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.

Mild COVID-19 and Impaired Blood Cell-Endothelial Crosstalk: Considering Long-Term Use of Antithrombotics?

BACKGROUND: Current coronavirus disease 2019 (COVID-19) pandemic reveals thrombotic, vascular, and endothelial dysfunctions at peak disease. However, the duration, degree of damage, and appropriate long-term use of antithrombotic strategies are unclear. Most COVID data are yielded from random clinical observations or autopsy of postmortem samples, while precise blood cellular data in survivors are insufficient. METHODS: We analyzed erythrocytes, circulating endothelial cells, and echinocytes by electron microscopy and flow cytometry in patients with confirmed COVID-19 (n = 31) and matched healthy controls (n = 32) on admission and at hospital discharge. RESULTS: All patients experienced mild disease, none required pulmonary support, and all survived. Admission number of circulating endothelial cells was significantly (40-100 times) higher in COVID-19 patients. Cells were massively damaged by multiple fenestrae in membranes with diameter comparable to the size of supercapsid in SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus. COVID-19 also provoked formation of stacked aggregated erythrocytes capable of clogging microvascular bed and of diminishing oxygen supply. In some patients, such abnormalities persisted at hospital discharge revealing remaining intracellular penetration of SARS-CoV-2 where it may be replicated and returned to circulation. CONCLUSION: These observational and descriptive data suggest that persistent viral cell injury may cause blood vessel damage; their increased permeability resulted in tissue edema, inflammation, platelet activation, and augmented thrombosis. There is a residual blood cell damage following the acute phase in some COVID-19 survivors. Controlled outcome-driven trials are urgently needed for exploring optimal use of long-term antithrombotics and vascular protection strategies even after mild COVID-19.

The relevance of macrocytosis induction during neoadjuvant dose-dense chemotherapy in breast cancer patients.

The aim of this study was to explore the red blood cell changes that occur during neoadjuvant dose-dense chemotherapy (NAC) of breast cancer. Also, we investigated the role of macrocytosis as a predictive biomarker for pathological complete response and disease-free survival (DFS) in these patients. A retrospective analysis of 82 breast cancer patients' data treated with anthracycline-cyclophosphamide-paclitaxel (AC-T) NAC in three oncology institutions in south Croatia from 2013 to 2020 was carried out. During chemotherapy mean corpuscular volume increased with time, with a median increase of 7.25 fl. Macrocytosis was induced in 38% of patients overall. Development of macrocytosis did not correlate with DFS [hazard ratio = 0.525; 95% confidence interval (CI), 0.074-3.768; P = 0.525]. Higher percentage of patients in macrocytosis group achieved PCR, 39% vs. 29% in no macrocytosis group, but this difference was not statistically significant. The relevance of macrocytosis induction during dose-dense neoadjuvant chemotherapy in breast cancer should be further explored.

Antisickling effect of chrysin is associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of functional chemistry and metabolic pathways of human sickle erythrocytes.

Sickle cell disease (SCD) treatment and management remain a challenging puzzle especially among developing Nations. Chrysin's sickling-suppressive properties in human sickle (SS) erythrocytes in addition to its effect on AA-genotype erythrocytes were evaluated. Sickling was induced (76%) with 2% sodium metabisulphite at 3 h. Chrysin prevented (81.19%) the sickling and reversed same (84.63%) with strong IC50s (0.0257 µM and 0.00275 µM, respectively). The levels of oxygenated haemoglobin in the two groups (before and after induction approaches) were similar but significantly (P < 0.05) higher than that of SS erythrocytes (the 'induced' control), with chrysin-treated AA-genotype showing no effects relative to the untreated. The level of deoxygenated haemoglobin in the 'induced' control group was significantly (P < 0.05) higher than those of the chrysin-treated SS erythrocytes. Normal and chrysin-untreated erythrocytes (AA-untreated) were significantly more resistant to osmotic fragility than the SS-untreated. However, treatment with chrysin significantly reduced the osmotic fragility of the cells relative to the untreated cells. Furthermore, chrysin treatment significantly lowers the high level of 2,3-diphosphoglycerate (2,3-DPG) observed in the sickle erythrocytes, with no effects on AA-genotype erythrocytes. Based on functional chemistry, chrysin treatment alters the functional groups in favour of its antisickling effects judging from the observed bends and shifts. From metabolomics analysis, it was observed that chrysin treatment favors fatty acid alkyl monoesters (FAMEs) production with concomitant shutting down-effects on selenocompound metabolism. Thus, sickling-suppressive effects of chrysin could potentially be associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of human sickle erythrocyte's functional chemistry and metabolic pathways implicated in SCD crisis.

