart, Lung, and Blood Institute and Cincinnati Children's Research Foundation.

Antioxidant supplementation for sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) refers to a group of genetic disorders characterized by the presence of an abnormal haemoglobin molecule called haemoglobin S (HbS). When subjected to oxidative stress from low oxygen concentrations, HbS molecules form rigid polymers, giving the red cell the typical sickle shape. Antioxidants have been shown to reduce oxidative stress and improve outcomes in other diseases associated with oxidative stress. Therefore, it is important to review and synthesize the available evidence on the effect of antioxidants on the clinical outcomes of people with SCD. OBJECTIVES: To assess the effectiveness and safety of antioxidant supplementation for improving health outcomes in people with SCD. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 15 August 2023. SELECTION CRITERIA: We included randomized and quasi-randomized controlled trials comparing antioxidant supplementation to placebo, other antioxidants, or different doses of antioxidants, in people with SCD. DATA COLLECTION AND ANALYSIS: Two authors independently extracted data, assessed the risk of bias and certainty of the evidence, and reported according to Cochrane methodological procedures. MAIN RESULTS: The review included 1609 participants in 26 studies, with 17 comparisons. We rated 13 studies as having a high risk of bias overall, and 13 studies as having an unclear risk of bias overall due to study limitations. We used GRADE to rate the certainty of evidence. Only eight studies reported on our important outcomes at six months. Vitamin C (1400 mg) plus vitamin E (800 mg) versus placebo Based on evidence from one study in 83 participants, vitamin C (1400 mg) plus vitamin E (800 mg) may not be better than placebo at reducing the frequency of crisis (risk ratio (RR) 1.18, 95% confidence interval (CI) 0.64 to 2.18), the severity of pain (RR 1.33, 95% CI 0.40 to 4.37), or adverse effects (AE), of which the most common were headache, nausea, fatigue, diarrhoea, and epigastric pain (RR 0.56, 95% CI 0.31 to 1.00). Vitamin C plus vitamin E may increase the risk of SCD-related complications (acute chest syndrome: RR 2.66, 95% CI 0.77 to 9.13; 1 study, 83 participants), and increase haemoglobin level (median (interquartile range) 90 (81 to 96) g/L versus 93.5 (84 to 105) g/L) (1 study, 83 participants) compared to placebo. However, the evidence for all the above effects is very uncertain. The study did not report on quality of life (QoL) of participants and their caregivers, nor on frequency of hospitalization. Zinc versus placebo Zinc may not be better than placebo at reducing the frequency of crisis at six months (rate ratio 0.62, 95% CI 0.17 to 2.29; 1 study, 36 participants; low-certainty evidence). We are uncertain whether zinc is better than placebo at improving sickle cell-related complications (complete healing of leg ulcers at six months: RR 2.00, 95% CI 0.60 to 6.72; 1 study, 34 participants; very low-certainty evidence). Zinc may be better than placebo at increasing haemoglobin level (g/dL) (MD 1.26, 95% CI 0.44 to 1.26; 1 study, 36 participants; low-certainty evidence). The study did not report on severity of pain, QoL, AE, and frequency of hospitalization. N-acetylcysteine versus placebo N-acetylcysteine (NAC) 1200 mg may not be better than placebo at reducing the frequency of crisis in SCD, reported as pain days (rate ratio 0.99 days, 95% CI 0.53 to 1.84; 1 study, 96 participants; low-certainty evidence). Low-certainty evidence from one study (96 participants) suggests NAC (1200 mg) may not be better than placebo at reducing the severity of pain (MD 0.17, 95% CI -0.53 to 0.87). Compared to placebo, NAC (1200 mg) may not be better at improving physical QoL (MD -1.80, 95% CI -5.01 to 1.41) and mental QoL (MD 2.00, 95% CI -1.45 to 5.45; very low-certainty evidence), reducing the risk of adverse effects (gastrointestinal complaints, pruritus, or rash) (RR 0.92, 95% CI 0.75 to 1.14; low-certainty evidence), reducing the frequency of hospitalizations (rate ratio 0.98, 95% CI 0.41 to 2.38; low-certainty evidence), and sickle cell-related complications (RR 5.00, 95% CI 0.25 to 101.48; very low-certainty evidence), or increasing haemoglobin level (MD -0.18 g/dL, 95% CI -0.40 to 0.04; low-certainty evidence). L-arginine versus placebo L-arginine may not be better than placebo at reducing the frequency of crisis (monthly pain) (RR 0.71, 95% CI 0.26 to 1.95; 1 study, 50 participants; low-certainty evidence). However, L-arginine may be better than placebo at reducing the severity of pain (MD -1.41, 95% CI -1.65 to -1.18; 2 studies, 125 participants; low-certainty evidence). One participant allocated to L-arginine developed hives during infusion of L-arginine, another experienced acute clinical deterioration, and a participant in the placebo group had clinically relevant increases in liver function enzymes. The evidence is very uncertain whether L-arginine is better at reducing the mean number of days in hospital compared to placebo (MD -0.85 days, 95% CI -1.87 to 0.17; 2 studies, 125 participants; very low-certainty evidence). Also, L-arginine may not be better than placebo at increasing haemoglobin level (MD 0.4 g/dL, 95% CI -0.50 to 1.3; 2 studies, 106 participants; low-certainty evidence). No study in this comparison reported on QoL and sickle cell-related complications. Omega-3 versus placebo Very low-certainty evidence shows no evidence of a difference in the risk of adverse effects of omega-3 compared to placebo (RR 1.05, 95% CI 0.74 to 1.48; 1 study, 67 participants). Very low-certainty evidence suggests that omega-3 may not be better than placebo at increasing haemoglobin level (MD 0.36 g/L, 95% CI -0.21 to 0.93; 1 study, 67 participants). The study did not report on frequency of crisis, severity of pain, QoL, frequency of hospitalization, and sickle cell-related complications. AUTHORS' CONCLUSIONS: There was inconsistent evidence on all outcomes to draw conclusions on the beneficial and harmful effects of antioxidants. However, L-arginine may be better than placebo at reducing the severity of pain at six months, and zinc may be better than placebo at increasing haemoglobin level. We are uncertain whether other antioxidants are beneficial for SCD. Larger studies conducted on each comparison would reduce the current uncertainties.

