Chronic Red Blood Cell Transfusion Research Articles

Grndad and Disease Modifying Therapy (DMT): Shifts in Dmt Are Seen at the Adolescent/Young Adult Transition in Sickle Cell Disease in a Multi-Site Prospective Registry

The ability to characterize the modern person living with SCD in the US has been limited by the lack of a well-curated longitudinal registry. The Globin Research Network for Data and Discovery ( GRNDaD) registry aims to overcome this challenge by collecting data from the now 53 IRB-approved centers across the US in collaboration with the National Alliance of Sickle Cell Centers (NASCC) and the HRSA-funded Sickle Cell Disease Treatment Demonstration Project Grant. Here, we describe the use of disease-modifying therapy in (actively consented) adults and children with Hgb SS/ SB 0 thalassemia (SCA) from 37 sites. Methods: Each site consents subjects and enters extensive baseline data including SCD complications and labs, subjects also complete patient-reported outcomes (PROs)-both at baseline and annually. Each subject is expected to have an annual follow-up where data is extracted from the Electronic Health Record (EHR). All data is stored in REDCap and, for this abstract, data were extracted into R on all active subjects (data entry in the last two years) who have complete minimum data collected. Median values were compared using Mann-Whitney U tests. Disease-modifying therapy (DMT) in this study comprises hydroxyurea (HU), chronic red blood cell (RBC) transfusions, crizanlizumab, L-glutamine, and voxelotor. Results: There are 2501 active patients in GRNDaD. Twenty-six percent of subjects are pediatric </= 18 years of age, 55.5% are female and 66.7% have HgbSS or HgbSB 0 thalassemia (SCA). Here, we report data on people with SCA only, whose minimum data were complete (n=1272). Table 1 shows DMT by age. 187 (21%) adults and 31 (8.5%) children were not on DMT. Figure 2 is a box plot of the absolute neutrophil count (ANC) comparing adults and children who are and are not taking HU. Adults on HU had significantly lower ANCs than those not on HU (median = 4.4 (IQR: 3.07, 6.3) v 6.6 (IQR:4.07, 8.14), p<0.001) (Figure). Similar findings were observed for children ( median = 3.5 (IQR:2.5, 5.7) Vs 8.1 (5.8, 12.8) , p =0.04) Both children and adults on HU had higher MCVs than those not on HU (median for peds: 91.5 (IQR: 85.1, 98.75) v 86 (IQR: 83.3, 86.1), p=0.05, median for adults: 96.5 (IQR: 89, 107) v 90 (IQR: 86.1, 95.1), p<0.001). We compared hemoglobin for those on and off HU, and on and off voxelotor, excluding patients on chronic transfusion therapy. There was a statistically significant difference in hemoglobin when comparing on or off of HU (median 8.9 g/dl (IQR 7.95, 9.9) v 8.2 g/dl (IQR: 7.2, 9.7, p =0.047)). There were no statistically significant differences in hemoglobin when comparing subjects on or off of voxelotor (median = 8.4 g/dl (IQR: 7.5, 9.6) vs 7.9 (IQR: 7.4, 9.8), p =0.57). In the adults, there were statistically significantly more males than females on HU, but there were no differences seen in the pediatric cohort. Examining DMT by age group, chronic RBC transfusion use doubled from the 11-17 age group to the 18-29-year age group (12% to 25%) and HU use decreased (83%-59%) over this same period. Limitations: This cohort may over-represent those on DMT as those enrolled in GRNDaD were more likely to have clinic visits. Discussion: GRNDaD has doubled the number of sites consenting subjects and entering data over the last year. The number is expected to increase again in the next 12 months, as an additional 15 sites are already IRB-approved with a goal to include all SCD centers in NASCC. This cross-sectional description of the current population of people living with SCD and treated in NASCC-recognized SCD centers in the US shows that the majority of those with SCA are receiving DMT. There is a noticeable decrease in HU use during the transition period with an increase in the use of chronic transfusion therapy. Newer DMT has had limited uptake in this cohort. The next steps will be to provide longitudinal data on this population and examine associations of DMT and important clinical outcomes including PROs.

Mitapivat Treatment Increases β-Thalassemic Erythroblasts Energy Production and Responsiveness to Oxidative Stress

β thalassemia (β thal) is a worldwide red blood cell (RBC) genetic disorder with limited therapeutic tools. Standard of care includes chronic RBC transfusions and iron chelation. In β thal, pathophysiologic studies have shown that oxidation plays a key role in both ineffective erythropoiesis and reduced survival of mature red cells in the peripheral circulation (Matte et al ARS 28: 1-14, 2018; Matte A et al. ARS 23: 1284, 2015). Recently, in a mouse model of β thal, we showed that the investigational pyruvate kinase (PK) activator mitapivat significantly improved anemia by targeting both ineffective erythropoiesis and hemolysis (Matte et al. JCI 131: e144206, 2021). We also found that mitapivat ameliorated the erythroid cell maturation index for in vitro CD34+ derived erythroblasts from β thal (cod b039) patients (Matte et al. JCI 131: e144206, 2021). The results of phase 2 proof-of-concept study (NCT03692052) in non-transfusion-dependent thalassemic patients support the beneficial effects of mitapivat on β thal (Kuo KHM et al Lancet 400: 493-501, 2022). Here, we study CD34+ derived erythroblasts from either healthy controls or β thal (cod b039) patients (n=10). Mitapivat (2uM) was added to the cell cultures as previously described (Matte et al. JCI 131: e144206, 2021). Compared to healthy controls, we found up-regulation of PKM gene expression in the late phase of β thal erythropoiesis (14 days of culture). Whereas the expression of PKLR gene declined during erythroid maturation in both healthy and β-thal erythroblasts. We found higher total PK activity in β thal erythroblasts than in healthy cells. Using recombinant PKR and PKM2 isoforms (R&D systems), we found that mitapivat activates both PKR and PKM2 (EC50 13.6±3.3 and 30.1±1.5, respectively) (Kung C et al Blood 2017). Studying erythropoiesis in vitro we confirmed that mitapivat significantly increased β thal erythroid maturation as supported by increased late-erythroblasts at 11 days of cell culture (Matte et al. JCI 131: e144206, 2021). The metabolomic analysis of β-thal erythroblasts at 14 days of culture confirmed that mitapivat activated PK, as gleaned by PGLY/DPG levels and increased fluxes through glycolysis, as inferred from steady-state measurements of glycolytic metabolites, DPG, ATP, and GTP. In addition, we observed higher levels of methionine oxidation in both mitapivat-treated healthy and β-thal erythroblasts, suggestive of a regulatory PTM effect secondary to higher ATP availability and, possible increased longevity of the cells. The improvement in the intracellular milieu by mitapivat is also supported by the downregulation of peroxiredoxin-2 (Prx2) and heme-oxygenase-1 (Hmox1) gene expression, which are up-regulated in response to oxidative stress and free heme accumulation in vehicle treated β-thal erythroblasts (Matte et al. ARS 23: 1284, 2015). Noteworthy, we also observed a reduction in heat shock protein-70 (Arlet JB et al Nature 514: 242, 2014) in mitapivat treated β-thal erythroblasts, further supporting the protective effect of mitapivat during β-thal erythropoiesis. In conclusion, our data show that both pklr and pkm isoforms are expressed in the late phase of β-thal erythropoiesis. The ability of mitapivat to activate both PKLR and PKM2 might represent an added value to limit oxidation during b-thal erythropoiesis, ensuring an improved β-thal erythroblasts maturation and survival.