Anti-sickling activities of two isolated compounds from the root of Combretum racemosum P. beauv. (Combretaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Evaluation of plants such as Combretum racemosum with claimed traditional use in the management of sickle cell anaemia in Nigeria and other parts of West Africa could serve as a useful research strategy in the search for potential anti-sickling drugs and templates. AIM OF THE STUDY: This study aimed at evaluating the antisickling potential of C. racemosum by activity-guided purification and isolation of its active constituents. MATERIALS AND METHODS: Crude methanol extract of the root of C. racemosum and the fractions obtained by partitioning with chloroform, ethyl acetate, and aqueous were investigated for anti-sickling activity against sodium metabisulphite induced sickling of sickle cell haemoglobin (HbSS). Repeated chromatographic separations were conducted on the most active chloroform fraction to purify and isolate bioactive compounds for further tests for anti-sickling activity. The characterization of the isolated compounds was done by mass spectrometry (FD+MS) and nuclear magnetic resonance (1HNMR) spectroscopy. RESULTS: The chloroform fraction (FA) (% sickled erythrocyte ranged from 3.0 to 34.1) exhibited better anti-sickling activity than aqueous (% sickled erythrocyte ranged from 38.9 to 51.5) as well as the crude methanol (% sickled erythrocyte ranged from 19.1 to 30.4). Hence, the phytochemical investigation was focused on the chloroform fraction, which led to the identification of two ellagic acid derivatives (3,3',4'-tri-O-methyl ellagic acid (A) and 3,3'-di-O- methyl ellagic acid (B). The two isolated compounds possessed good, comparable anti-sickling activities with compound A exhibiting a slightly better in vitro activity. CONCLUSION: This paper reports for the first time anti-sickling principles from C. racemosum and therefore, provided some justification for the ethnomedicinal use of the plant in the management of sickle cell disease.

Histoprotective effect of rutin against cisplatin-induced toxicities in tumor-bearing mice: Rutin lessens cisplatin-induced toxicities.

Cisplatin is an effective anticancer drug used against a variety of cancers. The full therapeutic potential of cisplatin is often hampered due to concurrent development of various side effects in the hosts. Rutin, a naturally occurring bioflavonoid shows several pharmacological activities. It has been earlier reported by us that rutin and cisplatin in combination show better antitumor activity against murine ascites Dalton's lymphoma. As cisplatin is given to cancer-bearing hosts only, the present study was undertaken to explore the histoprotective effect of rutin against some toxicities induced by cisplatin in tumor-bearing mice. Cisplatin treatment caused severe damages in tissue architecture such as degenerated hepatocytes with nuclear condensation and sinusoidal dilatation in the liver, glomerular deterioration, infiltration of cells, and tubular congestion in the kidney, and vacuolization of Sertoli cells or dense granules in the cytoplasm and damaged seminiferous tubules in the testes. In the rutin plus cisplatin combination-treated mice, all the abnormal tissue architectural features were decreased. Further, as compared to cisplatin treatment, combination treatment did not show any significant elevation in the liver functional biomarkers (serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase) and renal functional biomarkers (serum urea and creatinine levels). The combination treatment reduced the sperm abnormalities also as compared to the cisplatin alone treatment. The in vitro hemolysis assay of red blood cells and scanning electron microscopy revealed that combination treatment lessened the cisplatin-induced hemolysis and abnormalities in RBCs. Thus, the present findings demonstrate that rutin has histoprotective ability against cisplatin-induced toxicities in tumor-bearing mice.

