Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

BACKGROUND AND OBJECTIVES: Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. MATERIALS AND METHODS: Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). RESULTS: Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. CONCLUSION: Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fy(b) antigen expression: implication for genotyping interpretative algorithms.

BACKGROUND AND OBJECTIVES: An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fy(a) antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile. MATERIAL AND METHODS: Blood group genotyping was performed using a commercial SNP microarray platform. Sanger sequencing was performed using primer sets to amplify across exons 1 and 2 of the FY gene and using allele-specific primers. RESULTS: The propositus was genotyped as FY*A/B, FY*X heterozygote that predicted the Fy(a+b+(w) ) phenotype. Sequencing identified the 265T and 298A variants on the FY*A allele. This link between FY*A allele and 265T was confirmed by allele-specific PCR. CONCLUSION: The reduced Fy(a) antigen reactivity is attributed to a FY*A allele-carrying 265T and 298A variants previously defined in combination only with the FY*B allele and associated with weak Fy(b) antigen expression. This novel allele should be considered in genotyping interpretative algorithms for generating a predicted phenotype.

Detection, molecular characterization, and antibiogram of multi-drug resistant and methicillin-resistant Staphylococcus aureus (MRSA) isolated from pets and pet owners in Malaysia.

BACKGROUND: The emergence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA) in animals and humans with continuous contact are a great zoonotic concern. AIMS: This cross-sectional study was performed to investigate the carriage rate, genotypic characteristics, and to determine the antibiogram of S. aureus isolated from pets and pet owners in Malaysia. METHODS: Nasal and oral swab samples from 40 cats, 30 dogs, and 70 pet owners were collected through convenient sampling. Presumptive colonies on mannitol salt agar were subjected to biochemical identification. S. aureus and MRSA were confirmed by PCR detection of nuc and mecA genes, respectively. Molecular profiles for antimicrobial resistance and virulence genes in S. aureus were also determined. The antibiogram was carried out via Kirby-Bauer test using 18 antibiotics. RESULTS: 17.5% of cats, 20% of dogs, and 27% of pet owners were S. aureus positive. MRSA was also detected in dogs, and pet owners. S. aureus isolates displayed high resistance against penicillin (72.7%), and amoxicillin/clavulanate (66.7%). 39.4% of S. aureus isolates showed multidrug-resistance traits, phenotypically. Molecular characterization of S. aureus revealed the presence of mecA, tetk, tetL, ermA, ermB, ermC, msrA, scn, chp, sak, sep, and sea genes. CONCLUSION: This study showed the emergence of MRSA and MDRSA in pets and pet owners in Malaysia. The antibiogram findings showed resistance of S. aureus to multiple antibiotics. Furthermore, molecular analysis of immune evasion cluster (IEC) strongly suggests the spread of animal-adapted S. aureus lineages among pets and pet owners.

Monoclonal antibody recognizing a core epitope on mucin.

Monoclonal antibody TH1 (IgM) was prepared by immunizing mice with deglycosylated (TFMSA-treated) cystic fibrosis mucin. TH1 reacted strongly with TFMSA treated cystic fibrosis mucin but not with the fully glycosylated mucin, indicating reactivity with a core mucin epitope. TH1 showed no reactivity with ovine mucin (98% of glycans as sialyl-Tn) but reacted strongly with desialylated ovine mucin, indicating the epitope for this mab was the Tn-antigen (O-linked GalNAc). However, TH1 showed no reactivity with Tn-positive red blood cells, and the binding of TH1 was not inhibited by GalNAc at 2.5 mg/ml, illustrating the importance of the peptide sequence to which the GalNAc is attached. TH1 stained the majority of cancers of the colon, lung, stomach, ovary, breast, and cervix, and the cellular distribution of this antigen in normal tissue suggested reactivity with immature mucin. This antibody appears to be a useful reagent for the detection of immature mucin.

A Polynesian family showing co-dominant inheritance of normal glycophorin C and the Gerbich variant form of glycophorin C.

Two individuals with the rare Ge:-2-3,4 phenotype (Gerbich type of Gerbich negative) were identified in a family of Polynesian descent who reside in the Cook Islands. In initial serologic tests, all other family members typed as Ge-positive, and heterozygous individuals could not be identified. Further studies on blood samples from seven members of this Polynesian family by immunoblotting and hemagglutination tests on trypsin-treated red blood cells showed that normal glycophorin C and the product of the Gerbich allele were inherited in an autosomal dominant manner.

The elucidation of a Kell-related autoantibody using ZZAP-treated red cells.

A 61-year-old, nontransfused, Caucasian male was found to have a positive direct antiglobulin test (DAT) and an autoantibody in his serum prior to total hip replacement. Autoabsorption with the patient's ZZAP-treated red cells failed to absorb the autoantibody, giving a clue to its possible specificity, which was subsequently found to be Kell-system related. In addition, his red cells were found to have a slightly weakened expression of Kell system antigens. The patient was fit and healthy with normal hematological indices at the time of the operation. One year the hemlogical findings remained the same despite the persistence of the autoantibody and a positive DAT. This suggested that the autoantibody was benign, which was supported by a negative in vitro mactophage assay.

