• Rh serologic phenotype-matched transfusions from minority donors do not prevent all Rh alloimmunization in patients with SCD.• Variant RH genes are common in patients with SCD and contribute to Rh alloimmunization and transfusion reactions.Red blood cell (RBC) transfusion is a key treatment of patients with sickle cell disease (SCD) but remains complicated by RBC immunization. In the present study, we evaluated the effects of antigen matching for Rh D, C, and E, and K and transfusion from African American donors in 182 patients with SCD. Overall, 71 (58%) chronic and 9 (15%) episodically transfused patients were alloimmunized. Fifty-five (45%) chronic and 7 (12%) episodically transfused patients were Rh immunized. Of 146 antibodies identified, 91 were unexplained Rh antibodies, one-third of which were associated with laboratory evidence of delayed transfusion reactions. Fifty-six antibodies occurred in patients whose RBCs were phenotypically positive for the corresponding Rh antigen and 35 in patients whose RBCs lacked the antigen and were transfused with Rh-matched RBCs. High-resolution RH genotyping revealed variant alleles in 87% of individuals. These data describe the prevalence of Rh alloimmunization in patients with SCD transfused with phenotypic Rh-matched African American RBCs. Our results suggest that altered RH alleles in both the patients and in the donors contributed to Rh alloimmunization in this study. Whether RH genotyping of patients and minority donors will reduce Rh alloimmunization in SCD needs to be examined. (Blood. 2013;122 (6):1062-1071

Background: Red cell transfusions remain a mainstay of therapy for patients with sickle cell disease (SCD), but pose significant clinical challenges. Guidance for specific indications and administration of transfusion, as well as screening, prevention, and management of alloimmunization, delayed hemolytic transfusion reactions (DHTRs), and iron overload may improve outcomes. Objective: Our objective was to develop evidence-based guidelines to support patients, clinicians, and other healthcare professionals in their decisions about transfusion support for SCD and the management of transfusion-related complications. Methods: The American Society of Hematology formed a multidisciplinary panel that was balanced to minimize bias from conflicts of interest and that included a patient representative. The panel prioritized clinical questions and outcomes. The Mayo Clinic Evidence-Based Practice Research Program supported the guideline development process. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to form recommendations, which were subject to public comment. Results: The panel developed 10 recommendations focused on red cell antigen typing and matching, indications, and mode of administration (simple vs red cell exchange), as well as screening, prevention, and management of alloimmunization, DHTRs, and iron overload. Conclusions: The majority of panel recommendations were conditional due to the paucity of direct, high-certainty evidence for outcomes of interest. Research priorities were identified, including prospective studies to understand the role of serologic vs genotypic red cell matching, the mechanism of HTRs resulting from specific alloantigens to inform therapy, the role and timing of regular transfusions during pregnancy for women, and the optimal treatment of transfusional iron overload in SCD.

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid-primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo-and lymphopoiesis.transient myeloproliferative disorder | aneuploidy | human fetus C onstitutional trisomy 21 (T21) causes Down syndrome (DS), the most common syndrome-associated chromosomal anomaly in humans (1). As well as with neurodevelopmental, cardiac, and gut anomalies (2), there is a striking increase in childhood acute leukemia in DS, even though the risk of solid tumors is much lower than with the general population (3). Intriguingly, this susceptibility to hematopoietic tumors manifests as an increased risk both of acute megakaryocyte (MK)-erythroid leukemia (known as ML-DS) by 150-fold and of acute B-lymphoblastic leukemia (B-ALL) by 33-fold (3, 4).DS leukemias display distinct characteristics that support a crucial role for T21 in their pathogenesis. Hallmarks of ML-DS are the megakaryoblastic phenotype, clinical presentation confined to the first 5 y of childhood (5, 6), an antecedent clonally linked preleukemic condition (termed transient myeloproliferative disorder, TMD) in most cases, and acquired N-terminal truncating mutations in the erythroid-MK transcription factor GATA1 (7-9). Such mutations in GATA1 are present in both ML-DS and TMD (9) but are not found in patients without DS who develop megakaryoblastic leukemia (7) and are not leukemogenic in the absence of T21 (10).Molecular, biologic, and clinical data indicate that TMD is initiated before birth (9,(11)(12)(13)(14). We previously reported that by the second trimester, the T21 fetal liver (FL) myeloid progenitor compartment is abnormal and that this occurs in the absence of GATA1 mutation (11, 12). Specifically, the MK-erythroid progenitor (MEP) population is expanded with increased cell-intrinsic MK and erythroid lineage proliferation from CD34 + cells. These data suggest that T21-mediated developmental alterations to FL myeloid progenitor development provide a cell-specific substrate for...

