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Biomedical subjects

C A Hyland

Publications and source records attributed to C A Hyland.

At least 19 recordsLinked to original sources
PubMed2001

Prenatal RHD gene determination and dosage analysis by PCR: clinical evaluation.

BACKGROUND: Use of the polymerase chain reaction (PCR) for detection of the RHD gene can measure the RHD gene status for unborn babies at risk for hemolytic disease of the newborn (HDN). The occurrence of D gene variants has led to errors in prenatal typing. Previous reports have highlighted the danger of assigning a positive fetus as negative, resulting in intrauterine fetal deaths. OBJECTIVE: To evaluate the effectiveness of a testing strategy whereby PCR was not only performed to determine the presence/absence of the RHD gene, but also used to assess the D gene copy number (zero, one or two RHD genes) in family studies for at risk pregnancies. METHODS: Samples comprising maternal (57) and paternal (42) peripheral blood samples, amniotic fluid (64), and matching cord blood (64) were collected. Rhesus (Rh) serotyping was performed on all blood samples. For RHD genotyping, DNA was extracted from all samples except for 28 cord samples, where only serotyping was performed (total 199 DNA genotyping). RHD gene PCR amplified exon 4 and exon 7 regions of the RHD gene. The dosage of RHD gene was determined by comparing the intensity of the RHD gene to that of the RHCE gene. RESULTS: A total of 197/199 samples showed concordance between exon 4 and exon 7 PCR results. Two discrepant results occurred in one family: the father carried one normal D gene and one D gene variant where PCR was tested to be positive using exon 4 but negative using exon 7. One of a pair of dizygotic twins inherited this abnormal D gene and was mildly affected by HDN. This was correctly identified antenatally and the pregnancy successfully managed. The concordance rate between serotypes and genotypes for 135 blood samples was 100%. Amongst the family groups, 8/14 heterozygous fathers transmitted the D gene and 26/26 homozygous fathers transmitted the D gene to the babies. The concordance rate between RHD genotypes from amniotic fluid and Rh D serotypes from cord blood was also 100%. CONCLUSION: The present study demonstrates the effectiveness of using PCR in a clinical setting. It verifies the importance of testing more than one region of the gene, and also the need for a testing strategy where both maternal and paternal testing for RHD gene dosages are performed.

Amniotic Fluid↗
PubMed1998

A novel single missense mutation identified along the RH50 gene in a composite heterozygous Rhnull blood donor of the regulator type.

Rare individuals who lack all of the Rh blood group antigens are called Rhnull and may be classified as "regulator" or "amorph" types. The suppression of Rh antigen expression for regulator types may be attributed to mutations of the RH50 gene, which is independent of the RH locus. The RH50 gene encodes a glycoprotein that interacts with the Rh proteins to form a functional complex within the red blood cell membrane. This report describes an RH50 gene mutation for a previously unclassified Rhnull donor. Sequencing cDNA clones from Rh50 mRNA revealed a single base change (G836A) yielding a missense and nonconservative mutation (Gly279Glu) within a predicted hydrophobic domain for this membrane protein. Genomic DNA studies using polymerase chain reaction (PCR) restriction analysis and sequencing showed that the Rhnull propositus was a composite heterozygote for this mutation, carrying two alleles with the A and G at nucleotide 836, respectively. In contrast, cDNA studies showed that only the A836 sequence was present, suggesting that the second allele with G836 was apparently silent (no transcript detected). Family studies showed that the mutant RH50 allele (836A) was inherited maternally, whereas the silent RH50 allele (836G) was from paternal transmission. These findings provide further evidence that rare but diverse genetic alterations may occur along the RH50 gene where the Rhnull syndrome of the regulator type occurs. The single amino acid change (Gly to Glu) provides insight into the critical value of these residues for assembly of the Rh antigen complex within the membrane.

Blood Donors↗
PubMed1998

Exposure to GB virus type C or hepatitis G virus in selected Australian adult and children populations.