A miniaturized wash-free microfluidic assay for electrical impedance-based assessment of red blood cell-mediated microvascular occlusion.

The production of HbS - an abnormal hemoglobin (Hb) - in sickle cell disease (SCD) results in poorly deformable red blood cells (RBCs) that are prone to microcapillary occlusion, causing tissue ischemia and organ damage. Novel treatments, including gene therapy, may reduce SCD morbidity, but methods to functionally evaluate RBCs remain limited. Previously, we presented the microfluidic impedance red cell assay (MIRCA) for rapid assessment of RBC deformability, employing electrical impedance-based readout to measure RBC occlusion of progressively narrowing micropillar openings. We describe herein the design, development, validation, and clinical utility of the next-generation MIRCA assay, featuring enhanced portability, rapidity, and usability. It incorporates a miniaturized impedance analyzer and features a simplified wash-free operation that yields an occlusion index (OI) within 15 min as a new metric for RBC occlusion. We show a correlation between OI and percent fetal hemoglobin (%HbF), other laboratory biomarkers of RBC hemolysis, and SCD severity. To demonstrate the assay's versatility, we tested RBC samples from treatment-naïve SCD patients in Uganda that yielded OI levels similar to those from hydroxyurea (HU)-treated patients in the U.S., highlighting the role of %HbF in protecting against microcapillary occlusion independent of other pharmacological effects. The MIRCA assay could also identify a subset of HU-treated patients with high occlusion risks, suggesting that they may require treatment adjustments including a second-line therapy to improve their outcomes. This work demonstrates the potential of the MIRCA assay for accelerated evaluation of RBC health, function, and therapeutic effect in an ex vivo model of the microcapillary networks.

Evaluation of the effect of voxelotor and darbepoetin alfa on hemoglobin levels in patients with sickle cell disease.