Rationale An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. Objective To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDM) and their isogenic PBMC-derived macrophages (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. Methods and Results We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (LPS+IFN-γ) activation. RNA-Seq revealed that non-polarized (M0) as well as M1 or M2 (IL-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apoA-I and HDL3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease (TD), an autosomal recessive disorder due to mutations in ATP-binding cassette transporter A1. TD-IPSDM also revealed novel defects of enhanced pro-inflammatory response to LPS stimulus. Conclusions Our protocol-derived IPSDM are comparable to HMDM at phenotypic, functional and transcriptomic levels. TD-IPSDM recapitulated hallmark features observed in HMDM and reveal novel inflammatory phenotypes. IPSDM provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.

SummaryRed blood cell (RBC) transfusions can be life-sustaining in chronic inherited anaemias, such as thalassaemia, and the indications for blood transfusions in patients with sickle cell disease continue to expand. Complications of transfusions, such as allosensitization, can create significant medical challenges in the management of patients with haemoglobinopathies. This review summarizes key findings from the medical literature related to alloimmunization in haemoglobinopathies and examines potential measures to mitigate these risks. Areas where future studies are needed are also addressed.

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of β-thalassemia.To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain–focused CRISPR-Cas9–based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.

IntroductionTransient myeloproliferative disorder (TMD) occurs in 10% to 20% of newborns with Down syndrome (DS) and usually resolves after birth. However, approximately 30% of TMD patients develop acute megakaryoblastic leukemia (AMKL) within 4 years, suggesting that TMD is a premalignant disorder and that both diseases originate in the fetus. 1-4 DS TMD and AMKL blasts harbor somatic mutations of GATA1, which encodes an essential hematopoietic transcription factor. 5-7 These mutations occur in exon 2 and result in exclusive expression of an amino-truncated protein, termed GATA-1 short (GATA-1s).Presumably, GATA-1s cooperates with trisomy 21 (T21) to induce a preleukemic state that progresses to frank malignancy through additional mutations. TMD and AMKL blasts exhibit erythro-megakaryocytic features 8-10 and GATA-1 regulates the maturation, survival, and proliferation of these lineages. 11 In contrast, little is known about how T21 itself alters hematopoiesis, particularly in the fetus, where TMD/AMKL-initiating events occur. We analyzed fetal liver hematopoiesis in T21 and control abortuses. In vitro and in vivo transplantation studies show that T21 is associated with enhanced erythroid and megakaryocytic precursor expansion, independent of GATA1 mutations. These findings provide insight into the hematopoietic abnormalities of DS and indicate how T21 might synergize with GATA-1s to promote TMD and AMKL. MethodsFetal livers were obtained from pathology specimens of week 13 to 23 abortuses. Institutional Review Board-approval was obtained from The Children's Hospital of Philadelphia and The University of Pennsylvania. Informed consent was obtained in accordance with the Declaration of Helsinki. T21 was confirmed by karyotype analysis of fetal tissue. DNA sequencing, hematopoietic assays, and gene expression analysis were performed using standard methods described in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Approval from the Institutional Animal Care and Use Committee at the University of Pennsylvania was obtained for mouse studies. Results and discussionDS-associated TMD and AMKL initiate in utero, as evidenced clinically 1,3 and by GATA1 mutational analysis. 7,12,13 We analyzed fetal liver specimens from DS and control abortuses at 13 to 23 weeks' gestation. Histologically, T21 fetal livers were indistinguishable from controls (not shown). We isolated fetal liver hematopoietic mononuclear cells (MNCs), amplified GATA1 exon 2 by polymerase chain reaction (PCR), and subcloned the fragments. No mutations in 31 T21 or 10 control fetal liver MNC samples were detected by direct sequencing of the PCR product and 24 independent clones from each fetal liver, consistent with the relatively low incidence of GATA1 mutations (3.8%) identified by screening 585 DS newborns. 13 The absence of GATA1 mutations in our specimens allowed us to study the hematopoietic effects of T21 in isolation.We analyzed T21 and control fetal liver MNCs in methylcellulose...

Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 drives the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythromegakaryocytic differentiation. To investigate these functions further, we stimulated Gata1 ؊ murine embryonic stem-cell-derived hematopoietic cultures with thrombopoietin, a multistage cytokine. Initially, the cultures generated a wave of mutant megakaryocytes. However, these were rapidly overgrown by a unique population of thrombopoietin-dependent blasts that express immature markers and proliferate indefinitely. Importantly, on restoration of GATA-1 function, these cells differentiated into both erythroid and megakaryocytic lineages, suggesting that they represent bipotential progenitors.