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Red blood cell (RBC) transfusion is one of the most common medical treatments, with more than 10 million units transfused per year in the United States alone. Alloimmunization to foreign Rh proteins (RhD and RhCE) on donor RBCs remains a challenge for transfusion effectiveness and safety. Alloantibody production disproportionately affects patients with sickle cell disease who frequently receive blood transfusions and exhibit high genetic diversity in the Rh blood group system. With hundreds of RH variants now known, precise identification of Rh antibody targets is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen phenotypes. Using a combination of human-induced pluripotent stem cell (iPSC) reprogramming and gene editing, we designed a renewable source of cells with unique Rh profiles to facilitate the identification of complex Rh antibodies. We engineered a very rare Rh null iPSC line lacking both RHD and RHCE. By targeting the AAVS1 safe harbor locus in this Rh null background, any combination of RHD or RHCE complementary DNAs could be reintroduced to generate RBCs that express specific Rh antigens such as RhD alone (designated D--), Goa+, or DAK+. The RBCs derived from these iPSCs (iRBCs) are compatible with standard laboratory assays used worldwide and can determine the precise specificity of Rh antibodies in patient plasma. Rh-engineered iRBCs can provide a readily accessible diagnostic tool and guide future efforts to produce an alternative source of rare RBCs for alloimmunized patients.

Genomics is affecting all areas of medicine. In transfusion medicine, DNA-based genotyping is being used as an alternative to serological antibody-based methods to determine blood groups for matching donor to recipient. Most antigenic polymorphisms are due to single nucleotide polymorphism changes in the respective genes, and DNA arrays that target these changes have been validated by comparison with antibody-based typing. Importantly, the ability to test for antigens for which there are no serologic reagents is a major medical advance to identify antibodies and find compatible donor units, and can be life-saving. This review summarizes the evolving use and applications of genotyping for red cell and platelet blood group antigens affecting several areas of medicine. These include prenatal medicine for evaluating risk of fetal or neonatal disease and candidates for Rh-immune globulin; transplantation for bone marrow donor selection and transfusion support for highly alloimmunized patients and for confirmation of A2 status of kidney donors; hematology for comprehensive typing for patients with anemia requiring chronic transfusion; and oncology for patients receiving monoclonal antibody therapies that interfere with pretransfusion testing. A genomics approach allows, for the first time, the ability to routinely select donor units antigen matched to recipients for more than ABO/RhD to reduce complications. Of relevance, the growth of whole-genome sequencing in chronic disease and for general health will provide patients’ comprehensive extended blood group profile as part of their medical record to be used to inform selection of the optimal transfusion therapy.

NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Rh alloimmunization remains a challenge for patients with sickle cell disease despite transfusion of serologic Rh C, E and K antigen-matched red cells. Inheritance of altered RH alleles contributes to the prevalence of Rh antibodies following blood transfusion in patients with SCD and explains approximately one-third of cases. The remainder appear to be stimulated by altered Rh proteins on African-American donor red cells. Matching of patients with donors based on RH genotype may mitigate Rh alloimmunization, but the feasibility and resources required are not known. We compared RH allele frequencies between patients with SCD (n=857) and African-American donors (n=587) and show that RH allele frequencies are similar. Overall, 29% of RHD and 53% of RHCE alleles are altered in patients and African-American donors. We modeled RH genotype matching compared to serologic Rh D, C, and E, along with K antigen-matching, and found that approximately twice the number of African-American donors would be required for RH genotype versus Rh serologic matching at our institution. We demonstrate that African-American donor recruitment is necessary to maintain an adequate supply of C, E, and K negative donor units to avoid depleting the Rh negative (RhD-) blood supply. Our results suggest that prophylactic RH genetic matching for patients with SCD is feasible with a donor pool comprised primarily of African-Americans and would optimize utilization of our existing minority donor inventory. The current cost of RH genotyping all minority donors and management of the data remain limiting factors.

