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BACKGROUND: MER2 (RAPH1), the only antigen of the RAPH blood group system, is located on the tetraspanin CD151. Only four examples of alloanti-MER2 are known. We report here two new examples of alloanti-MER2, in women of Pakistani and Turkish origin, one of whom showed signs of a hemolytic transfusion reaction (HTR) after transfusion of 3 units of red cells (RBCs). STUDY DESIGN AND METHODS: Standard serologic methods were used. A monocyte monolayer assay (MMA) was used to assess the potential clinical significance of one of the antibodies. All exons and flanking intronic sequences of CD151 were amplified and sequenced. A homology model for CD151 second extracellular loop (EC2) was constructed based on the crystal structure of CD81. RESULTS: RBCs of both patients did not react with alloanti-MER2, and neither of their antibodies reacted with MER2-negative RBCs. The MMA results suggested that the antibody that appeared to have caused an HTR had the potential to be clinically significant. Both patients were homozygous for a 511C>T mutation in CD151 encoding an Arg171Cys change. This change did not result in any significant structural rearrangement in the protein model. CONCLUSIONS: Two MER2-negative patients with anti-MER2 are homozygous for the same novel mutation encoding an amino acid substitution in the EC2 of CD151. One of the antibodies may have been responsible for an HTR, and crossmatch-compatible RBCs should be recommended for transfusion to patients with anti-MER2.

BACKGROUND: In 1994 during the investigation of a case of hemolytic disease of the newborn, a new lowincidence red cell (RBC) antigen, LOCR, was described. Although the presence of LOCR was associated with altered expression of Rh antigens, its formal assignment to the Rh blood group system did not occur until haplotype and linkage analysis conducted in 2003 provided the necessary proof. The current study was undertaken in an attempt to define the underlying RH mutation in LOCR+ individuals. STUDY DESIGN AND METHODS: Genomic DNA from five unrelated LOCR+ individuals and three Rh-matched control individuals was amplified by polymerase chain reaction with intronic primers flanking all 10 exons of RH. Amplified products were separated on 1 percent agarose gels and isolated for DNA sequence analysis in both the forward and the reverse directions. RESULTS: DNA sequence analysis of the three LOCR+ D‐ individuals revealed a single heterozygous 286G>A nucleotide substitution resulting in a predicted Gly>Ser substitution at amino acid 96. DNA sequence analysis from the two LOCR+ D+ individuals revealed the identical mutation, as well as all of the changes associated with the common RHD gene. CONCLUSIONS: Based on our results, a Gly96Ser substitution in the Rhce polypeptide defines the lowincidence RBC antigen known as LOCR. This same amino acid change has previously been shown to be involved in the Rh:-26 phenotype, which suggests that LOCR and Rh26 are antithetical. Serologic investigations with various Rh:-26 cells and serum samples, however, reveal that only some c+ Rh:-26 phenotypes are LOCR+. ltered expression of primary (D, C, c, E, or e) Rh blood group system antigens in the presence of a low-incidence antigen is typically the first evidence that a “new” antigen is a member of the Rh system. The list of antigens that fall into this group include C w (Rh8), C x (Rh9), E w (Rh11), D w (Rh23), Go a

The Redelberger antigen (Rba) was first discovered in 1974 on the RBCs of a blood donor who was an employee of the Community Blood Center in Dayton, Ohio. The discovery was made as a result of the investigation of a reagent contamination problem. Two examples of the Rba antigen were subsequently identified in the United Kingdom, but no “new”examples have been identified in the United States or Europe. Anti-Rba is a commonly occurring antibody, often found in combination with other antibody specificities, especially in combination with other antibodies to low-incidence antigens. Immunohematology 2006;22:48–51.