Improved detection of weak, clinically significant antibodies by supplementation of polyethylene glycol with a low-ionic solution.

A comparative study of 164 serum samples was carried out to determine the specificity and sensitivity of the indirect antiglobulin test (IAGT) in three different formulations: physiologic saline, low-ionic solution (RAM), and RAM supplemented with polyethylene glycol (PEG). Serum samples containing mostly weak antibodies (anti-D, -C, -E, -c, -Jka, -Fya, -K, -S, -Lea, -Lua, -M, -Cob, -P1, -I, and -Kna) were used in a 10-minute IAGT in which PEG-IAGTs were compared with saline- IAGTs and RAM-IAGTs. With the exception of anti-P1, anti-I, and an anti-Lea, PEG-IAGTs detected all the antibodies tested compared with 72.3% and 77.4% for saline-IAGTs and RAM-IAGTs, respectively. The end-point titers of at least 82% of antibodies detected by PEG-IAGTs were 1-3 dilutions higher than those by saline- and RAM-IAGTs. When specificity of PEG-IAGTs was tested using 268 randomly selected, fresh (< 1 day old) blood samples, PEG-IAGT detected 11 out of 268 samples as positive compared with 7 out of 268 by both saline-IAGTs and RAM-IAGTs. The four antibodies that were not detected were identified as anti-D, anti-E, anti-Bga, and an autoantibody known previously to be only reactive with papain-pretreated red cells. No nonspecific reactions were detected by PEG-IAGTs and no hemolysis was evident in any of the IAGTs. PEG-IAGTs were more sensitive than saline- and RAM-IAGTs. PEG-IAGTs detected all weak, clinically significant antibodies as well as four antibodies that were otherwise undetected by saline-IAGT or RAM-IAGT. The overall sensitivity of the PEGIAGT was 96.3% compared with 84.1% and 73.2% for the RAM- and saline-IAGT, respectively.

Separable anti-T and anti-Tk lectins from the seeds of Vicia hyrcanica.

Separable anti-T and anti-Tk lectins can be extracted from the seeds of Vicia hyrcanica. The anti-T lectin is similar to that of the peanut, Arachis hypogaea; the anti-Tk lectin compares favourably with that of Griffonia simplicifolia II.

Serological and immunochemical specificity of a human autoanti-Gerbich-like antibody.

An 85-year-old male with cardiac failure secondary to anaemia had an apparent anti-Ge2 (Ge = Gerbich) in his serum which did not agglutinate his own red cells even though they were Ge-positive in tests with alloanti-Ge. The direct antiglobulin test was negative; however, an antibody with apparent anti-Ge2 specificity was eluted from his red cells. The patient's autoantibody was shown in immunoblotting experiments to react with an antigenic determinant on beta-sialoglycoprotein. This case illustrates that an autoanti-Ge can masquerade as an alloantibody, thereby complicating antibody identification, and implies that the immunochemical specificity of autoanti-Ge2 is different from that of alloanti-Ge2.

The red cell antigen JAL in the Swiss population: family studies showing that JAL is an Rh antigen (RH48).

The JAL antigen was found to have an overall frequency of 0.004% in the Swiss population and 0.06% in French-speaking Swiss. Family studies of 5 JAL+ individuals have shown that the JAL antigen is not part of the ABO, MNSs, Fy, Jk and Co blood group systems, or the Se system, nor is it X- or Y-linked. JAL is encoded by the RH locus or by a very closely linked locus. The number RH48 (4.48) has been assigned for JAL by the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens.

Enhanced reaction with Vicia graminea lectin and exposed terminal N-acetyl-D-glucosaminyl residues on a sample of human red cells with Hb M-Hyde Park.

A sample of polyagglutinable red cells was obtained from a healthy individual (group O, N) possessing a hemoglobin (Hb) variant called Hb M-Hyde Park. The sialic acid content of the individual's red cells is 90 percent of normal, and his cells are agglutinated by monoclonal but not lectin anti-Tn, a panel of lectins specific for N-acetylgalactosamine (or galactose), and N-acetylglucosamine. Enhanced agglutination reactions were obtained with Vicia graminea, Ulex europaeus, and human anti-I and -i. Using various enzyme treatments and different methods of labeling cell surface components, two defective cell membrane sites have been identified: one associated with the O-linked oligosaccharides on sialoglycoproteins and the other associated with exposed N-acetylglucosaminyl residues located on membrane components of apparent molecular weights 88,000 to 130,000 and 46,000 to 73,000 (probably the Band 3 and Band 4.5 regions, respectively).

A southern analysis of Rh blood group genes: association between restriction fragment length polymorphism patterns and Rh serotypes.