Background: Despite transfusion of Rh matched red cells for patients with sickle cell disease, Rh alloimmunization remains a persistent challenge. Rh specificities can be complex, resulting from RH genetic diversity found in patients and donors. Antibody identification is hampered by the lack of appropriate reagent red cells, especially those that can identify antibodies against high prevalence or low prevalence Rh antigens. We used human induced pluripotent stem cells (iPSCs) with the goal of producing renewable red cell reagents to both screen for Rh alloimmunization and to aid complex antibody identification. Methods: We generated a panel of iPSCs that include Rh null, D--, lack the high prevalence antigens hr S or hr B, or express uncommon Rh antigens such as V, VS, Go a, or DAK. For the Rh null line, we used CRISPR/Cas9 genetic engineering to disrupt RHCE via a large deletion in a D- iPSC. For D--, RHD was inserted into the AAVS1 safe harbor locus of an Rh null iPSC line using zinc finger nucleases resulting in a line that constitutively expresses RhD but no RhCE. iPSCs with uncommon variants were reprogrammed from RH genotyped donors or engineered similar to the generation of the D-- line. Hematopoietic differentiation by embryoid body formation was used to generate hematopoietic progenitors that were subsequently cultured towards the erythroid lineage. Mature iRBCs were ficin treated and tested with patient plasma with previously identified Rh antibodies using gel agglutination assays. Results: Rh null iPSC-derived RBCs (iRBCs) showed complete absence of cell surface Rh protein by flow cytometry, while D-- iRBCs showed Rh protein expression levels comparable to D-ce+ iRBCs using an anti-D/CE antibody. We assessed RBC agglutination of Rh null, D--, hr S-, hr B-, VVS+, Go a+, and DAK+ iRBCs using standard Rh typing reagents (Ortho). The reprogrammed uncommon donor iRBCs agglutinated with monoclonal anti-Rh antibodies as predicted by RH genotype, while the Rh null iRBCs showed no agglutination with all 5 common Rh antibodies and D-- iRBCs showed agglutination with anti-D reagents only. Rh null iRBCs showed no agglutination against patient plasma containing anti-D, while D-- iRBCs agglutinated. While D- RHCE*ce homozygous iRBCs showed strong agglutination against patient plasma containing anti-hr S, Rh null, D--, and hr S- iRBCs did not agglutinate. No iRBCs showed agglutination by plasma containing anti-V/VS while VVS+ iRBCs showed strong agglutination. Similarly, no iRBCs showed agglutination by plasma containing anti-Go a while Go a+ iRBCs showed strong agglutination. Detection of most antibodies against Rhce on iRBCs was enhanced by ficin treatment whereas antibodies with D specificity did not require ficin treated cells for detection. Conclusion: We suggest that genetically engineered iPSCs expressing uncommon Rh antigen phenotypes that are difficult or impossible to obtain from red cell donors can expedite antibody identification. Rh null and D-- iRBCs could be useful to discriminate antibodies against RhD versus RhCE. Customized iPSCs that lack high prevalence or express low prevalence Rh antigens could potentially standardize antibody evaluation in patients with complex Rh specificities. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood, Liu et al investigated the mechanism of action in antibody-mediated immune suppression (AMIS) and the fate of antigen in a KEL mouse model system.1

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.

About twelve million units of red blood cells (RBCs) are transfused yearly in the United States. Ex vivo cultured RBCs could serve as a supplemental source to treat alloimmunized patients requiring chronic transfusions. The limited proliferative capacity of erythroid precursors is a major obstacle to generating sufficient numbers of RBCs to constitute even one unit of blood. We previously determined that erythroblasts derived from the murine embryo have a unique ability to self-renew extensively ex vivo, and that Bmi-1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive the extensive self-renewal of murine bone marrow-derived erythroblasts. Here, we tested the hypothesis that Bmi-1 regulates the ex vivo self-renewal of human erythroid cells. Bmi-1 overexpression increased the proliferative capacity of adult human peripheral blood mononuclear cell-derived erythroblasts, which normally have restricted ex vivo self-renewal, more than 10 billion-fold. Bmi-1-induced self-renewing erythroblasts (iSREs) retained an immature erythroid morphology and cell surface phenotype throughout culture. Chemical inhibition of Bmi-1 led to collapse of the culture, with a massive reduction in cycling cells and an increase in apoptosis. Taken together, these data indicate that Bmi-1 in iSREs is both necessary and sufficient to increase the self-renewal capacity of human erythroid precursors. Importantly, Bmi-1 overexpression does not interfere with the ability of the iSREs to mature into reticulocytes in vitro. Serological analysis of multiple blood group antigens demonstrated that iSRE-derived reticulocytes express the same antigens as the donor's RBCs. Bmi-1 can act as part of canonical or non-canonical PRC1 and different complex members have been associated with hematopoietic self-renewal versus differentiation. Expression studies of self-renewing human erythroblasts revealed that the non-canonical PRC1 member, RYBP, was expressed at higher levels than canonical PRC1 members. In addition, maturation of erythroid precursors is associated with down-regulation of Bmi-1 and non-canonical PRC1 members, as well as the upregulation of several canonical members. Knockdown of RYBP reduced the proliferative capacity of the iSREs, suggesting that Bmi-1 regulates erythroid self-renewal through non-canonical PRC1 interactions. Consistent with this hypothesis, preliminary studies inhibiting canonical PRC1 members led to an additional several trillion-fold increase in the ex vivo proliferative capacity of human iSREs compared to vehicle-treated control cultures. These data suggest that Bmi-1 regulates erythroid self-renewal through non-canonical PRC1. Increasing the ex vivo self-renewal capacity of human erythroblasts ultimately paves the way for the generation of sufficient numbers of cultured RBCs for blood typing and transfusion therapy, as well as the establishment of in vitro models of RBC-intrinsic disorders. Disclosures Palis: Rubies Therapeutics: Consultancy.