Improvements in Health-Related Quality of Life after Exagamglogene Autotemcel in Patients with Transfusion-Dependent Beta-Thalassemia

Background: Transfusion-dependent β-thalassemia (TDT), an inherited hematological disorder which requires life-long, chronic red blood cell transfusion (RBC) and regular iron chelation therapy, has a substantial negative impact on health-related qualify of life (HRQoL). Exagamglogene autotemcel (exa-cel) is a one-time, non-viral, ex vivo CRISPR-Cas9 gene-edited cell therapy in phase 3 clinical trials that has been shown to eliminate the need for RBC transfusions and achieve transfusion independence. Here, we report HRQoL data from a pre-specified interim analysis of the exa-cel CLIMB TDT-111 study. Methods: CLIMB TDT-111 is an ongoing, 24-mo, phase 3 trial of a single dose of exa-cel in patients aged 12-35 years with TDT and a history of ≥100 mL/kg/year or ≥10 U/year of packed RBC transfusions for 2 years prior to screening. Changes in patient-reported outcome (PRO) measures including EuroQol Quality of Life Scale 5 dimensions 5 levels of severity (EQ-5D-5L, including descriptive system and visual analog scale [VAS]) and Functional Assessment of Cancer Therapy Bone Marrow Transplant (FACT-BMT, including FACT-General [FACT-G] and bone marrow transplant subscale [BMTS]) for adults and the EuroQol Quality of Life Scale 5 dimensions youth (EQ-5D-Y) and Pediatric Quality of Life Inventory (PedsQL) for adolescents were assessed through month 24 as a secondary endpoint in the trial. Data are presented as of 16 January 2023 for the 24 adults (aged ≥18-35 years) and 11 adolescents (aged ≥12 to < 18 years) who had been followed for ≥16 months after exa-cel infusion. Results: Clinically meaningful improvements, exceeding the minimal clinically important difference (MCID) thresholds, were observed in all PRO measures in adults. Baseline mean EQ-5D-5L health utility US index score (n=24, mean [SD]: 0.85 [0.18]) was near the general population norm and in line with baseline scores previously reported for adult patients with TDT. By month 24, both EQ-5D-5L health utility US index scores and EQ VAS scores showed substantial improvement (mean [SD] changes at month 24 [n=15]: 0.12 [0.26] and 10.2 [20.9] points; MCIDs 0.078 and 7 to 10, respectively). FACT-G Total Score improved from baseline by month 12 and was sustained through month 24 (mean [SD] change at month 24 [n=15] 10.3 [17.0] points; MCID 3 to 7), with improvements observed in all 4 subscales (physical, social/family, emotional, and functional well-being). BMTS score improved by month 12 and was sustained through month 24 (mean [SD] change at month 24 [n=15] 6.8 [4.7] points; MCID 2 to 3). For adolescents, EQ VAS scores improved through month 18 (mean [SD] change from baseline 4.8 [6.1] points). Total PedsQL score improved by month 6 and was sustained through month 18 (mean [SD] change from baseline [n=7] 14.1 [9.2] points; MCID 4.36). Physical functioning and psychosocial health scores, sub-components of PedsQL, both showed sustained improvement through month 18 (mean [SD] change [n=7] 19.2 [9.1] and 11.4 [10.9] points; MCID of 6.66 and 5.30 points, respectively). Improvement in psychosocial score was observed in all 3 subscales (social, emotional, and school functioning). Conclusion: Adults and adolescents infused with exa-cel reported sustained and clinically meaningful improvements in their HRQoL, with improvements observed across different instruments and domains, including physical, emotional, social/family and functional well-being and overall health status. These results demonstrate the broad clinical benefits of exa-cel in patients with TDT.

Going Going Gonad: Assessing Iron Staining Impact on Fertility for Patients with Sickle Cell Disease