VZHE-039, a novel antisickling agent that prevents erythrocyte sickling under both hypoxic and anoxic conditions.

Sickle cell disease (SCD) results from a hemoglobin (Hb) mutation ßGlu6 → ßVal6 that changes normal Hb (HbA) into sickle Hb (HbS). Under hypoxia, HbS polymerizes into rigid fibers, causing red blood cells (RBCs) to sickle; leading to numerous adverse pathological effects. The RBC sickling is made worse by the low oxygen (O2) affinity of HbS, due to elevated intra-RBC concentrations of the natural Hb effector, 2,3-diphosphoglycerate. This has prompted the development of Hb modifiers, such as aromatic aldehydes, with the intent of increasing Hb affinity for O2 with subsequent prevention of RBC sickling. One such molecule, Voxelotor was recently approved by U.S. FDA to treat SCD. Here we report results of a novel aromatic aldehyde, VZHE-039, that mimics both the O2-dependent and O2-independent antisickling properties of fetal hemoglobin. The latter mechanism of action-as elucidated through crystallographic and biological studies-is likely due to disruption of key intermolecular contacts necessary for stable HbS polymer formation. This dual antisickling mechanism, in addition to VZHE-039 metabolic stability, has translated into significantly enhanced and sustained pharmacologic activities. Finally, VZHE-039 showed no significant inhibition of several CYPs, demonstrated efficient RBC partitioning and high membrane permeability, and is not an efflux transporter (P-gp) substrate.

Inhibition of Band 3 tyrosine phosphorylation: a new mechanism for treatment of sickle cell disease.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

Quantitative prediction of erythrocyte sickling for the development of advanced sickle cell therapies.

Sickle cell disease is induced by a mutation that converts normal adult hemoglobin to sickle hemoglobin (HbS) and engenders intracellular polymerization of deoxy-HbS and erythrocyte sickling. Development of anti-sickling therapies requires quantitative understanding of HbS polymerization kinetics under organ-specific conditions, which are difficult to assess with existing experimental techniques. Thus, we developed a kinetic model based on the classical nucleation theory to examine the effectiveness of potential anti-sickling drug candidates. We validated this model by comparing its predictability against prior in vivo and in vitro experimental results. We used the model to quantify the efficacy of sickling inhibitors and obtain results consistent with recent screening assays. Global sensitivity analysis on the kinetic parameters in the model revealed that the solubility, nucleation rate prefactor, and oxygen affinity are quantities that dictate HbS polymerization. This finding provides quantitative guidelines for the discovery of intracellular processes to be targeted by sickling inhibitors.

Genotoxicity of textile dye C.I. Disperse Blue 291 in mouse bone marrow.

Color Index (C.I.) Disperse Blue 291 (DB291) is an azo dye used by the textile industry. After yarn dyeing, wastewater containing the dye, released into the aquatic environment, may pollute drinking water sources. We investigated the mutagenicity and genotoxicity of DB291 in male Swiss mice, following oral administration. Micronucleated cells, primary DNA damage (comet assay) in blood, liver, and kidney cells, and BAX, BCL2, SMAD4 and TNFA gene expression in leukocytes were evaluated. An increased frequency of micronucleated polychromatic erythrocytes (MNPCEs) was observed in animals treated with 50 mg/kg bw; no other genetic alteration was detected. Neither primary DNA damage nor changes in gene expression were observed.

Rheological properties of sickle erythrocytes in patients with sickle-cell anemia: The effect of hydroxyurea, fetal hemoglobin, and α-thalassemia.