Introduction RBC alloimmunization is common in patients with sickle cell disease (SCD). Despite serological matching RBCs for major Rh antigens, Rh alloimmunization remains problematic. The Rh blood group is encoded by two genes RHD and RHCE, which exhibit extensive nucleotide polymorphism and chromosome structural changes, resulting in the formation of Rh variant antigens. Rh variants can result in loss of protein epitopes or expression of neo-epitopes, and are common in SCD patients. Hence SCD patients harboring Rh variants can be predisposed to Rh alloimmunization. Given the limitation of traditional serologic antigen typing for detection of Rh variants, molecular genotyping has become required. A DNA microarray-based platform, BioArray RHCE and RHD BeadChip (Immuncor) is available for RH genotyping. However, it detects the most common, but not all, variants. Whole exome sequence data have been used for prediction of Rh variants (Chou, et. al, Blood Adv., 2017), offer some advantages, including detection of rare variants, structural rearrangements and copy number variation. However, whole genome sequence (WGS) analysis of RHD/RHCE is challenging due to difficulties in mapping next generation sequencing (NGS) reads to this duplicated gene family. We developed a computational algorithm to identify RH variants using WGS data. Methods The pipeline included three major components, RH allele database construction, RH variant calling, and classification of Rh blood group according the identified variants. The RH allele database was built based on NCBI Blood Group Antigen Gene Mutation (BGMUT) and International Society of Blood Transfusion (ISBT) database. Since the alleles in the BGMUT and ISBT databases were specified according to conventional RH genes (RHD, L08429; RHCE, DQ322275) that are different from those on reference human genome, we first called the variations based on the reference human genome. The positions of the identified variations were subsequently corrected to match with the BGMUT and ISBT annotation system. Next, the NGS reads with low base quality and/or mapping quality were discarded during the variation calling step. Synonymous and non-synonymous amino acid changes were characterized for each polymorphism. Haplotypes were constructed for the segments with NGS read support. Gene sequencing coverage was calculated to determine gene deletions or amplifications. Lastly, we implemented an algorithm to predict RH genotypes based on a selection of candidate alleles by read-mapping profile which considers both sequence variations and sequence consistency followed by a likelihood-based ranking of all pairwise combinations of the selected alleles. The allele combination with the highest likelihood is considered the most likely pair of alleles at a given locus. Patient specimens used in this study were from participants of the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer. 2018). Results We validated our method in a cohort of 58 SCD patients whose RH genotypes had been determined by BioArray RhCE and RhD BeadChip and supplementary molecular tests that identify the most common variants among individuals of African descent. In this validation cohort including a total of 11 RHD and 13 RHCE alleles, our approach achieved a concordance rate of 85.85% (91 of 106 alleles) for RHD and 83.02% (88 of 106 alleles) for RHCE genotyping. WGS was highly sensitive in distinguishing homozygosity from heterozygosity of genes. By comparing the numbers of NGS reads on RH regions and whole genome average coverage, heterozygous deletion can be determined. Since WGS provides comprehensive genotyping, our analysis identified single nucleotide polymorphisms that were not identified by the BeadChip and supplemental molecular testing. The final source of discordance was likely due to the short read length of NGS such that haplotype phases cannot be correctly predicted if the variations are separated by thousands of base pairs, for which long read DNA sequencing or RNA/cDNA sequencing are required. Evaluation of the identified discrepancies is ongoing. Conclusions We developed and validated a diagnostic method for RH genotyping that leveraged the accuracy and flexibility of RH genotyping based on WGS data. With further optimization of our method, this may be useful for RBC genotype matching sickle cell patients to blood donors in the future. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy.

