BACKGROUNDRHCE*ceAG has the nucleotide change c.254C>G, which encodes p.Ala85Gly associated with altered expression of e antigen. We analyzed serologic and DNA-based testing data on samples with RHCE*ceAG to determine its effect on antigen expression, linkage with RHD, and its prevalence in African Americans.STUDY DESIGN AND METHODSSerologic testing was performed by standard methods. Genomic DNA was used for polymerase chain reaction-restriction fragment length polymorphism, RH-specific exon sequencing, and RHD zygosity, and Rh-cDNA was sequenced. Samples from 32 individuals referred for serologic problems, 57 patients with sickle cell disease, and 44 donors positive for c.254C>G were investigated. Allele prevalence was determined in random African Americans.RESULTSRed blood cells from samples homozygous RHCE*ceAG/ceAG or in trans to RHCE*cE reacted variably with anti-e reagents and 17 samples from the 32 referred patients had alloanti-e in their plasma. The majority of samples with RHCE*ceAG, when tested for RHD zygosity gave discordant results between PstI-RFLP and hybrid box assay. Rare samples with 254C>G had additional allelic changes: one with c.697G (p.233Glu), three with c.733G, 941C (p.245Val, 314Ala), and two with c.307T (p.103Ser) encoding robust C antigen expression in the absence of other C-specific nucleotides. A total of 101 samples with RHCE*ceAG were encountered in 1159 randomly selected African Americans.CONCLUSIONSRHCE*ceAG (c.254G, p.85Gly) encodes a partial phenotype and the absence of the high-prevalence antigen RH59 (CEAG). The allele was present in one in 11 African Americans and is most often in cis to a RHD deletion associated with discordant RHD zygosity. To further determine clinical significance, detection of this allele should be part of routine RHCE genotyping in this population.

Abstract The JR blood group system (ISBT 032) consists of one antigen, Jra, which is of high prevalence in all populations. The rare Jr(a–) phenotype has been found mostly in Japanese and other Asian populations, but also in people of northern European ancestry, in Bedouin Arabs, and in one Mexican. Anti-Jrahas caused transfusion reactions and is involved in hemolytic disease of the fetus and newborn. The Jraantigen is located on ABCG2 transporter, a multipass membrane glycoprotein (also known as the breast cancer resistance protein, BCRP), which is encoded by the ABCG2gene on chromosome 4q22.1. The Jr(a–) phenotype mostly results from recessive inheritance of ABCG2null alleles caused by frameshift or nonsense changes. Immunohematology2013;29:63–68.

BACKGROUND:Anti-Lan has been implicated in hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The LAN blood group system is encoded by ABCB6, whose gene product, ABCB6, belongs to the ATP-binding cassette (ABC) efflux transporter superfamily. The purpose of this study was to characterize additional alleles by analyzing DNA from 14 (13 unrelated) subjects whose red blood cells were serologically defined as Lan-, Lan+(w) /-, or Lan+(w) . STUDY DESIGN AND METHODS:Genomic DNA was extracted from blood samples recovered from liquid nitrogen storage. Intronic primers flanking each of the ABCB6 coding exons were used for polymerase chain reaction amplification. Amplicons were sequenced and analyzed by standard methods. RESULTS:Among the study subjects, we identified five alleles (one with a nonsense change, three with frameshifts, one with a missense change) that encode the Lan- phenotype and four alleles (with missense changes) encoding either Lan+(w) or Lan+(w) /- phenotypes. CONCLUSIONS:Of the nine alleles we identified, three were novel and six were previously documented in the dbSNP. Of these six, only one allele was previously associated with Lan negativity. To date, 19 ABCB6 alleles that encode Lan- or Lan+(w) /-, or Lan+(w) phenotypes have been described.

Emily Cooley was a well-respected medical technologist and morphologist with a remarkable skill set. She was highly regarded both professionally and personally. The "Emily Cooley Lectureship and Award" was established to honor her in particular and medical technologists in general. This article first reviews the history of the Emily Cooley award and provides some of the reasons why it carries her name. Then, using two blood group systems, DO and JR, it illustrates how many discoveries regarding blood groups were dependent on access to techniques.

