RHD sequencing: a new tool for decision making on transfusion therapy and provision of Rh prophylaxis.

The serological differentiation of weak D from partial D, D-negative and D-positive is not always unequivocal. Therefore, sequencing of the RHD gene is required in some cases. Very recently, several new differences between RHD and RHCE have been identified which permitted us to design primers close to the exon/intron boundaries of the RHD-exons. We evaluated these primers in 83 D-positive and 18 D-negative blood donors and applied the new method for the characterization of the RHD gene in six individuals with weak D phenotype. The amplification reactions were concordant with serological findings in 100 of 101 donors (99.0%). In one D-positive donor the PCR for exons 2 and 5 gave a negative result, while the sequence of the remaining eight exons was unchanged. By sequencing samples with very weak D serological reactions, we identified weak D type 4.2.2 and weak D type 15, both previously reported to be associated with anti-D-alloimmunization. Consequently, we recommended the selection of D-negative blood in the weak D type 4.2.2 patient, and the provision of Rh prophylaxis for pregnant women with weak D type 15. In summary, a new RHD sequencing method was developed which can be applied if serological reactions are inconclusive.

MAM: a "new" high-incidence antigen found on multiple cell lines.

BACKGROUND: Three women have been identified with an antibody to a "new" high-incidence antigen found on multiple cell lines. CASE REPORTS: The proposita, M.A.M., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own. She delivered a thrombocytopenic infant with a 3+ DAT, but without symptoms of HDN. The second example, A.N., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own and those of M.A.M. She delivered a slightly thrombocytopenic but severely anemic infant. The third example, F.K., a sister of A.N., has an antibody reacting with all RBCs tested except her own and those of M.A.M. and A.N. CONCLUSION: This "new" high-incidence antigen has been named MAM and assigned high-incidence antigen number 901016 by the International Society of Blood Transfusion. The corresponding antibody, anti-MAM, has been shown to cause HDN and has the potential to shorten RBC survival after the transfusion of incompatible RBC units, as determined by monocyte monolayer assay. Immunoblotting and flow cytometry show that this new antibody reacts with various WBC lines in addition to RBCs. This antibody also appears to react with platelets in some assays.

The low-frequency MNS blood group antigens Ny(a) (MNS18) and Os(a) (MNS38) are associated with GPA amino acid substitutions.

BACKGROUND: Antigens of the MNS blood group system are located on two sialoglycoproteins, GPA and GPB, encoded by GYPA and GYPB. The molecular backgrounds of the low-frequency antigens Ny(a) and Os(a) are not known. STUDY DESIGN AND METHODS: Immunoblotting and a monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay were used to analyze Os(a). PCR-amplified products of the coding exons of GYPA were studied by single-strand conformation polymorphism analysis, and exon 3 was sequenced. Synthetic peptides were used in hemagglutination-inhibition tests. RESULTS: Sequencing of GYPA exon 3 of two unrelated Ny(a+) persons revealed heterozygosity for a T194A base change encoding an Asp27Glu substitution. Immunoblotting with anti-Os(a) and an MAIEA assay with MoAbs to GPA showed that Os(a) is on GPA. Sequencing exon 3 of an Os(a+) person from the only family with Os(a) revealed heterozygosity for a C273T base change encoding a Pro54Ser substitution. A synthetic peptide representing part of GPA with the Os(a) mutation (VRTVYPSEEETGE) completely inhibited anti-Os(a), whereas the control peptide (VRTVYPPEEETGE) did not inhibit anti-Os(a). CONCLUSION: Ny(a) and Os(a) are low-frequency antigens of the MNS blood group system that represent Asp27Glu and Pro54Ser substitutions in GPA, respectively.

The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis.

BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood. STUDY DESIGN AND METHODS: Red cells from 100 black South Africans and 43 black persons from Amsterdam, the Netherlands, were typed serologically for various Rh system antigens. Allele-specific polymerase chain reaction and sequencing of polymerase chain reaction products were used to analyze C733G (Leu245Val) and G1006T (Gly336Cys) polymorphisms in exons 5 and 7 of RHCE and the presence of a D-CE hybrid exon 3. RESULTS: The respective frequencies of all VS+ and of VS+ V-(r's) phenotypes were 43 percent and 9 percent in the South Africans and 49 percent and 12 percent in the Dutch donors. All VS+ donors had G733 (Val245), but six with G733 were VS- (4 V+w, 2 V-). The four VS- V+w donors with G733 appeared to have a CE-D hybrid exon 5. T1006 (Cys336) was present in 12 percent and 16 percent of donors from the two populations. With only a few exceptions, T1006, a D-CE hybrid exon 3, and a C410T (Ala137Val) substitution were associated with a VS+ V-phenotype ((C)ces or r's haplotype). Two VS+ V-individuals, with the probable genotype, (C)ces/(C)ces), were homozygous for G733 and for T1006. CONCLUSIONS: It is likely that anti-VS and anti-V recognize the conformational changes created by Val245, but that anti-V is sensitive to additional conformational changes created by Cys336.

Decay-accelerating factor (CD55) deficiency phenotypes in Japanese.

Decay-accelerating factor (DAF, CD55) is a complement regulatory glycoprotein that expresses the Cromer-system blood group antigens. Two, very rare, inherited DAF-deficiency phenotypes, Inab and Dr(a-), were identified in Japanese propositi. Red cells of the Inab phenotype propositus had no Cromer-system antigens and did not bind monoclonal anti-DAF. The Inab propositus was homozygous for a DAF non-sense mutation, converting the Trp53 codon to a stop codon; her parents were heterozygous for this mutation. This is the same mutation as that previously found in the original Inab phenotype propositus. Haemagglutination-inhibition titrations of the serum of the Inab propositus with soluble-recombinant DAF demonstrated that anti-IFC represents a mixture of antibodies to all four DAF short consensus repeat domains. The Dr(a-) individual had very low levels of Cromer-system antigens and DAF on her red cells. Loss of a TaqI restriction site from DAF exon 5 suggested that she has a previously detected mutation, encoding a Ser165Leu substitution. Red cells of the two propositi did not show abnormal levels of lysis in an acid lysis test, but after blocking of CD59 with monoclonal antibody, Inab phenotype red cells showed more lysis than Dr(a-) red cells, and Dr(a-) cells showed substantially more lysis than control cells.

Hemagglutination inhibition of Cromer blood group antibodies with soluble recombinant decay-accelerating factor.

BACKGROUND: Cromer blood group antigens are located on decay-accelerating factor (DAF, CD55), which contains four short consensus repeats (SCRs). Cromer system antibodies may be of clinical significance in blood transfusion. STUDY DESIGN AND METHODS: Soluble recombinant DAF (srDAF) constructs, consisting of all four SCRs or of only two SCRs, were expressed in the yeast Pichia pastoris. They are used in hemagglutination-inhibition tests with Cromer system antibodies and with DAF-specific monoclonal antibodies. RESULTS: The srDAF inhibited hemagglutination by all Cromer system alloantibodies in undiluted serum. Antibodies to antigens of other blood group systems were not inhibited by the srDAF. Hemagglutination-inhibition tests with domain-deleted srDAF showed that UMC is on SCR-4 and confirmed that Tca, TcaTcb, and WESb are on SCR-1; Dra is on SCR-3; and Cra is on SCR-4. CONCLUSIONS: Hemagglutination inhibition with srDAF is useful in the recognition of antibodies that belong to the Cromer blood group system and facilitates pretransfusion testing. This use of domain-deleted srDAF provides an easy method of determining epitope location on DAF and is an aid to more precise identification of Cromer system antibodies.

A clinically applicable method for determining the three major alleles at the Duffy (FY) blood group locus using polymerase chain reaction with allele-specific primers.