Introduction: Medical advances in the care of patients with sickle cell disease (SCD) and other hemoglobinopathies have allowed for the focus of care to shift and incorporate long-term quality of life metrics, such as fertility. Concerns for reproductive health in patients with SCD stem from the underlying disease itself as well as from medical therapies that are offered such as hydroxyurea, chronic red blood cell transfusions, and stem cell transplant (SCT). Iron deposition from chronic transfusions in other tissues is known to result in end organ damage and lifelong consequences. While little is known about iron overload and fertility, one study noted testicular iron deposition on MRI in pubertal males with transfusion-dependent beta-thalassemia; results were notable for lower sperm concentrations and a lower percentage of sperm with normal morphology, indicating that iron overload likely has an impact on fertility for patients. Iron deposition has not been studied in the pre-pubertal hemoglobinopathy population. Fertility counseling in this patient population does not often occur until patients are considering SCT as a curative therapy option, as the conditioning regimens for SCT are gonadotoxic. Without a good understanding of the impact of chronic transfusions on fertility for patients with hemoglobinopathies, it is difficult to determine the optimal time to offer fertility preservation to families. We aim to assess iron overload on testicular tissue to gain a better understanding of the impact that medical management and chronic transfusions can have on fertility for a patient with SCD. Methods: Our study is a cross-sectional evaluation of patients with SCD who underwent testicular tissue cryopreservation (TTC) as a part of a multicenter research protocol through the University of Pittsburgh Medical Center (UPMC) prior to SCT. As part of the TTC protocol, each patient provided a small piece of tissue for research, with the remaining cryopreserved for patient use. All samples were evaluated for the presence of germ cells. For this study, demographics, treatment data, imaging results, and labs pertinent to hormonal profile and iron status were obtained. All samples from patients with SCD and thalassemia will undergo additional pathology review to evaluate for the presence of germ cells and iron. At the time of this abstract, the tissue for two patients had been analyzed. Results: Banked research tissue from UPMC includes 77 patients with SCD and 21 patients with beta thalassemia major. Of the 77 SCD patient samples, 42 were found to have germ cells, 34 have not yet been analyzed and 1 had no germ cells present. Of the 21 samples from patients with beta thalassemia major, 17 were found to have germ cells, 3 were not yet analyzed and 1 had no germ cells present. To date, we have obtained pathology review on two patients, both with Hb SS genotype and were on the maximum tolerated dose of hydroxyurea prior to TTC. Patient 1 had been on hydroxyurea for 7 years and had 13 units of blood transfusions, and Patient 2 on hydroxyurea for 4 years and 6units of blood transfusions prior to TTC. Both patients were found to have normal testicular tissue histopathology with presence of germ cells and without evidence of iron on staining (Table 1). Conclusions: Based on our preliminary data it can be noted that patients without significant iron burden, based on low ferritin and a limited transfusion history may not have a significant impact on their testicular histopathology with their iron staining being negative. We will expand our patient cohort to include patients who have a severe disease course requiring more blood transfusions to determine whether this is associated with increased iron deposition in their testicular tissue and/or reduced germ cell numbers. This information will allow teams to optimize the timing of fertility discussions and possible preservation in this patient population, even outside of curative treatment.

Optimizing RBC Transfusion Outcomes in Patients with Acute Illness and in the Chronic Transfusion Setting

Red blood cell (RBC) transfusion is a common clinical intervention used to treat patients with acute and chronic anemia. The decision to transfuse RBCs in the acute setting is based on several factors but current clinical studies informing optimal RBC transfusion decision making (TDM) are largely based upon hemoglobin (Hb) level. In contrast to transfusion in acute settings, chronic RBC transfusion therapy has several different purposes and is associated with distinct transfusion risks such as iron overload and RBC alloimmunization. Consequently, RBC TDM in the chronic setting requires optimizing the survival of transfused RBCs in order to reduce transfusion exposure over the lifespan of an individual and the associated transfusion complications mentioned. This review summarizes the current medical literature addressing optimal RBC-TDM in the acute and chronic transfusion settings and discusses the current gaps in knowledge which need to be prioritized in future national and international research initiatives.

Iron Load Toxicity in Medicine: From Molecular and Cellular Aspects to Clinical Implications.

Iron is essential for all organisms and cells. Diseases of iron imbalance affect billions of patients, including those with iron overload and other forms of iron toxicity. Excess iron load is an adverse prognostic factor for all diseases and can cause serious organ damage and fatalities following chronic red blood cell transfusions in patients of many conditions, including hemoglobinopathies, myelodyspasia, and hematopoietic stem cell transplantation. Similar toxicity of excess body iron load but at a slower rate of disease progression is found in idiopathic haemochromatosis patients. Excess iron deposition in different regions of the brain with suspected toxicity has been identified by MRI T2* and similar methods in many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Based on its role as the major biological catalyst of free radical reactions and the Fenton reaction, iron has also been implicated in all diseases associated with free radical pathology and tissue damage. Furthermore, the recent discovery of ferroptosis, which is a cell death program based on free radical generation by iron and cell membrane lipid oxidation, sparked thousands of investigations and the association of iron with cardiac, kidney, liver, and many other diseases, including cancer and infections. The toxicity implications of iron in a labile, non-protein bound form and its complexes with dietary molecules such as vitamin C and drugs such as doxorubicin and other xenobiotic molecules in relation to carcinogenesis and other forms of toxicity are also discussed. In each case and form of iron toxicity, the mechanistic insights, diagnostic criteria, and molecular interactions are essential for the design of new and effective therapeutic interventions and of future targeted therapeutic strategies. In particular, this approach has been successful for the treatment of most iron loading conditions and especially for the transition of thalassemia from a fatal to a chronic disease due to new therapeutic protocols resulting in the complete elimination of iron overload and of iron toxicity.

Cardiac iron overload in pediatric oncology and bone marrow transplant survivors.