BACKGROUND: To date, there is limited evidence on patients utilizing both voxelotor and darbepoetin alfa and its impact on hemoglobin levels. The objective is to evaluate the effect of voxelotor and darbepoetin alfa on hemoglobin levels in patients with SCD. RESEARCH DESIGN AND METHODS: This was a retrospective chart review study that assessed the primary independent variable as the utilization of either voxelotor alone, darbepoetin alfa alone, or the concurrent administration of voxelotor and darbepoetin alfa. Descriptive statistics were utilized to obtain the mean standard deviation for numerical variables and proportions for categorical variables. RESULTS: A total of 23 participants were included in this study. When comparing baseline to 2 months and 3 months, participants on voxelotor alone experienced a 3% decrease and a 6.6% increase in hemoglobin, darbepoetin alfa alone group a 4.3% decrease and a 0.6% increase in hemoglobin and voxelotor and darbepoetin group a 4.4% decrease and a 0.5% decrease in hemoglobin levels. Fifty percent of the participants in the voxelotor group and 6 (66.7%) participants in the voxelotor plus darbepoetin alfa group experienced adverse drug events. CONCLUSIONS: Voxelotor resulted in a clinically significant difference in the percent change of hemoglobin from baseline to 3 months.

Survival of transfused red blood cells from a donor with alpha-thalassemia trait in a recipient with sickle cell disease.

BACKGROUND: Post-transfusion survival of donor red blood cells (RBCs) is important for effective chronic transfusion therapy in conditions including sickle cell disease (SCD). Biotin labeling RBCs allows direct in vivo measurement of multiple donor RBC units simultaneously post-transfusion. STUDY DESIGN AND METHODS: In an observational trial of patients with SCD receiving monthly chronic transfusion therapy, aliquots of RBCs from one transfusion episode were biotin-labeled and infused along with the unlabeled RBC units. Serial blood samples were obtained to measure RBC survival. Donor units were tested for RBC indices, hemoglobin fractionation, and glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. For microcytic donor RBCs (MCV < 70 fL), HBA1 and HBA2 genetic testing was performed on whole blood. RESULTS: We present one recipient, a pediatric patient with SCD and splenectomy who received two RBC units with aliquots from each unit labeled at distinct biotin densities (2 and 18 µg/mL biotin). One donor unit was identified to have microcytosis (MCV 68.5 fL after biotinylation); whole blood sample obtained at a subsequent donation showed 2-gene deletion alpha-thalassemia trait (ɑ-3.7kb/ɑ-3.7kb) and normal serum ferritin. G6PD activity was >60% of normal mean for both. The RBCs with alpha-thalassemia RBC had accelerated clearance and increased surface phosphatidylserine post-transfusion, as compared with the normocytic RBC (half life 65 vs. 86 days, respectively). DISCUSSION: Post-transfusion RBC survival may be lower for units from donors with alpha-thalassemia trait, although the impact of thalassemia trait donors on transfusion efficacy requires further study.

Estimating the serological underrecognition of patients with weak or partial RHD variants.

BACKGROUND: For patients with weak or discrepant RhD RBC phenotypes, RHD genotyping is employed to determine need for RhD-negative management. However, many RHD variants are type D-negative or D-positive. Serological recognition rates (RRs) of weak and partial RHD variants are poorly characterized. STUDY DESIGN AND METHODS: Four US studies employing RHD genotyping for weak or discrepant RhD phenotypes provided data for race/ethnicity-specific serological recognition. Three studies used microplate, and 1 used gel and tube; 2 had anti-D data. We obtained White and Hispanic/Latino allele frequencies (AFs) of weak D types 1, 2, and 3 single-nucleotide variants (SNVs) from the Genome Aggregation Database (gnomAD, v4.0.0) and devised Hardy-Weinberg-based formulas to correct for gnomAD's overcount of hemizygous RHD SNVs as homozygous. We compiled common partial RHD AF from genotyped cohorts of US Black or sickle cell disease subjects. From variant AF, we calculated hemizygous-plus-homozygous genetic prevalences. Serological prevalence: genetic prevalence ratios yielded serological RRs. RESULTS: Overall RRs of weak D types 1-3 were 17% (95% confidence interval 12%-24%) in Whites and 12% (5%-27%) in Hispanics/Latinos. For eight partial RHD variants in Blacks, overall RR was 11% (8%-14%). However, DAR RR was 80% (38%-156%). Compared to microplate, gel-tube recognition was higher for type 2 and DAU5 and lower for type 4.0. Anti-D was present in 6% of recognized partial RHD cases, but only in 0.7% of estimated total genetic cases. DISCUSSION: Based on AF, >80% of patients with weak or partial RHD variants were unrecognized serologically. Although overall anti-D rates were low, better detection of partial RHD variants is desirable.