G. L. Daniels (Chair), D. J. Anstee, J. P. Cartron, W. Dahr, A. Fletcher, G. Garratty, S. Henry, J. Jorgensen, W. J. Judd, L. K ornstad, C. Levene, M. Lin, C. Lomas-Francis, A. Lubenko, J. J. Moulds, J. M. Moulds, M. Moulds, M. Overbeeke, M. E. Reid, P. Rouger, M. Scott, P. Sistonen, E. Smart, Y. Tani, S. Wendel & T. Zelinski*

Typing for antigens in the Dombrock blood group system and identifying the corresponding antibodies are notoriously difficult tasks. The reagents are scarce and the antibodies are weakly reactive. When RBCs from family members of a patient with an antibody to a high-prevalence Dombrock antigen were tested for compatibility, an unusual pattern of inheritance was observed: RBCs from the patient’s children and one niece, in addition to those from some of the patient’s siblings, were compatible. This prompted the performance of DNA-based assays for DO alleles and the results obtained were consistent with and explained the compatibility test results. It was possible to study this large kindred because of the cooperation of family members, hospital personnel, and reference laboratory staff. Immunohematology 2006;22:69–71.

Investigation of a mild case of hemolytic disease of the newborn has led to recognition of a ‘new’ low-incidence red cell antigen, WARR (ISBT No. 700.55). Data gleaned from two kindreds, both with Native American heritage, exclude WARR from the MNS, Lutheran, Lewis, Duffy, Kidd, Xg, Chido/Rodgers, Kx and Gerbich blood group systems. Serologic or genetic evidence suggests it is not part of the Kell or Yt systems.

RH43 (Crawford) is encoded by RHCE*ce with nucleotide changes 48GC, 697CG, and 733CG (RHCE*ceCF). We investigated the Rh antigen expression and antibody specificities in four patients with this allele.Hemagglutination tests, DNA extraction, polymerase chain reaction (PCR)-restriction fragment length polymorphism, allele-specific PCR, reticulocyte RNA isolation, reverse transcription-PCR cDNA analyses, cloning, and sequencing were performed by standard procedures.Red blood cells (RBCs) from two patients typed D+C-E-c+e+/-, hrS-/+W, hrB- and their serum was reactive (3+) with all RBC samples of common Rh phenotype tested, but nonreactive with Rhnull or D-- RBCs (apparent alloanti-Rh17). At the RHCE locus, Patient 1 was homozygous for RHCE*ceCF, and Patient 2 inherited RHCE*ceCF in trans to a silenced RHCE*cE. Cross-testing of serum and RBCs from these two samples showed mutual compatibility, indicating that both antibodies define the same novel high-prevalence antigen on Rhce. Two additional patients, one whose serum contained alloanti-c but the RBCs typed C+c+ and one whose serum contained anti-e but the RBCs typed E+e+, also had RHCE*ceCF. RHCE*Ce was present in trans in the former and RHCE*cE in the latter patient.We report that amino acid changes on RhceCF (Trp16Cys, Gln233Glu, and Leu245Val) alter the protein to the extent that c and e antigens are partial, and a high-prevalence antigen, we have named CELO (provisional ISBT Number 004058; RH58) is not expressed. CELO is antithetical to RH43 (Crawford).

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

A multilaboratory investigation during several years has identified a low incidence antigen JAL on the red cells of 7 propositi. JAL appears to be associated with two unusual Rh complexes, one of which produces a depressed C antigen and the other a depressed c antigen. Family studies strongly suggest that the JAL antigen is encoded by the RH locus. Anti-JAL has been implicated in haemolytic disease of the newborn and is thus considered to be a clinically significant antibody.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder associated with absence of expression of phosphatidylinositol (PI)- linked membrane proteins from circulating hematopoietic cells of multiple lineages. Recent work demonstrated that decay accelerating factor, one such PI-linked protein, bears the Cromer-related blood group antigens. This study demonstrated that other high incidence antigens, including Cartwright (Yta/Ytb), Holley-Gregory (Hy/Gya), John Milton Hagen (JMH), and Dombrock (Doa/Dob), are absent from the complement-sensitive (PNH III) erythrocytes of patients with PNH. The relatively normal, complement-insensitive erythrocytes from the same patients express these antigens normally. Therefore, these antigens most likely reside on PI-linked proteins absent from PNH III, but not PNH I, erythrocytes.