e22022 Background: Cardiovascular late effects are a leading cause of mortality and morbidity in childhood cancer and Bone Marrow Transplant (BMT) survivors. In patients receiving chronic red blood cell transfusions, excess iron may be deposited in the liver, heart, and pituitary gland. Iron overload cardiomyopathy is characterized as a restrictive physiology with early diastolic dysfunction ultimately progressing to end-stage dilated cardiomyopathy. Iron loading in the heart can also lead to arrhythmias and increase myocardial ischemia reperfusion injury due to increased formation of reactive oxygen species. It is not yet known whether high transfused packed red blood cell (PRBC) volumes in cancer and BMT patients leads to cardiac iron deposition, and whether iron-induced cardiac injury contributes to the known cardiovascular late effects in these patients. Methods: 83 childhood cancer and BMT survivors with elevated serum ferritin levels and subsequent liver Magnetic Resonance (MR) imaging were identified. 18 patients (21.7%) who had liver iron content of > 12mg/g (mean 16.0 ± 7.36 mg/g) also underwent Cardiac MRI (CMR) to determine cardiac iron content. Results: Median transfused blood volume was 393 ml/kg [95% Confidence Interval (CI) 170-780 ml/kg]. All 18 patients had at least one echocardiogram showing normal ventricular ejection fractions [mean 65.1 ± Standard Deviation (SD) 3.53%] and normal shortening fractions (mean 35.4 ± 3.67%). All patients had myocardial iron content within normal ranges (T2* relaxation time mean 35.1 msec ± 7.1 [normal >20 msec], mean ± SD iron content 0.63± 0.2 mg/g dry weight, range 0.5-1.1). There was no association of cardiac iron content with transfused blood volume, anthracycline dose (mg/m2) or liver iron content (Pearson Correlation Coefficients 0.27, 0.29 and 0.89 respectively). Conclusions: Childhood cancer and BMT patients receive multiple PRBC transfusions to treat symptomatic anemia. It is currently unknown whether excess iron deposition in the heart contributes to the cardiac effects of impaired diastolic function and ultimately systolic dysfunction. In this study, 11 of 18 patients (61.1%) with high levels of liver iron had at least 1 other risk factor for the development of future cardiac dysfunction (either anthracycline exposure, radiation to the chest or total body irradiation or both). Larger studies are needed to determine whether oncology patients with high levels of iron deposition in their liver are also at risk for clinically significant cardiac iron loading, and what risk factors may exist for elevated myocardial iron content.

Modeling hemoglobin levels of patients chronically transfused with red blood cells.

Good models of chronic red blood cell (RBC) transfusion can improve care by accurately estimating time-dependent hemoglobin (Hb) levels and clinically relevant transfusion parameters. A previously derived mathematical model based on overall Hb mass balance (HMB model) with input parameters of transfused units, transfusion efficiency, RBC lifespan, endogenous Hb, and transfusion interval was applied to three separate clinical studies spanning six transfusion conditions across patient populations with myelodysplastic syndrome (MDS) or thalassemia. The HMB model accurately predicted mean pretransfusion Hb levels for each ensemble cohort of patients with thalassemia or MDS. Dynamic changes in Hb levels were modeled as a function of changes in key input parameters. Improving the 24-h post-transfusion RBC survival from 72% to 86% can be used to either (1) reduce RBC usage by 15%-20% through longer transfusion intervals or (2) increase pretransfusion [Hb] by 8%-11% while maintaining a constant transfusion interval. The endogenous Hb level is introduced in the HMB model which represents the patient's self-contribution to overall Hb levels via the autologous RBC lifecycle and was estimated to be 5.0 g/dL for patients with MDS or thalassemia. Transfusion therapy approaches and complementary therapeutics can target multiple, unique model inputs while monitoring net, overall impact on transfusion efficacy. Applying the HMB model to fit individual patient Hb fluctuations will be explored in the future.

Managing the Cerebrovascular Complications of Sickle Cell Disease: Current Perspectives.

The importance of protecting brain function for people with sickle cell disease (SCD) cannot be overstated. SCD is associated with multiple cerebrovascular complications that threaten neurocognitive function and life. Without screening and preventive management, 11% of children at 24% of adults with SCD have ischemic or hemorrhagic strokes. Stroke screening in children with SCD is well-established using transcranial Doppler ultrasound (TCD). TCD velocities above 200 cm/s significantly increase the risk of stroke, which can be prevented using chronic red blood cell (RBC) transfusion. RBC transfusion is also the cornerstone of acute stroke management and secondary stroke prevention. Chronic transfusion requires long-term management of complications like iron overload. Hydroxyurea can replace chronic transfusions for primary stroke prevention in a select group of patients or in populations where chronic transfusions are not feasible. Silent cerebral infarction (SCI) is even more common than stroke, affecting 39% of children and more than 50% of adults with SCD; management of SCI is individualized and includes careful neurocognitive evaluation. Hematopoietic stem cell transplant prevents cerebrovascular complications, despite the short- and long-term risks. Newer disease-modifying agents like voxelotor and crizanlizumab, as well as gene therapy, may treat cerebrovascular complications, but these approaches are investigational.

Understanding the Association between Red Blood Cell Transfusion Utilization and Humanistic and Economic Burden in Patients with β-Thalassemia from the Patients' Perspective.

We assessed the humanistic and economic burden of chronic red blood cell (RBC) transfusions on patients with β-thalassemia. This cross-sectional, US-based study included adults (≥18 years) who self-reported a β-thalassemia physician diagnosis and had received ≥1 RBC transfusion in the past 6 months. The outcomes included the Functional Assessment of Cancer Therapy-Anemia (FACT-An), Patient Health Questionnaire-9, Generalized Anxiety Disorder-7, and ad hoc questions about treatment experience, side effects, direct/indirect costs, and psychological burden. Overall, 100 patients completed the survey, of whom 70% experienced "moderate" to "extremely high" burden due to RBC transfusions, 81% reported iron overload, 42% reported compromised social lives. The mean FACT-An score was 132 (higher score indicates better outcomes; 0-188). Mean scores were 33/52 for fatigue and 20/28 for anemia symptoms in the previous 7 days. Health-related quality of life (HRQoL) temporarily improved after RBC transfusion, although patients continued to experience mild-to-severe depression and anxiety, substantial direct costs, compromised employment, and suboptimal quality of life. Over 6 months, patients dedicated a mean of 173 h to transfusion requirements and incurred out-of-pocket costs of USD 2239 for transfusions and USD 896 for additional care costs. These findings highlight the need for new treatment options to improve patient HRQoL and economic outcomes.

RBC Transfusion Strategy in Oncological Patients with Chronic RBC Transfusion Therapy.