Prevention of potential delayed hemolytic transfusion reaction in two sickle cell patients using intravenous immunoglobulins and steroids before and after red blood cell exchange with antigen positive units and review literature.

Emergent Red Blood Cell (RBC) exchange is indicated in sickle cell disease (SCD) patients with severe acute chest syndrome. However, fully matched RBC units may not be available for patients with multiple RBC antibodies. Intravenous immunoglobulin (IVIG) and steroids were reported for preventing potential delayed hemolytic transfusion reaction (HTR) in simple transfusion of antigen-positive RBCs. We investigated the efficacy and safety of IVIG and steroids in two SCD patients presented with acute chest syndrome receiving RBC exchange with multiple incompatible units. The first patient had multiple historical alloantibodies, including anti-Jsb, although none of them were reactive. IVIG (1 g/kg) was given before and after RBC exchange with methylprednisolone (500 mg IV) one hour before exchange. Her sickle hemoglobin (HbS) was reduced from 89.4% to 17.4% after the exchange with five Jsb-positive units. The patient improved clinically without acute or delayed hemolysis. The second patient had reactive anti-Jsb on two different admissions 18 months apart. Only one of the sixteen units used in the exchanges was Jsb negative. He received the same IVIG regimen during both admissions but 100 mg IV hydrocortisone instead of methylprednisolone. His HbS was reduced from 63.4% to 22.4% after the first exchange. Significant clinical improvements were achieved after both exchanges. No delayed HTR was observed. Our experience of these two patients suggested that IVIG and steroids may be used in preventing potential delayed HTR in some SCD patients with rare antibodies receiving large amounts of antigen-positive RBC products.

Utility of hemoglobin electrophoresis to distinguish between severe delayed hemolytic transfusion reaction versus hyperhemolysis syndrome.

Delayed hemolytic transfusion reaction (DHTR) and hyperhemolysis syndrome (HHS) are both complications of red blood cell transfusions in patients with sickle cell disease.Clinically, both present with hemolysis and can be difficult to differentiate. Hemoglobin electrophoresis may aid in the diagnosis. Herein we describe a case in which a patient with hemoglobin SC disease presented with features of severe hemolysis several days after initiation of red blood cell exchange. Increase in reticulocyte count and complete absence of hemoglobin A on electrophoresis during this event supported the diagnosis of severe DHTR, indicating a rapid and selective destruction of the transfused red blood cells. Ability to interpret the hemoglobin electrophoresis can help clinicians distinguish between these two severe transfusion complications in patients living with sickle cell disease. It is important to identify the presence or absence of concomitant HHS, as patients with HHS tend to have a worse prognosis and there is a higher rate of recurrence of HHS with subsequent transfusions. Accurate diagnosis can lead to prompt management and decrease morbidity and mortality.

Thrombin activatable fibrinolysis inhibitor plasma levels and TAFI Thr325Ile genetic polymorphism in a cohort of Egyptian sickle cell disease patients and impact on disease severity.