Polymorphisms within the Rh blood group system have been defined by serologic agglutination methods, but have not yet been defined at the DNA level. Two closely related genes associated with the Rh D antigen and with the Rh C/c and E/e antigens have been cloned. We used a Southern analysis incorporating probes to the 5' and 3' regions of the Rh C, E gene and D gene to identify polymorphisms associated with Rh C/c and E/e antigens, respectively. The D gene dosage could be determined by comparing the relative intensities of the D bands with bands from the 5' and 3' region of the Rh C, E gene. The concordance between restriction fragment length polymorphism (RFLP) patterns and serologic phenotypes for 102 randomly selected blood donors was 100% for C, e, and D, 94.8% for c, and 94.3% for E. The data are consistent with the sequences encoding the C/c epitopes residing on the 5' side of those for the E/e epitopes. All samples discordant for the 3' probe and E had the cE (r") serotype. These data show that the gene coding for the cE serotype is different in Rh-positive and -negative individuals. The study demonstrates that Rh DNA typing, including D gene dosage measurements and Rh gene haplotyping, may supplement traditional serotyping methods in transfusion medicine.

The MiIII phenotype among Chinese donors in Hong Kong: immunochemical and serological studies.

The incidence of the MiIII phenotype among Chinese blood donors in Hong Kong was found to be 6.28%. Eleven individuals apparently homozygous for the MiIII gene were detected by immunoblotting with monoclonal antibodies R1.3 and R18. R1.3 detects an identical epitope on both glycophorins A and B and R18 detects a different epitope on glycophorin A. Immunoblotting with R1.3 showed an absence of bands corresponding to normal glycophorin B. Immunoblotting with R18 showed an absence of a 58 K band, which corresponds to a heterodimer of normal glycophorin B complexing with the MiIII component, found in MiIII heterozygotes. In two families with apparent MiIII homozygous individuals, both parents of the propositi had the MiIII phenotype which implies normal autosomal inheritance of the MiIII gene. In another family, only one parent had the MiIII phenotype and the presence of an Su gene is postulated to explain the immunochemical and serological findings.

A specific antibody for cells with acquired B antigen.

An acquired B antigen, present on the red blood cells of a healthy male blood donor, was investigated. An anti-B typing serum which initially detected the weak antigen on the donor cells contained a separable antibody that was specific for cells with the acquired B antigen and did not react with normal group B cells. A similar, specific, immune antibody was produced in a rabbit by the injection of red blood cells with an acquired B antigen.

Biochemical studies on red blood cells from a patient with the Inab phenotype (decay-accelerating factor deficiency).

A 38-year-old Russian woman (KZ) has been identified as the fourth proposita with the Inab blood group phenotype. Like the first two propositi, she has a chronic intestinal disorder and, as shown for the third proposita, her Inab phenotype is demonstrably inherited. KZ's serum contained anti-IFC, which reacted with a red blood cell (RBC) membrane component with an Mr of 70,000, which is decay accelerating factor (DAF). Her RBCs lacked all Cromer-related blood group antigens and DAF. Her RBCs were no more susceptible than normal control RBCs to lysis in acid lysis or in rabbit or human antibody-initiated complement lysis tests. Northern blots of total RNA isolated from KZ's Epstein-Barr virus-transformed lymphoblasts showed a marked reduction of DAF mRNA when compared with normal. Polymerase chain reaction (PCR) amplification of cDNA confirmed this reduced level of DAF mRNA. Sequencing of the PCR product showed a 44-nucleotide deletion in the mRNA close to the short consensus repeats IIIa/IIIb intron/exon boundary. This deletion results in a change in the reading frame that places a termination codon six amino acids after the deletion. The putative translation product would lack a glycosyl phosphatidyl-inositol linkage site and, therefore, would not be membrane-bound in the RBC.

Characterization of a neuraminidase from Corynebacterium aquaticum responsible for Th polyagglutination.

Th polyagglutinability is characterized by the agglutination of the red blood cells (RBC) by Arachis hypogaea, Medicago disciformis, Vicia cretica but, in contrast to the T phenomenon, not by Glycine max (Glycine soja). Because Th transformation of RBC has been obtained in vitro, the mechanism of Th polyagglutinability expression has been studied and reproduced experimentally. An enzyme with neuraminidase specificity has been isolated from the culture supernatant of Corynebacterium aquaticum, and further characterized (MW = 55,600 kDa, pH = 5.5, Km = 0.138 microM, Kcat = 0.22 micrograms). Reversely, Th transformation of RBC could be obtained by using other neuraminidases but in very mild conditions of hydrolysis. From our results, it can be concluded that by the release of less than 20 micrograms of sialic acid per 10(10) RBC, Th reactivity can be induced whereas hydrolysis of greater amounts of sialic acid (greater than 20 micrograms/10(10) RBC) give the classical T polyagglutinability.