Red blood cell (RBC) transfusion therapy has greatly reduced mortality and morbidity in multiply transfused patients with oncological malignancies. The aim of this study was to underline the necessity of introducing a policy for extended RBC phenotyping of these patients and for the issuing of antigen-matched blood (at least for E antigen). Multivariate logistic regression analysis was used to evaluate the associations of age, gender, transfusion history, and various malignancies with the development of red cell alloimmunization. Given the results of antibody identification, we finally obtained 732 cases to be analyzed, designating them as the p group. The respiratory system (231/732; 31.6%), digestive system (273/732; 37.3%), and female reproductive system (127/732; 17.3%) had the three highest alloimmunization rates in the p group. We screened 81 cases from the p group for which antibody screening in our laboratory had historically yielded negative results. Among the 81 cases with antibody seroconversion, anti-E was the most frequently observed antibody (37%). The results related to multivariate logistic regression analysis of the Rh group indicate that, in contrast to the other variates, transfusion confers a strongly increased risk of Rh blood system-related red cell alloimmunization. To reduce alloimmunization in tumor patients, it will be essential to introduce a policy for extended RBC phenotyping of high-risk patients and for the issuing of antigen-matched blood (at least for E antigen).

Animal models of Diamond-Blackfan anemia: updates and challenges.

Diamond-Blackfan anemia (DBA) is a ribosomopathy that is characterized by macrocytic anemia, congenital malformations, and early onset during childhood. Genetic studies have demonstrated that most patients carry mutations in one of the 20 related genes, most of which encode ribosomal proteins (RP). Treatment of DBA includes corticosteroid therapy, chronic red blood cell transfusion, and other forms of immunosuppression. Currently, hematopoietic stem cell transplantation is the only cure for DBA. Interestingly, spontaneous remissions occur in 10-20% of transfusion-dependent DBA patients. However, there is no consistent association between specific mutations and clinical manifestations. In the past decades, researchers have made significant progress in understanding the pathogenesis of DBA, but it remains unclear how the ubiquitous RP haploinsufficiency causes the erythroid-specific defect in hematopoiesis in DBA patients, and why there is a difference in penetrance and spontaneous remission among individuals who carry identical mutations. In this paper, we provide a comprehensive review of the development of DBA animal models and discuss the future research directions for these important experimental systems.

Transcranial Doppler (TCD) with Transfusions Changing to Hydroxyurea (TWiTCH) in Sickle Cell Disease: A Clinical Practice Implementation of Study Findings and Outcomes on Study Subjects

BACKGROUND: Stroke is one of the most devastating complications of sickle cell disease. The groundbreaking National Institutes of Health (NIH)-funded TWiTCH study established hydroxyurea as an effective non-inferior alternative to chronic red blood cell transfusions for primary stroke prevention in children with sickle cell disease. The study findings, published in 2016, established a protocol for transitioning patients from transfusions to hydroxyurea. It is important to evaluate how well the protocol is being implemented in clinical practice. OBJECTIVE: The main objective of this study was to investigate the outcomes of patients who were involved in the TWiTCH study at one academic medical center and the degree of adherence to the transition protocol for patients transitioning from transfusions to hydroxyurea. METHODS: A retrospective chart review of TWiTCH study subjects in one academic medical center. Institutional Review Board (IRB) exemption was obtained. Data was collected for the time from the end of the TWiTCH study (2014) to the most recent (2021). Data collected included: Demographics, Magnetic resonance imaging (MRA), Magnetic resonance angiography (MRA), Ferriscan, TCD, blood chemistries, Ferritin, blood counts, and stroke prevention intervention strategies. RESULTS: Eight TWiTCH subjects were identified, seven of whom had full evaluable data. All seven patients were stroke-free and had normal TCDs as of their most recent imaging. Four patients transitioned from chronic transfusions during the TWiTCH study to hydroxyurea post-study. One patient who was on hydroxyurea stayed on hydroxyurea while two patients who were on hydroxyurea transitioned to chronic transfusions. The mean current hemoglobin F was 8.3% compared with 27% reported by the TWiTCH study for all centers. Mean current ferritin was 1981ng/mL compared to 2238.6ng/mL at end of the TWiTCH study for subjects who are on hydroxyurea, 5943ng/mL, and 1123ng/mL respectively for patients currently on transfusions. The TWiTCH study reported ferritin of 2624ng/mL and 1226ng/mL for transfusion and hydroxyurea arms, respectively. The mean hydroxyurea dose was 24.5 mg/kg compared to 27mg/kg reported from the TWiTCH study. The mean hemoglobin was 7.9g/dL compared to 8.7g/dL at end of the TWiTCH study. CONCLUSIONS: Most patients from the TWiTCH study have been safely transitioned to hydroxyurea as part of standard clinical care at our institution. Adherence with dosing and laboratory targets was suboptimal likely due to patient noncompliance and less rigorous follow up schedule. Suboptimal compliance places patients at risk of not benefiting from all the advantages of transitioning to hydroxyurea identified on the TWiTCH study. Continued monitoring of these patients to identify any complications is warranted. Strategies to improve adherence to the transitioning protocols need to be developed.