Background Oncogenic proteins can be stabilized either via genetic mutations or via defects in the ubiquitin ligating- and degradation machinery. NOTCH1 protein stability is affected by genetic mutations in approximately 10% of chronic lymphocytic leukemia (CLL) patients and mutations are associated with a worse prognosis. Yet, even in the absence of NOTCH1 mutations, NOTCH1 is activated in almost half of all CLL patients. Aims In order to shed light on NOTCH1 activation in CLL, we analyzed the impact of its modulator, the E3-ubiquitin ligase FBXW7 and the deubiquitinase USP28 that is a negative regulator of FBXW7 and is located in chromosomal band 11q23. Methods FBXW7 mutation analysis was performed via targeted next generation sequencing from a total of 905 patients. In silico modeling of potential substrate binding to the mutated FBXW7 was performed by the use of PolyPhen-2 and validated via co-immunoprecipitation of overexpressed mutated FBXW7 and NOTCH1 wild type proteins. Accumulation of FBXW7 substrates was tested via Western blot in CRISPR/Cas9 induced FBXW7 mutated HG-3 CLL cell lines and in FBXW7 mutated primary CLL cells, identifying the transcription factor NOTCH1 as FBXW7 target. Expression of NOTCH1 target genes were analyzed in FBXW7 mutated CLL cases via gene expression profiling (n=4) and RT-qPCR (n=19). In an independent cohort, USP28 and NOTCH1 target gene expression was analyzed in a total of 285 patients via gene expression profiling. Results Heterozygous FBXW7 mutations were identified in 41/905 (4.5%) CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain, while an additional 10% of mutations were located on the first exon of the abundantly expressed FBXW7 α-isoform. We identified substrate targets of FBXW7 in CLL via the generation of a truncation of the WD40 domain of FBXW7 in the CLL cell line HG-3 using CRISPR/Cas9, identifying NOTCH1 as an FBXW7 target. In silico modeling of FBXW7 mutations on protein binding predicted that novel mutations within the WD40 domain affected substrate recognition capacity of FBXW7. Interestingly, modeling predicted W425C and a new hotspot mutation G423V (found in 3/905 of CLL cases) to ablate NOTCH1 binding while the mutation A503V was predicted not to impair binding of NOTCH1 to FBXW7. The in silico modeling was confirmed by co-immunoprecipitation experiments of overexpressed NOTCH1 and FBXW7 and further revealed that mutations within the α-isoform specific N-terminus T15VR and V154I still enabled NOTCH1 binding. Intriguingly, in primary CLL cells FBXW7 mutations correlated with an increase in NOTCH1 levels that remained stable even upon inhibition of translation, underlining the enhanced protein stabilization by FBXW7 mutations. Furthermore, FBXW7 mutations in CLL resulted in an increased NOTCH1 target gene expression. FBXW7 activity is not only modulated by gene mutations, but also by the deubiquitinase USP28 which is localized in the recurrently deleted region 11q22-q23 close to the ATM tumor suppressor gene. We hypothesized that USP28 would impact on FBXW7 activity and thus on NOTCH1 stability and activity. In line with this concept we found that in primary cells from a cohort of 285 CLL patients, low USP28 expression significantly correlated with increased NOTCH1 target gene expression, independent of the 11q deletion status of the patients where USP28 is localized. Discussion In CLL patient cells we have identified novel mutations within the WD40 binding domain of FBXW7 in addition to the common hot spot mutations (R465, R479, R505). Amongst these novel mutations, G423V and W425C are recurrent and result in decreased binding of the FBXW7 substrate NOTCH1 and hence in an accumulation and induction of NOTCH1 activity. Furthermore, expression of USP28, the negative regulator of FBXW7, significantly correlated with increased NOTCH1 target gene expression. Hence, our findings uncover modulation of NOTCH1 in CLL via the FBXW7-USP28-NOTCH1 axis (Figure 1) in addition to genomic NOTCH1 modification, thus explaining the high proportion of CLL cases that harbor an activation of NOTCH1 leading to more aggressive disease. Disclosures Tausch: AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Döhner:AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding. Stilgenbauer:Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding.