BackgroundRHCE*ceMO has nucleotide changes 48G>C and 667G>T, which encode, respectively, 16Cys and 223Phe associated with altered expression of e antigen. RHD*DAU0 has Nucleotide 1136C>T, which encodes 379Met associated with normal levels of D. We compiled serologic and DNA testing data on samples with RHCE*ceMO to determine the red blood cell (RBC) antigen expression, antibody specificity, RHD association, and the prevalence in African‐American persons.Study Design and MethodsSerologic testing was performed by standard methods. Genomic DNA was used for polymerase chain reaction–restriction fragment length polymorphism and RH–exon sequencing, and for some, Rh‐cDNA was sequenced. Seventy‐seven (50 donor and 27 patient) samples with RHCE*ceMO were studied, and 350 African‐American persons were screened for allele prevalence.ResultsRBCs from RHCE*ceMO homozygotes (or heterozygotes with RHCE*cE in trans) were weak or nonreactive with some anti‐e and were nonreactive with polyclonal anti‐hrS and anti‐hrB. Twenty‐three transfused patients homozygous for RHCE*ceMO/ceMO or with RHCE*ceMO in trans to RHCE*cE or *ce had alloanti‐e, anti‐f, anti‐hrS/hrB, or an antibody to a high‐prevalence Rh antigen. Three patients with alloanti‐c had RHCE*ceMO in trans to RHCE*Ce. RHD*DAU0 was present in 30% of African‐American persons tested and in 69 of 77 (90%) of samples with RHCE*ceMO.ConclusionsRHCE*ceMO encodes partial e, as previously reported, and also encodes partial c, a hrS– and hrB– phenotype, and the absence of a high‐prevalence antigen (RH61). The antibody in transfused patients depended on the RHCE allele in trans. RHCE*ceMO was present in one in 50 African‐American persons with an allele frequency of 0.01, is often linked to RHD*DAU0, and is potentially of clinical significance for transfusion.

BackgroundThe ABCG2 gene encodes antigens of the JR blood group system. Red blood cells (RBCs) from individuals homozygous for ABCG2 null alleles are nonreactive with polyclonal and monoclonal anti-Jr(a). However, some RBCs have been defined as Jr(a+(W)/-) or Jr(a-), particularly when tested with polyclonal anti-Jr(a). In an effort to resolve these apparent serologic ambiguities, the current study was undertaken.Study Design and MethodsHemagglutination of RBCs from two individuals known to express a single copy of functional ABCG2 were compared to RBCs from eight unrelated, previously characterized, Jr(a+(W)/-) donors. Standard polymerase chain reaction-based methods were used to characterize ABCG2 alleles.ResultsTwo monoclonal anti-Jr(a) clones agglutinated RBCs from the eight Jr(a+(W)/-) study subjects. Two of these subjects were homozygous for a missense ABCG2 change (c.1858A; Asp620Asn). Two were heterozygous for two missense changes; one was c.1858G>A and c.421C>A (Asp620Asn; Gln141Lys), and the other was c.1714A>C and c.421C>A (Ser572Arg; Gln141Lys). The remaining four subjects were heterozygous for c.421C>A (Gln141Lys), and for one of four null alleles.ConclusionsWe have identified three ABCG2 alleles that are newly associated with weakened Jr(a) expression. One of these is novel, the missense allele c.1714A>C (Ser572Arg) and two that have been previously described c.421C>A (rs2231142; Gln141Lys) and c.1858G>A (rs34783571; Asp620Asn). In addition, we found a novel, presumed null allele, c.1017_1019delCTC (Ser340del).