BACKGROUND: Thrombin is a critical protease modulating thrombosis as well as inflammation, which are one of the main pathophysiological mechanisms in sickle vasculopathy, and its levels were reported to be high in sickle cell disease (SCD). The thrombin-thrombomodulin complex activates the TAFI inhibitor of fibrinolysis, which acts by reducing plasmin affinity for its substrate thus hindering fibrinolysis. OBJECTIVE: We aimed to determine the influence of the Thr325Ile single nucleotide polymorphism (SNP) on TAFI antigen levels and potential effects on the severity of SCD in a cohort of Egyptian patients. METHODS: Genotyping of Thr325lle polymorphism using Taq-Man SNP genotyping assay and TAFI level measurement using an enzyme-linked immunosorbent assay were performed for 80 SCD patients (45 homozygous HbSS, 16 S/ß0 and 19 Sß+) as well as 80 age- and gender-matched healthy control subjects. RESULTS: Plasma TAFI levels were higher in SCD patients with Thr325Ile polymorphism, yet the difference was not statistically significant (p = .204). SCD patients with polymorphic genotypes had a greater number of hospital admissions (p = .03). Ten patients with acute chest syndrome had the homozygous polymorphic genotype (GG), and all patients with pulmonary hypertension had the polymorphic genotype (six were homozygous [GG] and five were heterozygous [GA]). Patients with SCD complicated with pulmonary hypertension showed significantly higher plasma TAFI levels (p = .044). CONCLUSION: The analysis of Thr325Ile polymorphisms combined with plasma TAFI levels suggests that the analyzed SNP could influence plasma TAFL levels and SCD disease severity and hospitalization rates, which could be predictors for complex disease.

Post-transfusion biotin-labeled red blood cell survival studies in pediatric sickle cell disease with antibodies of uncertain significance.

BACKGROUND: Red blood cell (RBC) antibodies are common in multiply transfused patients with sickle cell disease (SCD). Unlike RBC alloantibodies, the potential of autoantibodies to cause post-transfusion hemolysis may be uncertain. Biotin-labeling provides a direct measurement of red cell survival (RCS) over time, thus can be used to assess the clinical significance of RBC antibodies. Antibodies to biotinylated RBC (B-RBC) occasionally are detected after exposure, which may impact B-RBC survival in subsequent RCS studies. STUDY DESIGN AND METHODS: Pediatric patients with SCD receiving monthly chronic transfusions underwent RCS studies, receiving aliquots of allogeneic RBC labeled at distinct densities of biotin (2-18 µg/mL). B-RBC survival was followed for 4 months post-transfusion, and B-RBC antibody screening for 6 months. Patients with warm autoantibodies (WAA) or B-RBC antibodies are reported here. RESULTS: RBC antibodies were detected during RCS in four patients: one with WAA, one with WAA followed by B-RBC-specific antibodies, and two with transient B-RBC antibodies within the first 5 weeks of exposure. B-RBC half-lives (T50) ranged 37.6-61.7 days (mean 47.8 days). There was no evidence of increased hemolysis or accelerated B-RBC clearance in the presence of WAA or B-RBC antibodies. DISCUSSION: Biotinylation of allogenic RBC can be used to assess the possible effects of RBC antibodies on transfusion survival in individual cases, particularly when it is uncertain if the detected antibodies may result in hemolysis. In the cases presented here, neither WAA nor B-RBC antibodies were associated with significant shortening of B-RBC survival in individuals with SCD.

Left ventricular global longitudinal strain and acute myocardial injury in patients with sickle cell disease admitted to the intensive care unit for vaso-occlusive crisis.

In patients with sickle cell disease (SCD), SCD-related cardiomyopathy may be partly due to repeated ischaemic events related to sickling during vaso-occlusive crises, but few clinical studies support this hypothesis. We evaluated the incidence of acute myocardial ischaemia during vaso-occlusive crises as assessed by the left ventricular global longitudinal strain (LVGLS) and high-sensitive cardiac troponin T (hs-cTnT). We included adult patients with SCD admitted to the intensive care unit (ICU) for vaso-occlusive crisis. We collected hs-cTnT and measured LVGLS with echocardiography at admission (day 1), day 2, day 3 and ICU discharge. Among 55 patients included, considering only the first hospitalization of patients admitted several times, 3 (5%) had elevated hs-cTnT at ≥1 time point of the ICU stay. It was ≤2 times the upper limit of normal in two of these patients. LVGLS was altered at ≥1 time point of the ICU stay in 13 (24%) patients. Both hs-cTnT and LVGLS were abnormal at ≥1 time point of the hospital stay in 2 (4%) patients. Acute myocardial injury as assessed by troponin elevation and LVGLS impairment was a rare event during vaso-occlusive crises.