BACKGROUND: Semaphorin 7A (Sema7A), the protein that carries the JMH blood group antigen, is involved in immune responses and plays an important role in axon growth and guidance. Because previous serologic studies on red blood cells (RBCs) suggested a considerable diversity of Sema7A, the present study was designed to elucidate the complex picture of the molecular diversity of this protein. STUDY DESIGN AND METHODS: The JMH antigen status was determined by serology, flow cytometry, and Western blot. Genomic and transcript analysis of SEMA7A was performed by nucleotide sequencing. Recombinant Sema7A proteins were used for genotype-phenotype correlation. A three-dimensional model of Sema7A was generated for topologic analyses. RESULTS: Our studies on 44 individuals with unusual JMH phenotypes and their family members revealed that aberrant Sema7A expression can be an inherited or an acquired phenomenon and is based on reduced surface expression or qualitative changes in Sema7A. These different phenotypes are caused by variations of the SEMA7A gene or seem to be generated by autoimmune-related or RBC lineage-specific mechanisms. The variant JMH phenotypes were related to the presence of missense mutations in SEMA7A, predicting amino acid changes in the semaphorin domain of Sema7A. Sequence analysis of the variant SEMA7A alleles revealed mutations affecting codons 207 and 460/461. Topologic analyses showed that Sema7A polymorphisms were prominently located on the top and bottom of the semaphorin domain, suggesting a functional relevance of these sites. CONCLUSION: These findings provide a basis with which to delineate the various ligand-binding surfaces of Sema7A.

1 Bristol Institute for Transfusion Sciences, Bristol, UK 2 Growing your Knowledge, Spit Junction, NSW, Australia 3 American Red Cross Blood Services, Los Angeles-Orange Counties Region, Los Angeles, CA, USA 4 Biotechnology Research Centre, Auckland University of Technology, Auckland, New Zealand 5 Regional Blood Transfusion Center, Department of Clinical Immunology, University Hospital, Arhus N, Denmark 6 Department of Pathology, University Hospitals UH-2G332, Ann Arbor, Michigan, USA 7 Reference Laboratory for Immunohematology and Blood Groups, National Blood Services Centre, Tel Hashomer, Israel 8 New York Blood Center, New York, NY, USA 9 Ortho-Clinical Diagnostics, Raritan, NJ, USA 10 Drexel University College of Medicine, Philadelphia, PA, USA 11 Gamma Biologicals Inc (subsidiary of Immunocor Inc), Houston, TX, USA 12 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands 13 Centre national de Reference pour les Groupes sanguines (CNTS), Paris, France 14 International Blood Group Reference Laboratory, Bristol, UK 15 Finnish Red Cross Blood Transfusion Service, Helsinki, Finland 16 South African National Blood Service, East Coast Region, Pinetown, South Africa 17 Osaka Red Cross Blood Center, Osaka, Japan 18 Blood Bank, Hospital Sirio-Libanes, Sao Paulo, Brazil 19 Rh Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada

Proteins G and A coated on agarose have been extensively used in affinity chromatography. Protein G will bind to all four subclasses of human IgG and protein A to the subclasses IgG1, IgG2, and IgG4. This IgG binding ability of protein G and protein A has been used in a red cell affinity column technology developed for the detection and identification of IgG red cell antibodies. When serum or plasma is incubated in a microcolumn with red blood cells (RBCs) that express the appropriate antigens, the antibodies become attached to the RBC surface. When the microcolumns are centrifuged, the RBCs pass through a viscous barrier into an active matrix containing proteins G and A. Positive tests adhere at the top of the gel and negative tests pass through, settling to the bottom. This study was undertaken to compare affinity column technology with low-ionic saline solution (LISS) tube tests in a reference laboratory setting. Over a 1-year period, 314 samples were tested in parallel by affinity column technology and by LISS tube technique. Both methods detected antibodies directed at common RBC antigens, high-incidence and low-incidence RBC antigens, and warm-reacting autoantibodies. IgM antibodies were not detected by affinity column technology. Affinity column technology compares favorably with the LISS tube technique for IgG antibody detection and identification.