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

The mouse double minute 4 (MDM4) negatively regulates p53 through the p53/MDM2/MDM4 regulatory network and is commonly overexpressed in several cancers. Here, we describe two unrelated pedigrees with germline MDM4 mutations with clinical features of bone marrow (BM) failure. Patient 1 (P1) presented at the age of 11 months with chronic pure red cell aplasia requiring chronic red blood cell transfusions and BM findings with absent erythroid precursors and dysplastic megakaryocytes. Patient 2 (P2) presented at age of 4 weeks with neutropenia with hypogranulation of neutrophils and a normocellular bone marrow with hypoplastic myelopoiesis and dysplasia of all three lineages. Both patients had normal chromosomal breakage and normal for age telomere length in peripheral blood. Whole genome and panel sequencing identified germline heterozygous variants in MDM4: p.Leu31Thrfs*15 in P1 and p.T454M in P2 as the only relevant candidate variants. Both mutations are absent in >140 thousand individuals in gnomAD database, and the constraint analysis indicates that MDM4 gene is intolerant to missense variation (observed to expected (O/E) ratio of 181/258, Z-score 1.71) and highly intolerant to protein truncating variation (O/E: 0/27 with a pLI score of 1). MDM4 protein consists of four key domains: p53 binding N-terminus, an acidic region, a zinc finger domain, and a C-terminal RING domain, which heterodimerizes with MDM2 to ubiquitinate and promote p53 degradation. MDM4 p.T454M missense mutation found in P2 was previously reported by Toufektchan et al. in 2020 in one patient with BM failure phenotype with short telomeres who carried additional TERT variant. The MDM4 p.T454M mutation was shown to result in lower protein levels (consistent with a loss-of-function effect) and had altered capacity to dimerize with the MDM2 RING domain, causing p53 hyperactivity. MDM4 p.Leu31Thrfs*15 mutation described in P1 is localized in the N-terminal p53 binding domain and results in RNA transcript loss, as confirmed by us in patient cells using RNA sequencing showing a two-fold reduction of the dominant transcript [ENST00000367182.8]. This was corroborated by Western blot demonstrating decreased levels of MDM4 protein. We sought to elucidate the impact of p.Leu31Thrfs*15 and p.T454M mutations on hematopoiesis and regulation of p53 expression. In two human induced pluripotent stem cell (iPSC) lines derived from male and female healthy donors, we knocked-in the MDM4 p.T454M and p.Leu31Thrfs*15 mutations by CRISPR/Cas9 engineering. Of note, MDM4 p.Leu31Thrfs*15 homozygous knock-in line could not be established despite multiple attempts. We discovered statistically significant decreased erythroid and myeloid differentiation capacity from MDM4-mutant iPSCs compared to isogenic control iPSCs (p <0.05). There were statistically significantly more AnnexinV+/DAPI+ mutant iPSC-derived myeloid and erythroid progenitors compared to isogenic control-derived progenitors (p <0.05), pointing to a higher rate of cell death in MDM4-mutant cells. We further demonstrated increased expression of p53 and p21 proteins in MDM4 p.Leu31Thrfs*15 iPSC clones. To better understand the loss-of-function effect of MDM4, we knocked out MDM4 in healthy CD34+ cells using CRISPR/Cas9 editing. Knock out of MDM4 with an efficiency of >80% was achieved in multiple biological replicates, resulting in increased p53 activity and fewer blast-forming erythroid (BFU-E) and myeloid colonies compared to control cells that were Cas9-edited at the AAVS1 safe harbor locus. This impairment was rescued by disruption of TP53 gene. Sequencing of individual BFU-E colonies did not reveal any cells with biallelic loss of MDM4, while similar percentages of unedited colonies and heterozygous disrupted MDM4 colonies were detected. Xenotransplantation studies using immunodeficient NBSGW mice are currently underway to explore the impact of MDM4-edited CD34+ cells in vivo. In summary, we describe two patients with germline loss-of-function MDM4 mutations that cause p53 hyperactivity which causes the unifying feature of hematopoietic defect across a BMF disorder spectrum without telomere shortening. Sharma and Bhoopalan are equal contributors

The Areal Project: How Virtual Reality Application Could Enhance Patient's Quality Time during Transfusion Therapy in Adult Patients with Thalassemia and Sickle Cell Disease

Background Chronic red blood cell transfusion therapy (CTT) is an integral component of the management of Transfusion Dependent Thalassemia (TDT) and Sickle Cell Disease (SCD). CTT requires time and significantly impacts the quality of life of patients with TDT and SCD. In some cases, the environment in which transfusions are performed is uncomfortable and doesn't offer adequate spaces and facilities. And as a result, the patient experiences a condition of discomfort and tries with his own means to get distracted or "estranged". Hence, attention should be paid to psychological, emotional and social needs of patients on CTT in order to reduce the burden of the procedure with activities that are capable of engaging more the person. Aims From this need and the opportunities offered by the digital technologies available today, the AREAL project was designed by the Italian startup Softcare Studios in partnership with the pharma company Novartis and in collaboration with hemoglobinopathy specialized medical staff. Objectives of this pilot project were: i) collect feedback from patients with TDT and SCD and from healthcare professional workers regarding utility and feasibility of AREAL; ii) detect and evaluate changing in states of patient's anxiety and depression. Methods AREAL consists of three gaming activities enjoyed in virtual reality thanks to the use of suitable Virtual Reality (VR) viewers (Figure 1A) developed to immerse the patient in a sensorial stimulating scenario and designed to stimulate cognitive skills: activity 1 for short-term memory, activity 2 for task switching, activity 3 for problem solving. AREAL also integrates elements of patient education in therapy and recommended lifestyle habits to promote their well-being. Anxiety and depression were assessed by a self-assessment questionnaire, the Hospital Anxiety and Depression Scale (HADS), administered after each virtual session. Results The pilot group was composed by 70 subjects on CTT followed in 3 hospital centers in Italy (Genova, Napoli, Padova). The 57% were males, median age was 31 years (IQR: 21-40), 53 patients were affected by TDT and 6 by SCD. The total number of sessions collected is 129; 59 patients experienced two sessions and were included in the analysis for HADS. The average duration of game sessions lasted 17 minutes.Activity 1 was the most played and most rarely interrupted, followed by 2 and finally by 3, where 50% of sessions are been interrupted due to its difficulty. In Activity 1 patients recognized the goal of the game, which is to stimulate memory, and they liked its immediacy. Excessive facility, especially considering the repetition over time during the therapy, was emphasized by patients who suggested making the game more complicated. Compared to Activity 1, Activity 2 is more complicated and engaging, emerging as the overall favorite of the study patients. Undoubtedly, Activity 3 emerged as the most difficult one, both to play and to understand, also given its focus on problem solving. Based on the feedback from healthcare professionals learning to use the virtual application wasn't particularly difficult. The experience of the staff confirmed some of the critical issues also communicated by patients (such as the high difficulty of the third game activity and request for greater variability over time).Anxiety and depression were detected by HADS respectively in 13 and in 9 pts after first session, in 10 and in 4 pts after second session, suggesting a possible benefit of VR; the number of patients with borderline profile reduced from 8 to 3 both for anxiety and depression (Figure 1B). There were no significant differences of scores and/or percentages between sessions. Conclusions There is a significant need to iterate AREAL on the basis of different age groups, each with specific needs, preferences and level of confidence with technologies. From the collected feedback, AREAL was perceived as an innovative project and an opportunity to pay attention to the critical issues of the patients' therapeutic experience on CTT. However, it is needed to optimize the experience in order to free the operator from extra commitments as much as possible that could impact the quality of hospital work routine. AREAL seems to be an opportunity to reduce discomfort and anxiety during CCT in the subset of pts with a profile borderline for anxiety and depression but needs to tailor the intervention to specific subsets of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cognitive Dysfunction Worsens with Age in Patients with Sickle Cell Disease in the Modern Treatment Era