Sickle cell disease iPSC-derived sensory neurons exhibit increased excitability and sensitization to patient plasma.

ABSTRACT: Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. Challenges to gaining mechanistic insight into pathogenic SCD pain processes include differential gene expression and function of sensory neurons between humans and mice with SCD, and extremely limited availability of neuronal tissues from patients with SCD. Here, we used induced pluripotent stem cells (iPSCs), derived from patients with SCD, differentiated into sensory neurons (SCD iSNs) to begin to overcome these challenges. We characterize key gene expression and function of SCD iSNs to establish a model to investigate intrinsic and extrinsic factors that may contribute to SCD pain. Despite similarities in receptor gene expression, SCD iSNs show pronounced excitability using patch clamp electrophysiology. Furthermore, we find that plasma taken from patients with SCD during acute pain associated with a vaso-occlusive event increases the calcium responses to the nociceptive stimulus capsaicin in SCD iSNs compared with those treated with paired plasma from patients with SCD at steady state baseline or healthy control plasma samples. We identified high levels of the polyamine spermine in baseline and acute pain states of plasma from patients with SCD, which sensitizes SCD iSNs to subthreshold concentrations of capsaicin. Together, these data identify potential intrinsic mechanisms within SCD iSNs that may extend beyond a blood-based pathology.

MALDI-MS in first-line screening of newborns for sickle cell disease: results from a prospective study in comparison to HPLC.

OBJECTIVES: Newborn screening (NBS) for sickle cell disease (SCD) requires a robust, high-throughput method to detect hemoglobin S (HbS). Screening for SCD is performed by qualitative methods, such as isoelectric focusing (IEF), and both qualitative and quantitative methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and tandem mass spectrometry (MS/MS). All these methods detect HbS, as well as low-level or absent HbA, and also other variants of hemoglobin. HPLC is considered as a reference method for NBS, because of its high sensitivity and specificity in detecting HbS. NeoSickle®, a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, combined with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation, has increased the throughput of SCD testing. The purpose of this study was to compare the performances of NeoSickle® and HPLC. METHODS: A prospective study was conducted including 9,571 samples from the NBS program to compare MALDI-MS using NeoSickle® with an HPLC method. Correlation between the two methods was studied. For the MALDI-MS method, sensitivity, specificity, NPV, and PPV were calculated. RESULTS: We found over 99.4 % correlation between the HPLC and MALDI-MS results. NeoSickle® showed 100 % of sensitivity and specificity in detecting SCD syndrome, leading to positive and negative predictive values of 100 %. CONCLUSIONS: NeoSickle® is adapted to NBS for SCD, and can be used in first-line high-throughput screening to detect HbS, and beta-thalassemia major warning. When HbS is detected, second-line use of another specific method as HPLC is necessary.

The influence of voxelotor on cerebral blood flow and oxygen extraction in pediatric sickle cell disease.

ABSTRACT: Voxelotor is an inhibitor of sickle hemoglobin polymerization that is used to treat sickle cell disease. Although voxelotor has been shown to improve anemia, the clinical benefit on the brain remains to be determined. This study quantified the cerebral hemodynamic effects of voxelotor in children with sickle cell anemia (SCA) using noninvasive diffuse optical spectroscopies. Specifically, frequency-domain near-infrared spectroscopy combined with diffuse correlation spectroscopy were used to noninvasively assess regional oxygen extraction fraction (OEF), cerebral blood volume, and an index of cerebral blood flow (CBFi). Estimates of CBFi were first validated against arterial spin-labeled magnetic resonance imaging (ASL-MRI) in 8 children with SCA aged 8 to 18 years. CBFi was significantly positively correlated with ASL-MRI-measured blood flow (R2 = 0.651; P = .015). Next, a single-center, open-label pilot study was completed in 8 children with SCA aged 4 to 17 years on voxelotor, monitored before treatment initiation and at 4, 8, and 12 weeks (NCT05018728). By 4 weeks, both OEF and CBFi significantly decreased, and these decreases persisted to 12 weeks (both P < .05). Decreases in CBFi were significantly correlated with increases in blood hemoglobin (Hb) concentration (P = .025), whereas the correlation between decreases in OEF and increases in Hb trended toward significance (P = .12). Given that previous work has shown that oxygen extraction and blood flow are elevated in pediatric SCA compared with controls, these results suggest that voxelotor may reduce cerebral hemodynamic impairments. This trial was registered at www.ClinicalTrials.gov as #NCT05018728.