OBJECTIVE: Determine the effect of fetal hemoglobin (HbF) and α-thalassemia on red blood cell (RBC) deformability of patients with sickle-cell anemia (SCA) with and without hydroxyurea (HU). METHODS: Adult patients were enrolled in the Sickle Cell Program of the Cardeza Foundation (Thomas Jefferson University) and were followed up prospectively during the period in which the Multicenter Study of Hydroxyurea (MSH) in patients with SCA was conducted. Ninety-one patients did not receive HU, 20 patients were enrolled in MSH, and 10 patients were enrolled in an open-label study of HU in SCA. Of the 20 patients enrolled in MSH, 11 took HU and nine took placebo. Control group included 113 normal individuals. Red blood cell deformability index (DI) was measured by ektacytometry. RESULTS: Patients with SCA taking HU (n = 21) had higher DI than those taking placebo (n = 9) or who were not taking this therapy (n = 91). In patients without therapy, those with α-thalassemia (n = 31) had higher DI than those without. We showed a significant positive correlation between the level of HbF and DI. SCA patients without α-thalassemia and HbF <10% (n = 48) had lower DI than patients with α-thalassemia and HbF <10% (n = 23) and patients with (n = 8) or without α-thalassemia but with HbF >10% (n = 12). DI measured in patients without α-thalassemia and HbF >10% was higher than in the three other subgroups. CONCLUSION: Elevated levels of HbF with or without HU and α-thalassemia improve sickle RBC rheology, which, in turn, improve the clinical picture of SCA.

Fragmentation of Red Blood Cells Caused Pseudothrombocytosis in a Patient.

BACKGROUND: Pseudothrombocytopenia, caused by platelet (PLT) clumping, is often found in clinical studies [1]. However, pseudothrombocytosis resulting from the fragmentation of red blood cells (RBCs) is a very rare phenomenon. METHODS: EDTA-K2 anticoagulation was used on a sample of venous blood extracted from the patient. A Symex XN9000 automatic blood analyzer was used to conduct CBC + DIFF mode and CBC + DIFF + RET mode tests, stained smear microscopy. RESULTS: The Symex XN9000 automatic blood analyzer was used to conduct CBC + DIFF mode test; PLTs were measured at 570 x 109/L. Stained smear microscopy revealed the number of PLTs did not conform to the instrument measured 570 x 109/L. "RET" alarm instrument, switch to CBC + DIFF + RET mode for testing. The second test result showed PLTs at 128 x 109/L, which accords with artificial microscopy. CONCLUSIONS: This was a case of a very rare phenomenon: the fragmentation of RBCs caused pseudothrombocytosis.

Is macrocytosis a potential biomarker of the efficacy of dose-dense paclitaxel-carboplatin combination therapy in patients with epithelial ovarian cancer?

The aims of this study were to investigate a clinical observation that patients with epithelial ovarian cancer treated with first-line platinum-paclitaxel chemotherapy combination (TP) develop macrocytosis and to explore the possible predictive role of macrocytosis in response rate, progression-free survival (PFS), and overall survival. A retrospective analysis of laboratory and clinical data on 184 consecutive ovarian cancer patients treated with first-line TP chemotherapy in a single oncology center from 2004 to 2015 was carried out. Macrocytosis was defined as an increase in mean corpuscular volume of peripheral red blood cells above 97.2 fl during the treatment and/or 30 days after the last chemotherapy cycle. One hundred and forty-one patients were treated with a conventional 3-weekly TP schedule, whereas 43 patients were treated with a dose-dense schedule. Macrocytosis was induced in 35% of patients overall. It was induced significantly more often in patients treated with the dose-dense schedule than in those treated with the 3-weekly schedule (67 vs. 26%, P=1.29×10). Macrocytosis did not correlate with PFS and overall survival in the overall patient population, nor in patients treated with the 3-weekly schedule. It correlated with PFS (hazard ratio=0.42, 95% confidence interval=0.18-0.94, P=0.036) and objective response on therapy in patients treated with the dose-dense schedule (P=0.0285). Dose-dense TP chemotherapy induces macrocytosis significantly more often than does a 3-weekly schedule in ovarian cancer patients. In patients treated with a dose-dense schedule, macrocytosis can potentially be predictive for longer PFS and better response rate. This finding needs further confirmation, preferentially in a prospective study.

The genotoxicity and cytotoxicity of tannery effluent in bullfrog (Lithobates catesbeianus).

Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.

Persistence of Macrocytosis after Discontinuation of Zidovudine in HIV-Infected Patients.