Introduction Patients with sickle cell disease (SCD) suffer from cognitive dysfunction compared to those unaffected by the disease. Such cognitive dysfunction in patients with SCD is further exacerbated by the occurrence of overt stroke or silent cerebral infarcts (SCIs). Fortunately, treatments such as hydroxyurea and red blood cell (RBC) transfusion for patients with SCD have been shown to be neuroprotective. Studies that predate guidelines recommending all patients with HbSS or HbS beta thalassemia null genotypes receive primary disease modification with hydroxyurea report that cognitive dysfunction worsens with age in patients with SCD. However, there is limited investigation into cognitive trajectories across the lifespan during the modern treatment era for patients with SCD. While neuroprotective interventions are widely recommended, we hypothesize that patients with SCD will continue to experience cognitive decline. Methods Patients >5 years of age with any genotype of SCD, excluding those receiving chronic RBC transfusions, were included in these analyses. Cognitive data, as measured by the NIH Toolbox-Cognitive Battery (NIHTB-CB) and the Wechsler Abbreviated Scale Intelligence 2nd edition (WASI-II), were combined from three prospective research studies at a single institution. A subgroup of participants had multiple timepoints of data collected. Multivariate general linear mixed-level models, accounting for multiple timepoints per participant, were used to predict each cognitive subdomain while controlling for the independent variables of age and sex. Age squared (age2) was included in models if necessary to account for a second-order polynomial fit. Significance was defined as α<0.05 and the Benjamini-Hochberg procedure was used to correct for multiple comparison. Results Data was collected from 123 participants with SCD between 2014-2022. The cohort was 45.4% male (56/123 participants). Thirty-four participants had 2 timepoints and 7 participants had 3 timepoints of cognitive data included in the analyses, for 164 total timepoints. First and last-timepoints were separated by 1-4 years for the NIHTB-CB and 1-8 years for the WASI-II tests. The average (SD, range) number of months between the first and last encounters was 31.2 (10.9, 14.2-47.7) for completion of NIHTB-CB testing and 43.9 (22.2, 14.2-89.3) for completion of WASI-II testing. Table 1 describes the cohort. The average (SD, range) hemoglobin at all timepoints was: 9.0 (1.6, 5.6-13.3). The number (percent) of participants taking hydroxyurea and the mean (SD, range) dosage for all timepoints was: 97/164 (59.1%), 380.9mg (649.4, 11.0-2000.0). Using multivariate general linear mixed-level models to predict each cognitive subdomain and composite score provided in the NIHTB-CB and WASI-II, the Flanker Inhibitory Control and Attention (age p=0.0164), Oral Reading (age p=0.0006, age2 p=0.0018), Pattern Comparison Processing Speed (age p=0.0210, age2 p=0.0206, sex p=0.0041), Crystallized Composite (age p=0.0038, age2 p=0.0063) and Total Composite (age p=0.0215, age2 p=0.0219) scores decreased with age while controlling for the effect of sex (Table 1, Figure 1). There was not a significant relationship between age and cognitive outcome measure for the Picture Vocabulary, List Sorting Working Memory, Dimensional Change Card Sorting, Picture Sequence Memory, Fluid Composite, Matrix Reasoning or Full-Scale IQ tests. Conclusion We conclude that cognitive abilities, encompassing executive function abilities (Flanker Inhibitory Control and Attention Test), non-executive function abilities (Oral Reading Test and Crystallized Composite) and processing speed (Pattern Comparison Processing Speed Test), decrease as patients with sickle cell disease age during the modern treatment era. Further analyses are required to determine the impact of age on cognitive outcomes while controlling for the presence of infarction, primary disease modification, hemoglobin and additional biologic covariates. These preliminary analyses support further investigation into the pathophysiology of SCD on both brain development and aging, and consideration of neurocognitive outcomes in the development of new therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of Blood Transfusion on Cerebral Hemodynamics and Vascular Topology Described by Computational Fluid Dynamics in Sickle Cell Disease Patients.

The main objective of this study was to demonstrate that computational fluid dynamics (CFD) modeling can be used to study the contribution of covert and overt vascular architecture to the risk for cerebrovascular disease in sickle cell disease (SCD) and to determine the mechanisms of response to therapy such as chronic red blood cell (cRBC) transfusions. We analyzed baseline (screening), pre-randomization and study exit magnetic resonance angiogram (MRA) images from 10 (5 each from the transfusion and observation arms) pediatric sickle SCD participants in the silent cerebral infarct transfusion (SIT) trial using CFD modeling. We reconstructed the intracranial portion of the internal carotid artery and branches and extracted the geometry using 3D Slicer. We cut specific portions of the large intracranial artery to include segments of the internal carotid, middle, anterior, and posterior cerebral arteries such that the vessel segment analyzed extended from the intracranial beginning of the internal carotid artery up to immediately after (~0.25 inches) the middle cerebral artery branching point. Cut models were imported into Ansys 2021R2/2022R1 and laminar and time-dependent flow simulation was performed. Change in time averaged mean velocity, wall shear stress, and vessel tortuosity were compared between the observation and cRBC arms. We did not observe a correlation between time averaged mean velocity (TAMV) and mean transcranial Doppler (TCD) velocity at study entry. There was also no difference in change in time average mean velocity, wall shear stress (WSS), and vessel tortuosity between the observation and cRBC transfusion arms. WSS and TAMV were abnormal for 2 (developed TIA) out of the 3 participants (one participant had silent cerebral infarctions) that developed neurovascular outcomes. CFD approaches allow for the evaluation of vascular topology and hemodynamics in SCD using MRA images. In this proof of principle study, we show that CFD could be a useful tool and we intend to carry out future studies with a larger sample to enable more robust conclusions.