The genetic dissection of fetal haemoglobin persistence in sickle cell disease in Nigeria.

The clinical severity of sickle cell disease (SCD) is strongly influenced by the level of fetal haemoglobin (HbF) persistent in each patient. Three major HbF loci (BCL11A, HBS1L-MYB, and Xmn1-HBG2) have been reported, but a considerable hidden heritability remains. We conducted a genome-wide association study for HbF levels in 1006 Nigerian patients with SCD (HbSS/HbSß0), followed by a replication and meta-analysis exercise in four independent SCD cohorts (3,582 patients). To dissect association signals at the major loci, we performed stepwise conditional and haplotype association analyses and included public functional annotation datasets. Association signals were detected for BCL11A (lead SNP rs6706648, ß = -0.39, P = 4.96 × 10-34) and HBS1L-MYB (lead SNP rs61028892, ß = 0.73, P = 1.18 × 10-9), whereas the variant allele for Xmn1-HBG2 was found to be very rare. In addition, we detected three putative new trait-associated regions. Genetically, dissecting the two major loci BCL11A and HBS1L-MYB, we defined trait-increasing haplotypes (P < 0.0001) containing so far unidentified causal variants. At BCL11A, in addition to a haplotype harbouring the putative functional variant rs1427407-'T', we identified a second haplotype, tagged by the rs7565301-'A' allele, where a yet-to-be-discovered causal DNA variant may reside. Similarly, at HBS1L-MYB, one HbF-increasing haplotype contains the likely functional small indel rs66650371, and a second tagged by rs61028892-'C' is likely to harbour a presently unknown functional allele. Together, variants at BCL11A and HBS1L-MYB SNPs explained 24.1% of the trait variance. Our findings provide a path for further investigation of the causes of variable fetal haemoglobin persistence in sickle cell disease.

Plasma sphingolipids in patients with sickle cell disease: Multiple-site vaso-occlusive crises could be associated with lower sphingolipid levels.

Although sickle cell disease (SCD) and its manifestations have been associated with various lipid alterations, there are a few studies exploring the impact of sphingolipids in SCD. In this study, we determined plasma ceramide (Cer) and sphingomyelin (CerPCho) species and investigated their association with the crisis in SCD. SCD patients (N = 27) suffering from vaso-occlusive crisis (VOC) or acute chest syndrome (ACS) were involved in this study. Blood samples were drawn at crisis and later at steady state periods. Clinical history, white blood cell count (WBC), C-reactive protein and lactate dehydrogenase (LDH) levels were recorded. 16:0, 18:0, 20:0, 22:0 Cer and 16:0, 18:0, 24:0 CerPCho were measured via LC-MS/MS. All measured Cer and CerPCho levels of SCD patients at crisis and steady-state were found to be similar. Inflammation-related parameters were significantly higher in patients with ACS compared to single-site VOC. Patients with multiple-site VOC were found to have significantly lower sphingolipid levels compared with those with single-site VOC, at crisis (16, 18, 24 CerPCho and 18, 22 Cer) and at steady-state (24:0 CerPCho and 18 Cer). Our results show that sphingolipid levels in SCD patients are similar during crisis and at steady state. However, lower sphingolipid levels appear to be associated with the development of multiple-site VOC. Since the differences were observed at both crisis and steady-state, sphingolipid level could be an underlying factor associated with crisis characteristics in patients with SCD.