BACKGROUND: The clinically significant antigens of the Duffy (Fy [FY]) blood group system are expressed on the red cell form of the FY glycoprotein, a promiscuous chemokine receptor and also a receptor for malarial parasites. After the cloning of cDNA coding for FY glycoprotein, the molecular basis of the three major alleles (Fya/Fyb/Fy) has been established. Because of the mistyping of the silent Fy allele as Fyb, the error rate of current genotyping methods is high in black populations. STUDY DESIGN AND METHODS: Two hundred blood donors (European whites and African Blacks) and some amniotic DNA samples were investigated by a new allele-specific primer polymerase chain reaction technique. Sense primers corresponding to normal and GATA-1-mutated FY gene promoter region sequences were combined with antisense primers discriminating the Fya/Fyb polymorphism. RESULTS: Complete correlation between FY phenotypes and genotypes was obtained in all samples studied, although, in two whites and one black, serology showed weak Fyb expression while polymerase chain reaction indicated a Fyb allele. Gene frequencies were calculated. CONCLUSION: This simple and rapid polymerase chain reaction method was shown to detect the three common alleles at the FY locus in two representative ethnic populations. Its future use as an independent technique in red cell FY investigations and for fetal genotyping in hemolytic disease of the newborn is predicted.

GIL: a red cell antigen of very high frequency.

A new high-frequency red cell antigen has been identified and named GIL. GIL differs from all high-frequency antigens included in the International Society of Blood Transfusion classification. There is very little family information and GIL has not been shown to be an inherited character. Five women with anti-GIL have been found. All had been pregnant at least twice. Red blood cells of two of the babies gave positive direct antiglobulin tests, but there were no clinical signs of hemolytic disease. Anti-GIL may have been responsible for a hemolytic transfusion reaction and results of monocyte monolayer assays of two of the anti-GIL suggested a potential to cause destruction of transfused GIL+ RBCs.

The monoclonal antibody-specific immobilization of erythrocyte antigens assay (MAIEA) in the investigation of human red-cell antigens and their associated membrane proteins.

The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) technique is an immunoassay devised primarily for locating blood group antigens on specific red-cell membrane proteins. The assay involves the incubation of intact red cells with two antibodies, one human alloantibody, the other a nonhuman antibody, usually a rodent monoclonal antibody, but polyclonal antibodies of rabbit origin have been utilized. For a positive result, both antibodies must bind to the same membrane protein. The red cells are lysed, the membrane solubilized and the trimolecular complex of two antibodies and membrane protein is captured in a well coated with goat antirodent (or rabbit) immunoglobulin. The immobilized complex is then detected by the use of peroxidase-conjugated goat antihuman (or rodent) immunoglobulin. Negative results, due to mutual blocking between the human and animal antibodies when their epitopes are close together on the same molecule, have permitted a degree of localization of epitopes on some proteins. This has been most effective in the mapping of Cromer blood group system antigens on the complement control protein domains of decay-accelerating factor (DAF, CD55), but has also proved informative in the clustering of antigens on the Lutheran and Kell glycoproteins. MAIEA is an effective tool for the identification of antibodies to Knops-system antigens on complement receptor 1 (CR1, CD35) in immunohaematology reference laboratories. These antibodies are clinically unimportant, but must be identified before they can be ignored for transfusion purposes.

Alternative splicing of a novel glycophorin allele GPHe(GL) generates two protein isoforms in the human erythrocyte membrane.

The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at -6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.

Use of domain-deletion mutants to locate Lutheran blood group antigens to each of the five immunoglobulin superfamily domains of the Lutheran glycoprotein: elucidation of the molecular basis of the Lu(a)/Lu(b) and the Au(a)/Au(b) polymorphisms.

Lutheran glycoprotein (Lu gp) has five predicted immunoglobulin superfamily (IgSF) domains. K562 cells were transfected with Lu cDNA and tested by flow cytometry with monoclonal antibodies and Lu blood group antisera. The results confirmed the identity of Lu cDNA. Deletion mutants lacking the regions encoding one or more IgSF domains were made by inverse polymerase chain reaction (PCR), expressed in K562 cells, and tested with the same antibodies. The Lu(b) and Lu5 antigens and the epitope recognized by monoclonal antibody BRIC 224 were mapped to the first, N-terminal, IgSF domain. Lu4 and Lu8 were mapped to domain 2; Lu20 to domain 3; Lu7 and BRIC 221 epitope to domain 4, and Lu13 and Au(b) to domain 5. The organization of the LU gene was determined. The region encoding the open reading frame is arranged in 15 exons extending over approximately 11 kb on chromosome 19q13.2. The Lu(a)/Lu(b) and Au(a)/Au(b) blood group polymorphisms were studied using genomic DNA from typed blood donors. The Lu(a) mutation is a base change in exon 3 (G252 to A) encoding an Arg77 (Lu(b)) to His (Lu(a)) change on the CFG face of domain 1. The Au(a)/Au(b) polymorphism is an A1637 to G substitution in exon 12 encoding a Thr539 (Au(a)) to Ala (Au(b)) change on the G strand of domain 5.