The duration of macrocytosis after stopping zidovudine (ZDV) is unknown. Among 104 HIV-infected patients treated with ZDV for more than 1 year, 84 patients had macrocytosis at ZDV discontinuation. The median mean corpuscular volume (MCV) was 114.6 fL (range 100-128 fL). Patients were divided into 2 groups: those who did (resolved macrocytosis, n = 36) and did not (persistent macrocytosis, n = 48) normalize MCV at 3 to 6 months after ZDV discontinuation. Alcohol use ( P = .02), smoking ( P = .03), and lower (but within normal range) folic acid levels ( P = .05) were related to the persistence of macrocytosis. A persistence of macrocytosis was observed in 57% at 3 to 6 months, 38% at 1 year and 37% at 2 years after ZDV therapy had stopped. Duration of ZDV therapy did not have an effect on the persistence of macrocytosis ( P = .73). The median time for the MCV to normalize after stopping ZDV was 12.5 months.

Macrocytosis during sunitinib treatment predicts progression-free survival in patients with metastatic renal cell carcinoma.

Sunitinib, a multi-targeted receptor tyrosine kinase inhibitor, is a first-line treatment for metastatic renal cell carcinoma (mRCC) in patients in 'low' and 'intermediate' Memorial Sloan Kettering Cancer Center and Heng risk groups. Disruptions of hematopoiesis, such as anemia, neutropenia, and thrombocytopenia, are typically observed during sunitinib treatment. When it comes to RBC parameters, an increase in mean cell volume (MCV) tends to occur, meeting the criteria for macrocytosis in some patients (MCV > 100 fL). We examined changes in RBC parameters of 27 mRCC patients treated with sunitinib (initial dose of 50 mg/day, 6-week treatment: 4 weeks on, 2 weeks off) and correlated them with progression-free survival time (PFS). Patients who had macrocytosis after 3 treatment cycles had significantly longer PFS than those whose MCV stayed less than 100 fL (not reached vs. 11.2 months, p < 0.001). We also found a correlation between MCV values after the first and third treatment cycles and the risk of progression: HR of 0.9 (0.81-0.99) and 0.76 (0.65-0.90) per 1 fL increase in MCV, respectively. The mechanism of MCV elevation during sunitinib treatment has not yet been fully explained. One of the probable causes is sunitinib's inhibitory influence on c-Kit kinase, as is the case with imatinib. For mRCC patients, this phenomenon could help predict PFS, but since our sample was small, further studies are essential.

GBT440 increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease.

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end-organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N-terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half-life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease-modifying agent in sickle cell patients.

Biological parameters predictive of percent dense red blood cell decrease under hydroxyurea.

BACKGROUND: Dense red blood cells (DRBCs) are associated with chronic clinical manifestations of sickle-cell-disease (SCD). Hydroxyurea (HU) decreases the percent (%) DRBCs, thereby improving its therapeutic benefits, especially the prevention of SCD clinical complications, but parameters influencing %DRBCs remain unknown. The purpose of this study was to determine predictive biological parameters of %DRBC decline under HU. METHODS: Factors affecting the %DRBC decrease in SCD patients HU-treated for ≥6 months were analyzed. Biological parameters and the %DRBCs were determined before starting HU and after ≥6 months of HU intake. Bivariate analyses evaluated the impact of each biological parameter variation on %DRBC changes under treatment. Multivariate analyses assessed the correlations between the decreased %DRBCs and biological parameters. RESULTS: The %DRBCs declined by 40.95% after ≥6 months on HU. That decrease was associated with less hemolysis, however in several analyses on this group of patients we did not find a statistically significant correlation between decrease in %DRBCs and increase in HbF. Initial %DRBC values were the most relevant parameter to predict %DRBC decline. CONCLUSION: Our results strengthen the known HU efficacy in SCD management statistically independently of the classical HbF biological response. Decreasing %DRBCs is essential to limiting chronic SCD symptoms related to DRBCs and predictive factors might help prevent those manifestations. The results of this study provide new perspectives on indication for HU use, i.e., to prevent SCD-induced organ damage.