How I Treat Challenging Transfusion Cases in Sickle Cell Disease.

Transfusion of red blood cells (RBCs) can be lifesaving for individuals living with sickle cell disease (SCD). However, alloimmunization following transfusion is more common with SCD than other patient populations, resulting in morbidity and mortality. Management of complications related to RBC alloantibodies, including delayed hemolytic transfusion reactions (DHTRs) and identifying compatible RBCs for future transfusions, remains a challenge for hematologists and transfusion medicine providers. Although transfusion guidelines from organizations including the American Society for Hematology provide general recommendations, individual cases remain challenging. Antibody evanescence and the lack of widespread RBC alloantibody data sharing across hospitals pose unique challenges, as do RH variants in both transfusion recipients and blood donors. Further, as potentially curative therapies require RBC transfusions to lower the hemoglobin S prior to cellular therapy collections and infusions, highly alloimmunized patients may be deemed ineligible. The cases described are representative of clinical dilemmas the authors have encountered and the approaches are as evidence-based as the literature and the authors' experiences allow. A future desired state is one in which RBC alloantibody data are efficiently shared across institutions, Rh alloimmunization can be mitigated, better treatments exist for DHTRs, and a label of "difficult to transfuse" does not prevent desired therapies.

Meeting the transfusion needs of a patient with anti-Ena requires an international effort.

This extraordinary case showcases the identification of a rare anti-Ena specificity that was assisted by DNA-based red blood cell antigen typing and collaboration between the hospital blood bank in the United States, the home blood center in Qatar, the blood center Immunohematology Reference Laboratory, as well as the American Rare Donor Program (ARDP) and the International Society for Blood Transfusion (ISBT) International Rare Donor Panel. Ena is a high-prevalence antigen, and blood samples from over 200 individuals of the extended family in Qatar were crossmatched against the patient's plasma with one compatible En(a-) individual identified. The ISBT International Rare Donor Panel identified an additional donor in Canada, resulting in a total of two En(a-) individuals available to donate blood for the patient.

Machine learning to optimize automated RH genotyping using whole-exome sequencing data.

Rh phenotype matching reduces but does not eliminate alloimmunization in patients with sickle cell disease (SCD) due to RH genetic diversity that is not distinguishable by serological typing. RH genotype matching can potentially mitigate Rh alloimmunization, but comprehensive and accessible genotyping methods are needed. We developed RHtyper as an automated algorithm to predict RH genotypes using whole-genome sequencing (WGS) data with high accuracy. Here, we adapted RHtyper for whole-exome sequencing (WES) data which are more affordable but challenged by uneven sequencing coverage and exacerbated sequencing read misalignment, resulting in uncertain prediction for 1) RHD zygosity and hybrid alleles, 2) RHCE*C versus RHCE*c alleles, 3) RHD c.1136C>T zygosity, and 4) RHCE c.48G>C zygosity. We optimized RHtyper to accurately predict RHD and RHCE genotypes using WES data by leveraging machine learning models and improved the concordance of WES with WGS predictions from 90.8% to 97.2% for RHD and 96.3 to 98.2% for RHCE among 396 patients in the Sickle Cell Clinical Research and Intervention Program (SCCRIP). In a second validation cohort with 3030 cancer survivors (15.2% Black or African Americans) from the St. Jude Lifetime Cohort Study (SJLIFE), the optimized RHtyper reached concordance rates between WES and WGS predications to 96.3% for RHD, and 94.6% for RHCE. In conclusion, machine learning improved the accuracy of RH predication from WES data. RHtyper has the potential, once implemented, to provide a precision medicine-based approach to facilitate RH genotype-matched transfusion and improve transfusion safety for patients with SCD.

BMI1 regulates human erythroid self-renewal through both gene repression and gene activation.