EVALUATION OF THE COST AND EFFECT OF PROPHYLACTIC GENOTYPIC MATCHING ON ALLOIMMUNIZATION AND AUTOIMMUNIZATION IN PATIENTS WITH SICKLE CELL DISEASE (SCD)

Blood transfusion is one of the mainstays of treatment for patients with SCD but despite of the beneficial effects, transfusion therapy can lead to red blood cell (RBC) alloimmunization with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and the hyperhemolysis syndrome. In addition, the presence of alloantibodies, which is often associated with the concomitant presence of autoantibodies, can lead to difficulty in finding compatible units, which can cause transfusion delays. To reduce alloimmunization some strategies have been implemented to provide antigen matched RBC transfusions to patients with SCD who need chronic transfusion support. In this context, phenotypic and genotypic matching protocols have been used. Based on this, the aim of this study was to evaluate the cost and the effect of prophylactic RBC transfusion genotypic matching on alloimmunization and autoimmunization in patients with SCD. Our study included 79 (33 male and 46 female) patients with SCD, homozygous for HbS, on chronic RBC transfusion therapy receiving prophylactic genotypic matching RBC transfusions in the last two years. In this period, patients received RBC units genotypic matched for Rh, K, Fya/Fy/b, Jka/Jkb, S/s, Doa/Dob and Dia antigens. Molecular matching was performed according to previous molecular typing results performed by the HEA BeadChip (Immucor) in patients and donors. Adsorption onto autologous RBC was also performed to aid the differentiation of autoantibodies from alloantibodies. The cost and availability of compatible units by performing genotypic matching were compared to serologic matching. The patients received a range of 5-215 units. And the median age in this group was 39 years old. Of the 79 patients, 17 (21.5%) were alloimmunized before they started receiving genotypic matched RBC transfusions. During this study-period only 2 patients (2.5%) with Rh variants developed alloantibodies (1 anti-C, 1 anti-e) and no patient produced autoantibody. Although the availability of compatible units has decreased when compared to serological matching, it was possible to find compatible units for most patients. A cost reduction of 30% was observed in the genotypic matching compared to the serological matching for the selected matched antigens. RBC transfusions with extended genotypic matching had significant effects on autoimmunization and alloimmunization rates in chronically transfused patients with SCD over two years. Alloimmunization rates have shown to decrease from a range of 21.5 to 2.5% in these patients. SCD patients may benefit from receiving prophylactic genotypic matching RBC transfusions as demonstrated by the reduction on the rates of alloimmunization, autoimmunization and cost.

Normalization of cerebral hemodynamics after hematopoietic stem cell transplant in children with sickle cell disease

AbstractChildren with sickle cell disease (SCD) demonstrate cerebral hemodynamic stress and are at high risk of strokes. We hypothesized that curative hematopoietic stem cell transplant (HSCT) normalizes cerebral hemodynamics in children with SCD compared with pre-transplant baseline. Whole-brain cerebral blood flow (CBF) and oxygen extraction fraction (OEF) were measured by magnetic resonance imaging 1 to 3 months before and 12 to 24 months after HSCT in 10 children with SCD. Three children had prior overt strokes, 5 children had prior silent strokes, and 1 child had abnormal transcranial Doppler ultrasound velocities. CBF and OEF of HSCT recipients were compared with non-SCD control participants and with SCD participants receiving chronic red blood cell transfusion therapy (CRTT) before and after a scheduled transfusion. Seven participants received matched sibling donor HSCT, and 3 participants received 8 out of 8 matched unrelated donor HSCT. All received reduced-intensity preparation and maintained engraftment, free of hemolytic anemia and SCD symptoms. Pre-transplant, CBF (93.5 mL/100 g/min) and OEF (36.8%) were elevated compared with non-SCD control participants, declining significantly 1 to 2 years after HSCT (CBF, 72.7 mL/100 g per minute; P = .004; OEF, 27.0%; P = .002), with post-HSCT CBF and OEF similar to non-SCD control participants. Furthermore, HSCT recipients demonstrated greater reduction in CBF (−19.4 mL/100 g/min) and OEF (−8.1%) after HSCT than children with SCD receiving CRTT after a scheduled transfusion (CBF, −0.9 mL/100 g/min; P = .024; OEF, −3.3%; P = .001). Curative HSCT normalizes whole-brain hemodynamics in children with SCD. This restoration of cerebral oxygen reserve may explain stroke protection after HSCT in this high-risk patient population.

Measurement of post-transfusion red blood cell survival kinetics in sickle cell disease and β-Thalassemia: A biotin label approach.

Red blood cell (RBC) transfusions are an important treatment modality for patients with sickle cell disease (SCD) and β-thalassemia. A subgroup of these patients relies on a chronic RBC transfusion regimen. Little is known about RBC survival (RCS) of the transfused allogeneic RBCs. In this study, we aimed to study the RCS kinetics of transfused RBCs in SCD and β-thalassemia and to investigate factors that determine RCS. We performed a prospective cohort study on fourteen adults with SCD and β-thalassemia disease receiving a chronic transfusion regimen. RCS and the influence of donor and patient characteristics on RCS were assessed by simultaneous transfusion of two allogeneic RBCs using RBC biotinylation. Phenotyping of well-known RBC markers over time was performed using flow cytometry. RCS of the two transfused RBC units was similar in most patients. Although intra-individual variation was small, inter-individual variation in RCS kinetics was observed. Most patients demonstrated a non-linear trend in RCS that was different from the observed linear RCS kinetics in healthy volunteers. After an initial slight increase in the proportion of biotinylated RBCs during the first 24 h, a rapid decrease within the first 10-12 days was followed by a slower clearance rate. These are the first data to demonstrate that patient-related factors largely determine post-transfusion RCS behavior of donor RBC in SCD and β-thalassemia, while donor factors exert a negligible effect. Further assessment and modeling of RCS kinetics and its determinants in SCD and β-thalassemia patients may ultimately improve transfusion therapy.