Activating mutations of c-KIT lead to ligand-independent growth. Internal tandem duplications (ITDs) of exon 11, which encodes the juxtamembrane domain (JMD), are constitutively activating mutations found in 7% of gastrointestinal stromal tumors (GISTs) but have not been described in childhood acute myeloid leukemia (AML). DNA and cDNA from 60 children with AML were screened by polymerase chain reaction (PCR) for mutations of the JMD. A complex ITD (kit cITD) involving exon 11 and exon 12 was identified with a relative frequency of 7% (4/60). The human kit cITDs were inserted into the murine c-Kit backbone and expressed in Ba/F3 cells. KIT cITD induced factorindependent growth and apoptosis resistance, and exhibited constitutive autophosphorylation. KIT cITD constitutively activated the PI3K/AKT pathway and phosphorylated STAT1, STAT3, STAT5, and SHP-2. Imatinib (IM) or rapamycin (Rap) led to complete inhibition of growth, with IC50 values at nanomolar levels. IM and Rap synergistically inhibited growth and surmounted KIT cITD-induced apoptosis resistance. IM but not LY294002 inhibited phosphorylation of STAT3 and STAT5, suggesting aberrant cross talk between PI3K- and STAT-activating pathways. The findings presented may have immediate therapeutic impact for a subgroup of childhood AML-expressing c-KIT mutations.

The reduction in expression of the integral membrane protein CD47 in human red blood cells (RBCs) deficient in protein 4.2 suggests that protein 4.2 may mediate a linkage of CD47 to the membrane skeleton. We compared the fractions of membrane skeleton-attached CD47, Rh-associated glycoprotein (RhAG), Rh, and band 3 in normal and protein 4.2-deficient cells using fluorescence-imaged microdeformation. We found that CD47 attachment decreases from 55% in normal cells to 25% to 35% in 4.2-deficient cells. RhAG, which has been shown to have no significant variation in expression among the cells studied, shows a significant decrease in membrane skeleton attachment in 4.2-deficient cells from 60% to 40%. Both Rh and band 3, which have also been shown to have no change in expression, show a smaller decrease from 75% attached in normal RBCs to 55% attached in 4.2-deficient cells. In normal cells, Rh phenotype influences CD47 expression but not the level of membrane skeleton attachment of CD47. In contrast, the results indicate that protein 4.2 strongly influences CD47 levels as well as the extent of membrane skeleton attachment in the RBC, whereas protein 4.2 affects membrane skeletal attachment of RhAG, Rh, and band 3 to a lesser extent. (Blood. 2004;103:1131-1136)

Background: Sickle cell trait (SCT) is more common in minority African-Americans (AA) and Hispanic blood donors. Screening donors for Sickle Hemoglobin (HbS) carrier status (SCT) can be useful, especially in managing the transfusion needs of neonates and patients with sickle cell disease. Currently, the Sickledex test, based on the principle of relative insolubility of HbS in phosphate buffers, is routinely used for HbS donor screening. However, there are several limitations including the inability to perform high-throughput testing by automation as donor centers target larger numbers of minority blood donors. False positives results due to hyperlipidemia, dysglobulinemia and higher peripheral blood hematologic counts have also been reported. Hence alternative methods to identify SCT donors are highly desirable in blood centers. The purpose of this study was to validate testing, and to identify rates of False Positive (FP) and False Negative (FN) sickledex screening in blood donors by verifying the presence or absence of the most common mutation seen with HbS production using molecular methods. The comparison could offer evidence to use molecular methods as a test of record to manage HbS carriers at blood centers. Methods and Results Between Jan 2012 till Dec 2014, 200 sickledex screening tests were done daily, totaling approximately 90,000 donors self-identifying as Caucasians and 60,000 donors identifying as African American or Hispanic tested during the study period. Amongst Caucasians tested, 154 had a positive sickledex screen result. Since the prevalence of SCT is less common in the Caucasian population, we hypothesized several of these Caucasian donors could have a false positive screening test. All 154 sickledex positive Caucasian donor samples were prospectively tested for the HbS mutation detection with molecular methods. BioArray Human Erythrocyte Antigen (HEA) Bead Chip DNA assay with PCR multiplex based technology was used to detect the HbS mutation (β-globin change -173 A>T, more commonly designated 20A>T). Of the 154 sickledex + Caucasians, 108 (70%) donors tested negative (FP Screen) and 46 (30%) tested positive for the HbS mutation. A retrospective review of sickledex results in our electronic database for minority donors (n-3431) who also had HEA BeadChip assay was performed to determine concordance. Of 3431 minority donors tested, only 2 (0.0006%) had a positive sickledex screening and were negative by HEA, with the remaining negative by both the sickledex