Background: Red cell (RBC) blood group alloimmunization remains a major problem in transfusion medicine. Patients with sickle cell disease (SCD) are at particularly high risk for developing alloantibodies to RBC antigens compared to other multiply transfused patient populations. Hemagglutination is the classical method used to test for blood group antigens, but depending on the typing methods and reagents used may result in discrepancies that preclude interpretation based on serologic reactivity alone. Molecular methods, including customized DNA microarrays, are increasingly used to complement serologic methods in predicting blood type. The purpose of this study was to determine the diversity and frequency of RH alleles in African Americans and to assess the performance of a DNA microarray for RH allele determination.Material and methods: Two sets of samples were tested: (i) individuals with known variant Rh types and (ii) randomly selected African American donors and patients with SCD. Standard hemagglutination tests were used to establish the Rh phenotype, and cDNA- and gDNA-based analyses (sequencing, PCR-RFLP, and customized RHD and RHCE microarrays were used to predict the genotype).Results: In a total of 829 samples (1658 alleles), 72 different alleles (40 RHD and 32 RHCE) were identified, 22 of which are novel. DNA microarrays detected all nucleotides probed, allowing for characterization of over 900 alleles.Conclusions: High-throughput DNA testing platforms provide a means to test a relatively large number of donors and potentially prevent immunization by changing the way antigen-negative blood is provided to patients. Because of the high RH allelic diversity found in the African American population, determination of an accurate Rh phenotype often requires DNA testing, in conjunction with serologic testing. Allele-specific microarrays offer a means to perform high-throughput donor Rh typing and serve as a valuable adjunct to serologic methods to predict Rh type. Because DNA microarrays test for only a fixed panel of allelic polymorphisms and cannot determine haplotype phase, alternative methods such as Next Generation Sequencing hold the greatest potential to accurately characterize blood group phenotypes and ameliorate the clinical course of multiply-transfused patients with sickle cell disease. (c) 2013 Elsevier Inc. All rights reserved.

Blood groups are antigenic determinants on the surface of blood cells, encoded either by a single gene or by a cluster of closely linked, homologous genes. There are 30 blood group systems, some of which contain only one determinant while others contain many. The genes controlling blood group systems have been cloned and sequenced and located on specific chromosomes. There are additional blood group antigens, mostly of very high or very low prevalence, which have not been assigned to a system due to insufficient genetical evidence. Antibodies to blood groups are usually formed in response to antigen-positive red cells as the result of transfusion or pregnancy. Some of these antibodies are clinically relevant in terms of transfusion reactions and hemolytic disease of the fetus and newborn. The structure and function of most components carrying blood group antigens have been elucidated.

Terminal erythroid differentiation starts from morphologically recognizable proerythroblasts that proliferate and differentiate to generate red cells. Although this process has been extensively studied in mice, its characterization in humans is limited. By examining the dynamic changes of expression of membrane proteins during in vitro human terminal erythroid differentiation, we identified band 3 and α4 integrin as optimal surface markers for isolating 5 morphologically distinct populations at successive developmental stages. Functional analysis revealed that these purified cell populations have distinct mitotic capacity. Use of band 3 and α4 integrin enabled us to isolate erythroblasts at specific developmental stages from primary human bone marrow. The ratio of erythroblasts at successive stages followed the predicted 1:2:4:8:16 pattern. In contrast, bone marrows from myelodysplastic syndrome patients exhibited altered terminal erythroid differentiation profiles. Thus, our findings not only provide new insights into the genesis of the red cell membrane during human terminal erythroid differentiation but also offer a means of isolating and quantifying each developmental stage during terminal erythropoiesis in vivo. Our findings should facilitate a comprehensive cellular and molecular characterization of each specific developmental stage of human erythroblasts and should provide a powerful means of identifying stage-specific defects in diseases associated with pathological erythropoiesis.

