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Background: Adult sickle cell patients on chronic transfusion therapy (CTT) garnered special concern during the COVID-19 pandemic, when mass cancellation of blood drives threatened the national blood supply. In response, the American Society of Hematology proposed several strategies to decrease blood utilization, while maintaining adequate disease control for sickle cell patients on CTT. These included targeting a higher end hemoglobin S%, switching patients to simple transfusions when appropriate, and transitioning to alternative disease modifying therapies. There is little evidence to support the safety of altering exchange therapy regimens. Accordingly, at the Johns Hopkins Sickle Cell Center for Adults, a multidisciplinary team of clinicians and transfusion medicine specialists evaluated patients on a case by case basis to determine how their exchanges could be modified to accommodate for anticipated blood supply shortages. We describe our blood conservation efforts during the COVID-19 pandemic and resulting clinical outcomes for adult patients with sickle cell anemia (SCA). Methods: For inclusion in this IRB-approved retrospective study, patients received at least 7 monthly exchange transfusions between March 2019 and February 2020 and continued care through March 2021. Decisions regarding CTT were made prior to data collection. Modifications to chronic exchanges included increasing the fraction of cells remaining (FCR), decreasing the end hematocrit, or switching to a hemodilution method. Additionally, select patients transitioned to monthly simple transfusions if they were clinically stable, had a hemoglobin ≤ 7 g/dL, and had a persistently suppressed hemoglobin S% (≤ 30). We collected basic demographics, pre-exchange laboratory studies, and exchange parameters before and after each transfusion encounter in the year before (March 2019 - February 2020) and during (March 2020 - February 2021) the COVID-19 pandemic. Lastly, we recorded presentations to urgent care and the emergency department; and hospital admissions during each time period. We reported descriptive statistics for the cohort and compared outcomes using the Wilcoxon signed-rank test and Mann Whitney U test. We analyzed data using Stata/SE Version 16.1. Results: We identified 58 patients with SCA who qualified for inclusion (Table 1). Fifty-three patients remained on chronic exchange transfusions during the pandemic, and five were switched to simple transfusions. For patients who remained on chronic exchanges, most received conventional automated red cell exchange (RCE) prior to (85%) and during the pandemic (77%). Use of hemodilution increased (15% to 23% of patients). Forty-three patients experienced an increase in mean FCR (33.6 (SD 11.6) vs 37.7 (4.7), p = .00). Of those, 22 patients saw a concomitant decrease in mean end hematocrit (30.8 (1.7) vs 29.6 (1.1), p = .00). These changes resulted in a decline in the average number of units per procedure (8.6 (1.9) vs 7.3 (1.7), p = .00), which corresponded to 890 units conserved. Mean pre-transfusion hemoglobin values declined (9.4 (1.3) vs 9.2 (1.3), p = 0.01), but hemoglobin S%, reticulocyte count, and ferritin values were unchanged (p > 0.05). Acute care presentations and hospital admissions declined, which were likely spurred by concerns about COVID-19 infection (Table 2, p < 0.05). During the pandemic, three patients died, one of whom had been switched to simple transfusions. This patient experienced a consistent rise in hemoglobin S% until death. Two of the remaining patients on simple transfusions were switched back to automated RCE due to an increase in hemoglobin S% above goal (Figure 1). Conclusions: During the COVID-19 pandemic, we conserved red blood cell units through expanded use of hemodilution, higher FCR values, and switching some patients to simple transfusions. Patients who remained on exchanges maintained hemoglobin S% values near a goal of 30% without increasing iron burden. In contrast, the majority of patients who were switched to simple transfusions were unable to maintain goal hematologic parameters, and one patient died. Our data suggest that in a blood shortage crisis, changing the exchange procedure itself may be the safest means of conserving blood in a population of adult sickle cell patients; however longer follow-up is needed to ensure that these changes are safe. Figure 1 Figure 1. Disclosures Lanzkron: GBT: Research Funding; Shire: Research Funding; Novo Nordisk: Consultancy; CSL Behring: Research Funding; Pfizer: Current holder of individual stocks in a privately-held company; Teva: Current holder of individual stocks in a privately-held company; Novartis: Research Funding; Imara: Research Funding; Bluebird Bio: Consultancy.

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype. The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C). We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mutvs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H). Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wtEP cells (Fig. K). Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O). In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. Figure 1 Figure 1. Disclosures Dai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.