screen and HEA tests. Among minority donors (n= 254) testing + for HbS by HEA, 3% (8/254) were negative by sickledex screening. To establish concordance of HEA assay with high performance liquid chromatographic (HPLC) assay, two separate cohorts were evaluated. Initially 12 sickle screen and HEA positive donors was subject to HPLC analysis, which confirmed SCT status in all 12 samples. Subsequently sickledex screen positive but HEA negative samples (n= 40), were subject to HPLC. All 40 showed absence of SCT by HPLC. Finally, 58 random sickledex + samples had gene sequencing and HEA performed in parallel. Out of 58 samples, 54 showed a positive HbS mutation by HEA with A>T (SCT) nucleotide change noted with gene sequencing. In the remaining 4 samples, HEA was negative with gene sequencing showing (3 T>T and in 1C>T) change at nucleotide 163 associated with Hemoglobin Okayama. Conclusion: The HEA bead chip assay testing for the HbS mutation showed 100% concordance with HPLC and gene sequencing in samples studied. Molecular methods can process multiple samples in a single setting to detect SCT. FP sickledex screening results in Caucasians needs confirmatory testing for the HbS mutation to avoid unnecessary donor notification and deferral. The association, if any, of false positive sickledex screening in Caucasian donors with the Hgb Okayama mutation is under further investigation. Disclosures No relevant conflicts of interest to declare.

[Graphic][1] Introduction: The clonal selection of a mutant BCR-ABL positive clone can be observed in about one of two patients with imatinib-resistant chronic myeloid leukemia (CML). The early detection of BCR-ABL kinase domain mutations is crucial, since it allows to change the tyrosine kinase inhibitor (TKI) regimen in a timely manner and may therefore prevent disease progression and the accumulation of further genetic lesions. European LeukemiaNet (ELN) recommendations suggest a mutation analysis if optimal response criteria are not achieved at 3, 6, 12 or 18 months, or whenever a loss of optimal response occurs (Soverini et al., Blood 2011). Several attempts have been made to derive this indication from a specific increase of BCR-ABL levels. Here we report on the correlation of a rise in BCR-ABL transcript levels and the prevalence of BCR-ABL kinase domain mutations in imatinib-treated patients of the CML-Study IV. Methods: A total of 1,173 patients were enrolled until 2009 and randomized to one of four imatinib-based treatment arms. BCR-ABLIS of 988 patients was determined in 7,876 samples by quantitative RT-PCR in the central laboratory (median sample number per patient: 8.4, range 1-37; median follow up: 34 months, range 0-86), representing the eligible patients for the study. Thereby, the estimated intra-laboratory variance is assumed to be about 20%. A first rise of BCR-ABLIS to at least two-fold and >0.1% between two samples of a patient's molecular course defined a sample suspected of bearing a mutant BCR-ABL positive clone. A mutation analysis was performed on this critical sample by direct sequencing of ABL exons 4 to 10. Results: A critical rise in BCR-ABLIS was observed in 231 of 988 patients (23%) after a median of 15.2 months on treatment (range 2.8-59.4). In the corresponding sample 33 mutant clones could be detected in 31 patients (13%). Thereby a steeper rise of BCR-ABLIS was correlated with a higher incidence of BCR-ABL mutations in the respective group (table). A total of 18 different mutations could be detected, the most frequent were: M244V, n=7 (21%); E255K, n=4 (12%); T315I, n=3 (9%); L248V, G250E, L387M and F486S, n=2 (6%), respectively. Mutations occur in a substantial proportion (8%) of patients with an only 2 to 3-fold rise of BCR-ABLIS transcript levels (table). Therefore, the most sensitive cut-off should be applied and mutation analysis may be triggered by a doubling of BCR-ABL transcripts at levels >0.1% IS. Conclusion: BCR-ABL kinase domain mutations occur already in a substantial proportion of patients with a doubling of BCR-ABL transcript levels, which should determine mutation analysis. | Rise of BCR-ABL expression | Patients (n) | Patients with BCR-ABL mutations (n) | Patients with BCR-ABL mutations (%) | Inter-sample interval (median, days) | | -------------------------- | ------------ | ----------------------------------- | ----------------------------------- | ------------------------------------------- | | 2 to 3-fold | 72 | 6 | 8.3 | 98 | | 3 to 5-fold | 50 | 3 | 6.0 | 100 | | 5 to 10-fold | 39 | 4 | 10.3 | 99 | | 10 to 100-fold | 49 | 10 | 20.4 | 98 | | > 100-fold | 21 | 8 | 38.1 | 125 | | > 2-fold (total) | 231 | 31 | 13.4 | 101 | Table 1. Disclosures Hanfstein: Novartis: Research Funding; Bristol-Myers Squibb: Honoraria. Hehlmann: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding. Saussele: Novartis: Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria, Travel, Travel Other. Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Neubauer: MedUpdate: Honoraria, Speakers Bureau. Kneba: Novartis: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pfirrmann: Novartis: Consultancy; Bristol-Myers Squibb: Honoraria. Hochhaus: Pfizer: Consultancy, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Muller: Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. [1]: /embed/inline-graphic-2.gif