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient numbers of in vitro-derived red blood cells (RBC) for clinical purposes. We and others have determined that BMI1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts (SREs). However, the mechanisms of BMI1 action remain poorly understood. BMI1 overexpression led to 10 billion-fold increase BMI1-induced (i)SRE self-renewal. Despite prolonged culture and BMI1 overexpression, human iSREs can terminally mature and agglutinate with typing reagent monoclonal antibodies against conventional RBC antigens. BMI1 and RING1B occupancy, along with repressive histone marks, were identified at known BMI1 target genes, including the INK-ARF locus, consistent with an altered cell cycle following BMI1 inhibition. We also identified upregulated BMI1 target genes with low repressive histone modifications, including key regulator of cholesterol homeostasis. Functional studies suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. These findings support the hypothesis that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand the pool of immature erythroid precursors for eventual clinical uses.

Generation of red blood cells from induced pluripotent stem cells.

PURPOSE OF REVIEW: Human induced pluripotent stem cells (iPSCs) are an attractive source to generate in-vitro-derived blood for use as transfusable and reagent red cells. We review recent advancements in the field and the remaining limitations for clinical use. RECENT FINDINGS: For iPSC-derived red blood cell (RBC) generation, recent work has optimized culture conditions to omit feeder cells, enhance red cell maturation, and produce cells that mimic fetal or adult-type RBCs. Genome editing provides novel strategies to improve cell yield and create designer RBCs with customized antigen phenotypes. SUMMARY: Current protocols support red cell production that mimics embryonic and fetal hematopoiesis and cell yield sufficient for diagnostic RBC reagents. Ongoing challenges to generate RBCs for transfusion include recapitulating definitive erythropoiesis to produce functional adult-type cells, increasing scalability of culture conditions, and optimizing high-density manufacturing capacity.

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells.

ABSTRACT: During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

BACKGROUND: Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. STUDY DESIGN AND METHODS: We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. RESULTS: Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. DISCUSSION: These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

Involvement of Diverse Populations in Transfusion Medicine Research.

Communities of color and diverse communities (eg, race, socioeconomic status, language, sexual orientation etc.) have not been recruited and enrolled equitably to participate in research studies in transfusion medicine. The exclusion of diverse communities in transfusion research can lead to health disparities lack of access to approved therapeutics and unequal allocation of interventions, resulting in missed opportunities to optimize health for individuals and communities. Involvement of diverse populations in research goes beyond inclusion as research subjects. Strategies should include specific studies on health conditions of importance to diverse communities with stable funding sources and specific funding announcements to develop projects led by diverse researchers, mentorship of diverse researchers, and openness to various ways of communicating research plans. Qualitative approaches and interdisciplinary collaboration should be supported to enhance inclusivity.

Synergistic roles of DYRK1A and GATA1 in trisomy 21 megakaryopoiesis.

Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild-type GATA1 (wtGATA1) or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and derepression of its downstream targets. Notably, DYRK1A loss had no effect in T21 iPSCs or megakaryocytes with wtGATA1. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in T21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.

Tropomyosin 1 deficiency facilitates cell state transitions to enhance hemogenic endothelial cell specification during hematopoiesis.

Tropomyosins coat actin filaments and impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. Prior work suggested that TPM1 regulated blood cell formation in vitro, but it was unclear how or when TPM1 affected hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, TPM1 knockout was found to augment developmental cell state transitions, as well as TNFα and GTPase signaling pathways, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses showed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Indeed, analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced the formation of HE during embryogenesis. These findings illuminate novel effects of TPM1 on developmental hematopoiesis.

Generation of CHOPi-008-B, a euploid iPSC line from a patient with Trisomy 21 and a GATA1 mutation.

Transient myeloproliferative disorder (TMD) is a pre-leukemic condition that occurs only in neonates with Trisomy 21 (T21), and is attributed to a genetic interaction between the third copy of chromosome 21 (HSA21) and a mutation in the transcription factor GATA1 that results in a truncated protein (GATA1s). We generated a euploid iPSC line with a GATA1s mutation that is isogenic to a previously published pair of T21 lines with and without a GATA1 mutation. The line was characterized for pluripotency, differentiation potential, and genomic stability. This line is a valuable isogenic control for studying the T21 hematopoietic phenotype.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

In vivo hematopoietic stem cell modification by mRNA delivery.

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 ( TPM1 ) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

Generation of 2 isogenic clones from a patient with Trisomy 21 and a GATA1 mutation.