BackgroundAntibodies to Kell antigens can be clinically important but only limited data are published regarding anti‐Ku. Missense nucleotide changes in KEL account for the numerous Kell antigens, the Kmod phenotype, and even the Knull phenotype.Study Design and MethodsDNA and RNA were extracted from white blood cells and polymerase chain reaction–based assays, cloning, and sequencing were done using standard protocols.ResultsThe anti‐Ku in Proband 1, which caused hemolytic disease and anemia of the fetus and newborn, was a mixture of immunoglobulin (Ig)G1 and IgG2 and gave macrophage indexes ranging from 47.8 to 59.3 (>20 is clinically significant) in a monocyte monolayer assay. The proband, her daughter, and compatible sister had a heterozygous deletion of a G in Exon 18 (Nucleotide c.1972_1975delG) in a KEL*02 allele causing a frameshift. The mechanism for silencing of the other KE*02 allele was undetermined. Proband 2 was heterozygous for a nonsense change (KEL*382C/T; Arg128Stop), a missense change (KEL*244T/C; Cys82Arg), and KEL*578T/C (KEL*01/KEL*02). Direct sequencing of cDNA and cloning showed that the KEL*01 allele had 244C, 382C, 578T and the KEL*02 allele carried 244T, 382T, 578C.ConclusionsWe report a novel single‐nucleotide deletion, a novel nonsense allele, and a novel missense allele all resulting in the Knull phenotype. The anti‐Ku from Proband 1 was clinically important.

BackgroundThe numerous antigens in the Kell blood group system result from missense nucleotide changes in KEL. Antibodies to antigens in this system can be clinically important. We describe six probands whose plasma contained antibodies to high-prevalence Kell antigens and discuss their relationship.Study Design and MethodsPolymerase chain reaction amplification, direct sequencing, restriction fragment length polymorphism assays, hemagglutination, flow cytometry, and protein modeling were performed by standard methods.ResultsProband 1 (KUCI) and her serologically compatible sister were heterozygous for a nucleotide change in Exon 11 (KEL*1271C/T; Ala424Val). Proband 2 (KANT) was heterozygous for KEL*1283G/T (Arg428Leu) and KEL*1216C/T (Arg406Stop) in Exon 11. Red blood cells (RBCs) from Proband 1 and her sister were not agglutinated by plasma from Proband 2; however, RBCs from Proband 2 were agglutinated by plasma from Proband 1. Probands 3, 4, 5, and 6 had the KEL*1391C>T change associated with the previously reported KETI- phenotype. Proband 5 was also homozygous for KEL*905T>C encoding the K11-K17+ phenotype. Hemagglutination studies revealed an association between KUCI, KANT, KETI, and K11. Protein modeling indicated that whereas Ala424 and Arg428 are clustered, Val302 and Thr464 are not.ConclusionAla424 in the Kell glycoprotein is associated with the high-prevalence Kell antigen, KUCI (ISBT 006032), which is detected by the antibody of Proband 1. Arg428 is associated with the high-prevalence Kell antigen, KANT (ISBT 006033). The association between KUCI, KANT, KETI, and K11 and the results of protein modeling are discussed.

Antigens in the SC blood group system are expressed by the human erythrocyte membrane-associated protein (ERMAP).Two molecular bases have been reported for the Sc,un phenotype:SC*307del2 and SC*994C>T. We report our investigation of the molecular background of five Sc,n1 individuals from the Pacific Islands and describe the successful transfusion of Sc3+ blood to a patient with anti-Sc3 in her plasma. SC (ERMAP) exons 2,3, and 12 and their flanking intronic regions were analyzed. TheSC*994C>T change introduces a restriction enzyme cleavage site for Tsp45I, and polymerase chain reaction (PCR) products from exon 12 were subjected to this PCR-restriction fragment length polymorphism (RFLP) assay. The five samples had the variant SC*994T/T. One sample, from a first cousin of one Marshallese proband, was heterozygous for SC*1514C/T (in the 3' untranslated region); the other four samples were SC*1514C/C(consensus sequence). Samples from white donors (n = 100) and African American donors (n = 99) were tested using the Tsp45IPCR-RFLP assay; all gave a banding pattern that was consistent with the SC*994C/C consensus sequence. In all five samples,our analyses showed homozygosity for the nonsense nucleotide change SC*994C>Tin an allele carrying the nucleotide associated with SLd. Further investigation determined that one of the probands reported previously with the SC*994C>T change was from the Marshall Islands (which form part of the Micronesian Pacific Islands) and the other was from an unspecified location within the large collection of Pacific Islands. Taken together, the five known probands with the SC*994C>T silencing nucleotide change were from the Pacific Islands.