Interactions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)– tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced clusters, whereas Band 3 and glycophorin C remain more homogeneously dispersed on the cell periphery. In a second study, red cells are aspirated into a micropipette, and immunofluorescent maps of the surface gradients that develop for CD47 and RhAG determine cytoskeletal connectivity. CD47 and RhAG gradients on normal red cells prove to be nearly identical and also appear intermediate to those found for the fluid bilayer and network-linked glycophorin C. Similar gradients are obtained for CD47 on Rhnull cells, suggesting that linkage of CD47 to the spectrin-actin skeleton is independent of Rh or RhAG and is not affected by CD47's reduced surface expression on these cells. The results show that CD47 colocalizes with Rh and RhAG but is fractionally attached to the red cell membrane skeleton independent of these and other major integral membrane proteins involved in cytoskeletal attachment. The results imply a homogeneous base distribution of CD47, restrained by cytoskeleton linkages, plus a smaller fraction of CD47, which is able to diffuse in the membrane.

Abstract 2287 High throughput genomic testing for blood groups allows large scale antigen typing and assessment of donor pool compatibility with chronic transfusion dependent populations, particularly thalassemia or sickle cell disease (SCD) patients, in order to decrease RBC alloimmunization. Thus, it is crucial to determine if the quantity and antigen diversity of the donor pool meets the demands to sustain these patients on phenotype/genotype extended matched chronic transfusion protocols. We calculated the most common extended RBC predicted phenotypes for the 12 major clinically significant blood group antigens, D, CcEe, K, Jka/b, Fya/b, and Ss, in patients undergoing chronic transfusion for sickle cell disease (n=203) or thalassemia (n=98). The most common phenotypes in each diagnostic group were used to determine the prevalence of these phenotypes in a single day donor inventory (n=5,000) and stratified by ethnic group (70% Caucasian, 10% African-American, 13% Hispanic, 5% Asian, and 2% Other). All patient samples were tested for the presence of the GATA mutation which disrupts erythroid expression of Fy(b), and if present deemed not at risk for Fy(b) alloimmunization. The majority of patients with SCD and thalassemia are RhD positive (97% and 90% respectively), but differ in extended Rh phenotype, with Ro (Dce) prevalent in SCD pa tients (61%), and R1 (DCe) in thalassemia patients (79%). For patients with SCD, the most prevalent antigen-negative phenotypes were 17% C-E-K-,Fy(a-),Jk(b-), S-; 9% C-E-K-, Fy(a-),S-; 5% E-K-,Fy(a-),S-; and 4% C-K-,Fy(a-), Jk(b-), S-. In patients with thalassemia, no minor antigen profile exceeded 5% of individuals. The most prevalent antigen-negative profiles were 5% E-c-K-, Fy(b-),Jk(b-),S-, and 4% E-K-. Comparison of the most prevalent antigen-negative phenotype in patients with SCD with the donors revealed only 0.06% Caucasian (n=2), but 20% of the African-American donors (n=90) were antigen-negative matches. For the second most prevalent phenotype, 0.08% Caucasian (n=3) and 33% of African-American (n=167), 2% of Hispanic (n=13), and 5% other (n=5) were antigen-negative matches. For the third and fourth prevalent phenotypes, 47% of African-American (n=233) and 23% (n=115) respectively, were antigen-negative matches, while only 0.14% (n=5) and 0.06% (n=2) of Caucasians, but 5% (n=31) and 2% (n=13) of Hispanic donors were appropriate matches, respectively. For the thalassemia patients, antigen matches for the most common phenotype were found most often in Asian (13%) and donors identifying as “other” (6%). Matches were present in only 2% of the Caucasians, 2% of Hispanics, and 1.8% of African-American donors for thalassemia patients. These results confirmed the importance and impact of African-American donors for extended antigen-matching for patients with SCD. Less than 1% of our Caucasian donors could serve as extended matching for SCD. Nearly 20% of patients with SCD are negative for a common group of antigens, which allows future donor recruitment efforts focused on extended antigen profiles of the donor. Patients with thalassemia do not have a common antigen-negative profile, but extended matching for these patients can be improved by increase recruitment of Asian donors. High throughput genotyping enables typing of large numbers of donors, and potentially the majority of the donor inventory. Analysis of antigen-negative phenotypes in the donor pool with analysis of patient groups is important for inventory management, focused donor recruitment, and improved transfusion practice by avoiding alloimmunization. Disclosures: Stassinopoulos: Cerus: Employment, Equity Ownership, Patents & Royalties.