Since the cloning in 1990 of complementary DNA corresponding to messenger RNA transcribed at the blood group ABO locus, polymorphisms and phenotype-genotype correlations have been reported by several investigators. Exons 6 and 7, constituting 77% of the gene, have been analyzed previously in samples with variant phenotypes but for many subgroups the molecular basis remains unknown. This study analyzed 324 blood samples involved in ABO grouping discrepancies and determined their ABO genotype. Samples from individuals found to have known subgroup alleles (n = 53), acquired ABO phenotypes associated with different medical conditions (n = 65), probable chimerism (n = 3), and common red blood cell phenotypes (n = 109) were evaluated by ABO genotype screening only. Other samples (n = 94) from apparently healthy donors with weak expression of A or B antigens were considered potential subgroup samples without known molecular background. The full coding region (exons 1-7) and 2 proposed regulatory regions of the ABO gene were sequenced in selected A (n = 22) or B (n = 12) subgroup samples. Fifteen novelABO subgroup alleles were identified, 2 of which are the first examples of mutations outside exon 7 associated with weak subgroups. Each allele was characterized by a missense or nonsense mutation for which screening by allele-specific primer polymerase chain reaction was performed. The novel mutations were encountered in 28 of the remaining 60 A and B subgroup samples but not among normal donors. As a result of this study, the number of definable alleles associated with weak ABO subgroups has increased from the 14 previously published to 29.

A survey of sera containing antibodies to multiple low-incidence antigens revealed a variety of patterns of reactions with NFLD+, BOW+, and Wu+ red cell samples. Although NFLD, BOW, and Wu are distinct antigenic determinants (International Society of Blood Transfusion numbers 700.37, 700.46, and 700.13, respectively), their ability to absorb and elute "crossreactive" antibodies indicates a serologic and possibly a genetic relationship among the three.

BACKGROUND: Reports published in 1976 and 1980 described the low- incidence red cell antigen Wu. Distinction of Wu from all other known low-incidence antigens and from the ABO, Rh, Lutheran, Duffy, Kidd, P, and X-linked blood group systems allowed Wu to be placed in the International Society of Blood Transfusion's 700 series, designated as 700013. Recently, a blood donor apparently homozygous for Wu has been identified. This report documents the serologic and molecular findings of samples from this individual and the members of his family. STUDY DESIGN AND METHODS:Blood samples from 26 members of a kindred of Dutch descent segregating for Wu were collected and analyzed. Red cells were subjected to titration and enzymatic tests, while DNA was analyzed by polyacrylamide gel electrophoresis for single-strand conformational polymorphism (SSCP) and nucleotide differences by DNA sequencing. RESULTS:Serologic investigations conducted on red cells of the propositus and two of his siblings consistently revealed higher titers with various sera containing anti-Wu than did cells from their parents or children. Treatment of intact red cells with alpha-chymotrypsin completely abolished Wu recognition. Because erythroid band 3 is cleaved by alpha-chymotrypsin, the possible relationship between Wu and AE1 (the gene controlling erythroid band 3 expression) was investigated by molecular methods. SSCP analysis of DNA revealed that all Wu+ family members exhibited a mobility shift in exon 14 of AE1. The nature of the SSCP was defined by DNA sequencing as a G–>C mutation that resulted in a Gly565–>Ala substitution in human erythroid band 3. CONCLUSIONS:Three members of the kindred are homozygous for the low-incidence red cell antigen Wu. A G–>C mutation in AE1 gives rise to a Gly565–>Ala substitution in band 3, thereby accounting for the Wu red cell polymorphism. In light of these findings, the International Society of Blood Transfusion Working Party has provisionally assigned Wu to the Diego blood group system (designated 010009 or D19).

Concordance of reactions of Wu+ and ‘Hov’+ cells with 153 sera containing multiple specificities to low-incidence antigens indicates that the ‘two’ antigens are identical. This conclusion is confirmed by absorption and elution studies.

Allo-anti-Chido (Ch) was detected in a patient whose red cells typed as Ch+. The C4 allotype of the patient was A4,B2 which associates strongly with the Ch phenotype Ch:1,-2,3,4,-5,6. Anti-Ch2 + Ch5 were the Ch specificities identified. Absence of only Ch2 and Ch5 determinants on the C4B protein allowed this unique immune response to blood transfusion.