Analysis of Knops blood group antigens on CR1 (CD35) by the MAIEA test and by immunoblotting.

Kna, McCa, Sla and Yka are red cell antigens of relatively high frequency, located on complement receptor 1 (CR1, CD35). Antibodies to these Knops system antigens are not uncommon. They are not haemolytic and do not reduce the survival of transfused incompatible red cells, but they are a nuisance in transfusion laboratories as they can cause an incompatible crossmatch and must be identified before they can be dismissed as clinically insignificant. Human red cell alloantibodies can be shown to be Knops system antibodies by the monoclonal-antibody-specific immobilization of erythrocyte antigens (MAIEA) test, using murine monoclonal anti-CR1. In addition to confirming that Kna, McCa, Sla and Yka are located on CR1, the MAIEA test was used to confirm that Csa is not on CR1. Red cells of the Helgeson phenotype, the null phenotype of the Knops system by conventional serological methods, have levels of Kna, McCa, Sla and Yka intermediate between those of alpha-chymotrypsin-treated cells (which lack Knops system antigens) and those of positive control cells. Level of expression of Knops system antigens is very variable and intensity of staining of immunoblots probed with monoclonal anti-CR1 correlated with strength of Knops system antigens, as determined by the MAIEA test. In individuals heterozygous for alleles producing different allotypes, separate bands representing each allotype on an immunoblot showed identical intensity of staining, suggesting that the quantity of CR1 on red cells is controlled, at least in part, by a locus independent of CR1. An analysis of CR1 on red cells of individuals who have made Knops system antibodies suggested that the Knops system antigens and the antibodies that detect them are complex and heterogeneous.

A combination of the effects of rare genotypes at the XK and KEL blood group loci results in absence of Kell system antigens from the red blood cells.

The 22 antigens of the Kell blood group system are located on a red blood cell (RBC) membrane glycoprotein that shows sequence homology with a family of metalloendopeptidases. Expression of the Kell system antigens is partially governed by XK, an X-linked gene that encodes the Kx protein; absence of Kx results in reduced Kell antigen expression. Almost total absence of Kell antigens from the RBCs of a German man with no symptoms of neuroacanthocytosis could not be due to the Kell-null phenotype, Ko, because his RBCs had very weak expression of Kx antigen and his three children were Kp(a + b+). Kell antigens were normal on the RBCs of his son but weak on those of his two daughters. An Nla III restriction fragment-length polymorphism within the KEL gene showed the Kpa/Kpa genotype in the propositus. Sequencing of his XK gene showed a single base change within the donor splice consensus sequence of intron 2. A BsaAl restriction fragment-length polymorphism showed the mutation in both of his daughters but not in his son. The extreme depression of the Kell antigens of the propositus must be due to a combination of effects, ie, homozygosity for Kpa and deficiency of Kx protein, each of which is capable of causing some degree of weakening of Kell antigens.

Rhmod phenotype: a parentage problem solved by denaturing gradient gel electorphoresis of genomic DNA.

Initial Rh phenotyping of a man with hemolytic anemia, his wife, and son appeared to exclude paternity. No exclusion was found in other blood groups or in the human leukocyte antigen (HLA) system; excluding Rh, the paternity index was 98.58 percent. Samples from these three family members, and two other family members, were tested with additional Rh antisera. The results indicated that the propositus has an Rhmod phenotype with expression of c, weak e, and very weak D, E, and G antigens. To support this hypothesis, DNA analysis of the RHD and RHCE genes was performed on the five family members. Polymerase chain reaction (PCR) products from exons 2 and 5 were analyzed by denaturing gradient gel electrophoresis (DGGE). The DNA results corroborated the serologic findings and refuted the exclusion of paternity.