BACKGROUND: The epidemiology and disease association for the GB virus type C (GBV-C) or hepatitis G virus (HGV) are poorly understood. STUDY DESIGN AND METHODS: This study describes the exposure rates to GBV-C/HGV in diverse Australian population groups by testing for current infection and evidence of past infection with a reverse transcriptase polymerase chain reaction and an anti-E2 enzyme-linked immunosorbent assay, respectively. Subjects included volunteer blood donors, hepatitis C antibody (anti-HCV)-positive donors, children, hemodialysis patients, pregnant women attending a prenatal clinic, injecting drug users (IVDUs), and adult hemophiliacs. RESULTS: Combined GBV-C RNA and E2 antibody prevalence was 6.5 percent (6/93) in children, 13.3 percent (75/565) in blood donors, 14 percent (14/99) in pregnant women, 22.5 percent (18/80) in hemodialysis patients, 80 percent (56/70) in anti-HCV-positive donors, 88.6 percent (31/35) in IVDUs, and 85.7 percent (54/63) in adult hemophiliacs. Children had the lowest antibody rate, 1.1 percent, whereas the rate was 10.8 percent for blood donors and rose to 45.7 percent for IVDUs, 57.1 percent for anti-HCV-positive donors, and 74.6 percent for hemophiliacs. In contrast, current infection rates were comparable for children, blood donors, and pregnant women (5.4, 2.6, and 6%, respectively), rising to 11.1 percent for hemophiliacs, 24.3 percent for anti-HCV-positive donors, and 48.6 percent for IVDUs. Ten of 12 blood donors had persistent viremia, while 2 had recent infections, 1 with apparent resolution. CONCLUSION: Exposure to GBV-C can commence at an early age, although ongoing exposure may also occur among adults with no apparent risk factors. GBV-C RNA positivity was not associated with abnormal plasma alanine aminotransferase levels among blood donors.

Adult↗
PubMed1997

Prevalence of hepatitis C virus and genotype distribution in an Australian volunteer blood donor population.

BACKGROUND: This study was undertaken to assess the prevalence of hepatitis C virus (HCV) antibody and RNA in first-time blood donors and to examine the HCV genotype distribution. STUDY DESIGN AND METHODS: A third-generation enzyme-linked immunosorbent assay (ELISA) was used to screen 34,725 donors for HCV antibodies. Donors who were repeatably reactive were tested in two immunoblot assays-a second-generation and a third-generation recombinant immunoblot assay-as well as by a polymerase chain reaction (PCR) assay. PCR-positive donors were genotyped. All samples were screened for alanine aminotransferase levels. RESULTS: The ELISA repeat reactivity rate was 0.55 percent. PCR testing showed that 69 (38%) of the 183 ELISA-reactive samples contained HCV RNA. The third-generation recombinant immunoblot assay identified all 69 viremic samples as antibody positive; however, only 63 tested positive on the second-generation immunoblot. The remaining six PCR-positive donors tested antibody-indeterminate to the core peptide. All six of these donors had HCV subtype 3a infections. Genotype distribution among 58 samples showed that 34 were type 1, of which 22 could be further subtyped as 1a (16) and 1b (6); 2 were 2a; 5 were 2b; and 17 were subtyped as 3a. Donors infected with 2b and 3a had reduced antibody reactivity to the NS4 and NS3 peptides only on the second-generation immunoblot. CONCLUSION: The prevalence of confirmed anti-HCV and viral RNA in new donors is 0.29 and 0.2 percent, respectively. The third-generation recombinant immunoblot assay was more sensitive than the second-generation immunoblot assay in detecting 2b and 3a HCV subtypes. The inclusion of the NS5 peptide in the third-generation recombinant immunoblot did not result in positive tests in any additional donors. Rather, the improvement was due to the increased detection of NS3 and, to a lesser extent, NS4 antibodies. Subtypes 1a and 3a were most prevalent in this population.

Antigen-Antibody Reactions↗
PubMed1996

Prevalence of hepatitis G virus in Queensland blood donors.