For some time, anomalous serological reactions have been observed when the same anti-Swa sera are tested against red cells from different individuals reported as Sw(a+). A comparative collaborative study using the same collection of Sw(a+) cells and anti-Swa sera was undertaken by 4 reference laboratories, and it was found that Swa represents a heterogeneous group of antigens that can be subdivided into two categories. Both categories, Sw(a+) 700:41 and Sw(a+) 700:-41, were shown to be inherited.

Red cell membranes from patient NE, Mr. Dantu and 16 additional Black individuals, positive for the low-frequency MNSs-system antigen Dantu, were studied by dodecylsulfate polyacrylamide gel electrophoretic techniques. The content of the major, blood group M- or N-active sialoglycoprotein (glycophorin A, GP A) was found to be decreased by about 57%. The blood group S- or s-active sialoglycoprotein (GP B) was decreased by about 51% in membranes from proven Dantu/U heterozygotes and not detectable in those from patient NE and other Dantu + U − individuals. Donor NE was shown to exhibit the genotype Dantu/u. Dantu-positive cells exhibit a proteinase-resistant GP B-GP A hybrid with an apparent molecular mass of 29 KDa whose intramembraneous and cytoplasmic domains were shown to be similar to those of GP A. The molar hybrid: GP A ratio in all cells was found to be about 2.4: 1, indicating that the NE variety of the Dantu phenotype is much more frequent than the Ph or MD types. The significance of an additional minor ‘new’ component (molecular mass 21 KDA) in Dantu + membranes and the minor component J (molecular mass 22 KDa) occurring in normal and Dantu + U + GP preparations, but not in those from Dantu + U − cells, has not been resolved. The apparent molecular mass of the anion channel protein (band 3) in all cells of the NE variety was shown to be decreased by about 3 KDa, due to a shortening of carbohydrate chains. This suggests that the hybrid, just like GP A, might form a complex with band 3.

Three examples of an antibody were found to be detecting a red cell polymorphism probably identical to MER2. The antibodies were made by Jews originating from India and living in Israel. Two of them were sibs and the third was unrelated. All 3 had kidney disease requiring renal dialysis and regular blood transfusion. In 2 cases the antibodies were detected before dialysis was started and before the patients had been transfused. The human antibodies reacted with red cells of 90% of Israeli blood donors tested. In tests on selected blood donors, 82 English and 56 Israeli, one of the human antibodies gave almost identical reactions to those given by monoclonal anti-MER2. Anomalous reactions were probably due to anti-Bga. Two of the human antibodies completely blocked, and one partially blocked, the reaction of monoclonal anti-MER2 with MER2+ red cells.

The sialoglycoproteins (glycophorins) in human red cell membranes of rare individuals lacking totally (Ge-1,-2,-3 phenotype) or partially (Ge-1,-2,3 phenotype) the Gerbich (Ge) blood group antigens and two Ge-1,-2,-3 heterozygotes were studied by dodecylsulfate polyacrylamide gel electrophoretic techniques. Two sialoglycoproteins (components D and E) were not detectable in the membranes from the homozygotes and found to be decreased by about 50% in those from the heterozygotes. Ge--1,-2,-3 and Ge-1,-2,3 cells were found to contain a 'new' component (mol. masses about 29 and 30 kDa, respectively) possibly representing a D/E hybrid molecule. This sialoglycoprotein was not detectable in membranes from the Ge-1,-2,-3 heterozygotes, suggesting that the Ge-1,-2,-3 phenotype may be caused by at least two different alleles at the Ge blood group antigen locus. Hemagglutination or hemagglutination inhibition tests involving anti-Ge 1,2,3 and -Ge 1,2 as well as native and enzyme-treated normal red cells (phenotype Ge 1,2,3) or membrane and sialoglycoprotein fractions from normal erythrocytes indicate that the receptors of these sera are located within the glycosylated domain(s) of the D and/or E sialoglycoprotein(s). Our data suggest that the Ge locus encodes the polypeptide sequences of the D and E sialoglycoproteins.

Anti-Au, the first example of the antithetical antibody to anti-Au, was identified in the serum of a blood donor who had been transfused 16 years previously. Au has a gene frequency of 0.4326 in an American black population and 0.2994 in a southern English donor population. The expression of Au is suppressed by In(Lu). XS2 also suppresses Auberger antigen expression.