This chapter discusses the Gerbich blood group system. The terminology, expression, database accession numbers, and the carrier molecule related to the Gerbich blood group system are also reviewed. The Gerbich blood group became a system in 1990. The function and disease association of this blood group system is also discussed. This blood group aids in the maintenance of the red blood cell (RBC) membrane integrity via interaction with protein 4.1. It also contributes to the negatively charged glycocalyx. Glycophorin C (GPC) and glycophorin D (GPD) are markedly reduced in protein 4.1-deficient RBCs and can be associated with hereditary elliptocytosis. The majority of RBC samples with Leach or Gerbich phenotypes have a weak expression of the Kell blood group system antigens. The chapter also discusses the terminology, occurrence, and expression of Ge2-, Ge3-, and Ge4-antigens.

The Dombrock (Do) glycoprotein is a glycosylphosphatidylinositol(GPI)-linked membrane protein carrying Dombrock blood group antigens. There are no standardized typing reagents for Do(a) or Do(b). We have developed ten different monoclonal antibodies(MoAbs) that are specific for Dombrock. The objectives of this study were to characterize these MoAbs serologically and determine the epitopes they recognize. MoAbs were generated by standard fusion methods. Mice were immunized with transfected human embryonic kidney 293T cells expressing high levels Do(a) or Do(b). The MoAbs were tested serologically with untreated and enzymatically or chemically modified red blood cells (RBCs).Serologic inhibition studies were performed with synthetic peptides corresponding to Do(a) and Do(b) amino acid sequences.Pepscan epitope analysis was done on an array of immobilized tridecapeptides corresponding to the full-length polypeptide. All ten antibodies were serologically specific for Dombrock. Eight of the antibodies recognized epitopes that were resistant to treatment with ficin, pronase, a-chymotrypsin, and neuraminidase,but sensitive to trypsin and 0.2 M dithiothreitol (DTT). Five have anti-Do(b)-like specificity. The epitope recognized by MIMA-52 was neuraminidase sensitive, and MIMA-127 epitope recognized a DTT-resistant, linear epitope (90)QKNYFRMWQK(99) of the Dombrock polypeptide. MIMA-127 was the only one of the ten Dombrock MoAbs mapped to a specific sequence of the Dombrock glycoprotein; the other nine MoAbs did not provide aspecific peptide binding pattern. The other MoAbs could not be mapped as they most likely recognize nonlinear, conformation-dependent epitopes, as is evident by their sensitivity to reduction of disulfide bonds by DTT. The dependence of some epitopes on antigen glycosylation is also a possibility.

Publisher Summary This chapter discusses the Knops blood group system. The terminology, expression, database accession numbers, and the carrier molecule related to the Knops blood group system are also reviewed. The complement receptor 1 (CR1) *1 allotype has 30 complement control protein repeats (CCPs) each comprising about 60 amino acids with sequence homology (also called short consensus repeats (SCRs) ). The function and disease association of the Knops blood group system is also discussed. CR1 binds C3b and C4b and has an inhibitory effect on complement activation by classical and alternative pathways, protecting red blood cells (RBCs) from autohemolysis. Erythrocyte CR1 is important in processing immune complexes by binding them for transport to the liver and spleen for removal from the circulation. Low levels of CR1 on RBCs may result in deposition of immune complexes on blood vessel walls with subsequent damage to the walls.

This chapter discusses: (i) the blood group systems; (ii) the blood group collections; (iii) the 700 series of low incidence antigens; and (iv) the 901 series of high incidence antigens. Within each system, facts are presented for individual antigens, listed in an international society of blood transfusion (ISBT) numerical order. The format for the facts sheets displaying the data about the systems and about the antigens is explained. The commonly used name for the blood group system is used at the top of each page. Some of the components carrying blood group antigens have been detected on blood cells and tissues by use of various methods including testing with polyclonal antibodies and monoclonal antibodies or Northern blot analysis. The chromosome number, arm and band number are discussed. If the presence of a blood group antigen is detected by serological means, the gene is named by the corresponding ISBT system symbol followed by the antigen number. The organization of the gene in terms of number of exons, kilobase pairs of gDNA, and a map is provided.