OBJECTIVE: To determine the prevalence of hepatitis G virus (HGV) carriage in Queensland blood donors. DESIGN: Cross-sectional survey with retrospective longitudinal study of HGV-positive donors. SETTING: Brisbane Red Cross Blood Bank, 1995. SUBJECTS: 100 consecutive blood donors attending the Blood Bank on two days in October 1995 and 20 blood donors with a raised plasma alanine aminotransferase (ALT) level on their last donation. OUTCOME MEASURES: Presence of HGV RNA by reverse transcription polymerase chain reaction (RT-PCR) in currently donated blood and in blood samples archived for up to 34 months. RT-PCR used two different reverse transcription methods and three different specific sets of primers and probes. RESULTS: Five of the 120 blood donors were positive for HGV RNA by all RT-PCR methods (four of the 100 with normal ALT levels [4%] and one of the 20 with raised ALT levels [5%]). Retrospective testing of archived samples showed that four of these five had been persistently HGV RNA-positive for at least two years, while the fifth had been HGV RNA-negative on two donations before becoming HGV RNA-positive. No risk factors were identified for this donor. CONCLUSIONS: A relatively large number of Queensland blood donors (4%) are persistently HGV RNA-positive.

Adult↗
PubMed1994

Three unrelated Rh D gene polymorphisms identified among blood donors with Rhesus CCee (r'r') phenotypes.

Human red blood cells are traditionally typed as Rhesus (Rh)-positive or -negative depending on the presence or absence of the Rh D antigen. A recent report demonstrated that the Rh D gene is completely absent in Rh D-negative individuals. In this study, Rh D-negative blood donors with ccee (n = 25) and CCee (n = 3) phenotypes were examined for the presence of absence of the D gene. Polymerase chain reaction (PCR) probes that hybridize to the 5' and 3' regions of the Rh CcEe gene and the closely related D gene were used in a Southern analysis. The D gene was absent in all ccee phenotypes examined. The CCee phenotypes showed three Rh D polymorphisms: one donor lacked the D gene, one donor had a partial deletion on one D gene at the 3' region, and the remaining donor appeared to have one normal D gene within the intron/exon regions examined. We conclude that, while the D gene may be absent in the majority of Rh D-negative phenotypes, rarer polymorphisms also occur that prevent expression of the D antigen resulting in the Rh D-negative phenotype.

Base Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1992

Predictive markers for hepatitis C antibody ELISA specificity in Australian blood donors.

The hepatitis C antibody reactivity rate in 91,748 blood donors tested using the ORTHO HCV C-100 ELISA system was 0.51%. Specificity of ELISA positive reactions was measured using a recombinant immunoblot assay (RIBA). The aim of this study was to identify markers in ELISA positive donors which were predictive of a RIBA positive result. Samples from 430 ELISA positive donors were tested by the first generation RIBA, RIBA-1, which incorporates two HCV peptides C-100 and 5-1-1. Fifty-five per cent (236) were positive and 19% (83) indeterminate. Multivariate analysis of gender, age, HCV ELISA OD ratio, alanine aminotransferase (ALT) status and hepatitis B core antibody (anti-HBc) status identified age, magnitude of HCV ELISA OD ratio and anti-HBc status as the only independent predictors of a positive RIBA-1 result. The relative odds of being RIBA-1 positive were 4.6-fold (95% CI 1.3-16.4) higher among donors aged 25-34 years compared with donors less than 25 or greater than 44; 6.1-fold (2.1-17.9) higher if the donor was anti-HBc positive and 273.4-fold (30.9-2417) higher if the HCV ELISA OD ratio was greater than 5.98 compared to those with a ratio less than 1.77. Seventy-eight of the 83 RIBA-1 indeterminates were tested on the second generation RIBA, RIBA-2, which includes two additional HCV peptide, C22 and C33c. Thirty-one per cent (24) were positive and 41% (32) were negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Surrogate testing for non-A, non-B hepatitis in Queensland, Australia: an ALT microtitre tray method for screening blood donors.

The estimation of plasma alanine aminotransferase (ALT) has been proposed as a surrogate test to identify potential non-A, non-B hepatitis carriers in blood donor populations. This report describes an ALT screening procedure which uses wells of microtitration trays as reactant vessels. The method utilizes a rate reading photometer, is economical and conveniently fits into the routine workflow. Within-batch and between-batch precision was 4.1% and 6.3% at enzyme concentrations of 49 IU/I. Results of testing 29,675 healthy blood donors gave values which ranged between 1.0 IU/I and 214 IU/I. A study of 762 donations showed a significant difference in mean ALT values between males and females (p less than 0.01). When a cut-off value of 46 IU/I was used, 2.5 percent of donations were considered unsuitable for transfusion. The medico-legal implications that may arise from the introduction of this screening test into the routine work flow are discussed.

Alanine Transaminase↗