Trisomy 21 (T21), or Down Syndrome (DS), is a common chromosomal disorder resulting from a third copy of chromosome 21 (HSA21). Transient myeloproliferative disorder (TMD) is a pre-leukemic condition that occurs only in neonates with DS and is characterized by a mutation in the transcription factor GATA1 that results in a truncated protein (GATA1s). We generated a pair of isogenic T21 lines derived from a patient with TMD that differ only in GATA1 status. The iPSC lines were characterized for pluripotency, differentiation potential, and genomic stability. These lines are a valuable resource for studying T21 hematopoietic diseases.

Variant RHD alleles and Rh immunization in patients with sickle cell disease.

RH diversity among patients and donors contributes to Rh immunization despite serologic Rh-matched red cell transfusions. Anti-D can occur in D+ patients with RHD variants that encode partial D antigens. Anti-D has also been reported in patients with conventional RHD transfused primarily with units from Black donors who frequently have variant RHD. We report 48 anti-D in 690 D+ transfused individuals with sickle cell disease, categorized here as expressing conventional D, partial D or D antigen encoded by RHD*DAU0. Anti-D formed in a greater proportion of individuals with partial D, occurred after fewer D+ unit exposures, and remained detectable for longer than for those in the other categories. Among all anti-D, 13 had clinical or laboratory evidence of poor transfused red cell survival. Most individuals with anti-D were chronically transfused, including 32 with conventional RHD who required an average of 62 D- units/year following anti-D. Our findings suggest that patients with partial D may benefit from prophylactic D- or RH genotype-matched transfusions to prevent anti-D. Future studies should investigate whether RH genotype-matched transfusions can improve use of valuable donations from Black donors, reduce D immunization and minimize transfusion of D- units to D+ individuals with conventional RHD or DAU0 alleles.

Proinflammatory state promotes red blood cell alloimmunization in pediatric patients with sickle cell disease.

We examined risk factors for red blood cell (RBC) alloimmunization in pediatric patients with sickle cell disease, focusing on the recipients' inflammatory state at the time of transfusion and anti-inflammatory role of hydroxyurea (HU). Among 471 participants, 55 (11.70%) participants were alloimmunized and formed 59 alloantibodies and 17 autoantibodies with an alloimmunization rate of 0.36 alloantibodies per 100 units. Analysis of 27 participants in whom alloantibodies were formed with specificities showed 23.8% (30/126) of units transfused during a proinflammatory event resulting in alloantibody formation compared with 2.8% (27/952) of units transfused at steady state. Therefore, transfusion during proinflammatory events increased the risk for alloimmunization (odds ratio [OR], 4.22; 95% confidence interval [CI], 1.64-10.85; P = .003). Further analysis of all the 471 participants showed that alloimmunization of patients who received episodic transfusion, mostly during proinflammatory events, was not reduced with HU therapy (OR, 6.52; 95% CI, 0.85-49.77; P = .071), HU therapy duration (OR, 1.13; 95% CI, 0.997-1.28; P = .056), or HU dose (OR, 1.06; 95% CI, 0.96-1.16; P = .242). The analysis also identified high transfusion burden (OR, 1.02; 95% CI, 1.003-1.04; P = .020) and hemoglobin S (HbSS) and HbSß0-thalassemia genotypes (OR, 11.22, 95% CI, 1.51-83.38; P = .018) as additional risk factors for alloimmunization. In conclusion, the inflammatory state of transfusion recipients affects the risk of RBC alloimmunization, which is not modified by HU therapy. Judicious use of transfusion during proinflammatory events is critical for preventing alloimmunization.

Adverse events of red blood cell transfusions in patients with sickle cell disease.

Blood transfusion is a common medical intervention for patients with sickle cell disease (SCD) and disease related complications. While patients with SCD are at risk for all transfusion related adverse events defined by the National Healthcare Safety Network (NHSN) Biovigilance Component Hemovigilance Module Surveillance Protocol, they are uniquely susceptible to certain adverse events. This review discusses risk factors, mitigation strategies, and management recommendations for alloimmunization, hemolytic transfusion reactions, hyperviscosity and transfusion-associated iron overload in the context of SCD.