Abstract 3239 Red blood cell (RBC) transfusion is a key treatment for patients with sickle cell disease (SCD), but remains complicated by the high incidence of RBC immunization. Despite provision of phenotypic Rh D, C, and E antigen-matched donor RBCs, patients continue to develop Rh antibodies. In many cases, these antibodies are considered autoantibodies with specificities for D, C, and e antigens because the patient's own RBCs type serologically positive for the corresponding antigen. Recent evidence is emerging that Rh alloimmunization within populations of African origin is complicated by the genetic diversity of this locus. Individuals of African ancestry often carry RH alleles that differ from those defined as conventional alleles that are common in Europeans and other ethnic groups. These “variant” alleles encode Rh proteins, often with multiple amino acid changes, that either lack common epitopes or produce novel immunogenic epitopes. The clinical significance of Rh alloimmunization in patients with SCD with variant RH genes is largely unknown. In the present study, we performed RH genotyping in 212 patients with SCD to determine prevalence of RH variants, the association with Rh alloimmunization, and the clinical significance measured by changes in hematologic parameters at time of antibody detection. RH genotyping was performed by polymerase chain reaction (PCR) amplification using RHD-specific and RHCE-specific primers designed in the flanking intronic regions and analyzed by direct sequencing of exons, and/or a combination of multiple PCR-restriction fragment length polymorphism (RFLP) assays and by RHD and RHCE BeadChip arrays. We identified variant RH alleles in 88.7% (188/212) of patients with SCD. Twelve different RHD and 13 RHCE alleles encoding variant Rh D, C, and e antigens were represented in this cohort of patients. In 172 patients with >1 RBC transfusion (median 125 units), 55 antibodies were identified with Rh specificity despite antigen-positive status (28 anti-D, 16 anti-e, 9 anti-C, and 2 anti-E). RH genotypes revealed 47.3% of these antibodies developed in patients who lacked the corresponding conventional RH allele, and would be considered Rh alloantibodies. In 43.6%, RH genotypes predicted expression of the conventional antigen to which the antibody was directed, suggesting these were potentially autoantibodies. However, these patients had at least one other RH allele that was altered. This may suggest that the presence of one variant protein potentially changes the Rh complex in the membrane, and carrying at least one conventional RH allele is not necessarily protective against production of Rh specific antibodies. In the remaining 9.1%, we detected no variant RHD or RHCE alleles, and complete gene sequencing is in progress to confirm the absence of novel mutations. Importantly, to determine clinical significance, we evaluated whether Rh antibodies in patients carrying variant alleles caused decreased transfused RBC survival by comparing the patient's hematologic parameters at the time of antibody detection with the baseline pre-transfusion parameters. In all but 4 cases, Rh antibodies in antigen-positive patients occurred in chronically transfused patients. Therefore, baseline values were determined from the average pre-transfusion hemoglobin, hematocrit and % hemoglobin S level for the 6–12 months preceding antibody detection. Compromised transfused RBC survival was determined by a lower hemoglobin/hematocrit or higher % hemoglobin S level at time of antibody formation compared to the patient's baseline. Forty percent of antibodies were associated with delayed hemolytic transfusion reactions or decreased transfused RBC survival. Our data suggest that the high prevalence of variant RH alleles in patients with SCD is associated with clinically significant immunization. Discrimination of allo- versus auto- antibodies in this chronically transfused patient population presents a significant technical challenge and suggests a role for RH genotyping in the clinical evaluation of Rh antibodies and to improve RBC matching. Importantly, in this study Rh antibodies in patients with variant RH often compromised transfused RBC survival and, therefore, were clinically significant and may be targets for prevention strategies analogous to standard phenotype matching for C, E, and K. Disclosures: No relevant conflicts of interest to declare.