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W R Mayr

Publications and source records attributed to W R Mayr.

At least 73 records · Page 4Linked to original sources
PubMed1997

Demonstration by flow cytometry of the numbers of residual white blood cells and platelets in filtered red blood cell concentrates and plasma preparations.

BACKGROUND AND OBJECTIVES: New-generation polyester filters provide significant depletion of white blood cells (WBC) and platelets (PLT) in filtered red blood cell concentrates (FRCC) and in filtered plasma preparations (FP). The aim of this study was to elaborate a sensitive flow cytometric method for monitoring residual WBC and PLT in FRCC and FP. MATERIALS AND METHODS: We determined the number of WBC in 500 microliters FRCC of FP using 50 microliters of a combination of monoclonal antibodies (MAB) against CD45 (FITC labeled) and CD19 (PE labeled). After lysis of red blood cells, we mixed a specific number of reference beads with the remaining WBC. The number of residual WBC related to the acquisition volume was defined by the acquired reference beads. Using this method, the detection limit (DL) was 3 WBC/microliter. Alternative methods used MAB against CD45 (FITC and PerCP labeled) and CD14 (PE labeled) or lymphocyte subsets such as CD3 (FITC labeled) and CD19, CD4, CD8, CD16 and CD56 (PE labeled) in combination with CD45 (PerCP labeled). The DL values were 10 WBC/microliter for the CD45/CD14 staining and 0.1 WBC/microliter for the determination of both CD3+ and CD19+ lymphocytes. For residual PLT in FRCC or FP, we used an FITC-conjugated MAB against CD41, with reference beads to determine the acquisition volume. PLT were demonstrated in a green-fluorescence (FL1) single histogram after gating in the forward light scatter x 90 degrees light scatter signal dot plot. PLT counting was as described for WBC. The DL value was about 2 PLT/microliter. RESULTS: Filtration with Pall WBF-1 filters reduces WBC by 4 log and PLT by 3-4 log, resulting in cell counts which are below the critical limit for causing adverse transfusion reactions. CONCLUSIONS: Flow cytometry techniques provide a reproducible and objective tool for counting residual WBC and PLT in blood preparations compared with the Nageotte hemocytometer. Absolute numbers of leukocyte and lymphocyte subpopulations are obtainable.

Antibodies, Monoclonal↗
PubMed1997

Prestorage inline filtration of whole blood for obtaining white cell-reduced blood components.

BACKGROUND: Preliminary studies have indicated that the inline filtration of whole blood is a feasible method of obtaining white cell (WBC)-reduced packaged red cells (RBCs) and WBC-reduced fresh-frozen plasma (FFP) while using only one filter. STUDY DESIGN AND METHODS: An inline WBC-reduction filter, specially designed for this purpose and integrated in a "top-top" system, was used in the preparation of 24 units of WBC-reduced RBCs (RBC-F) and FFP (FFP-F) in each of two transfusion centers (Vienna and Göttingen). Twelve conventionally prepared units of RBCs (RBC-C) and FFP (FFP-C) served as controls. WBC contamination was assessed in each unit with the Nageotte chamber. Several coagulation measures were evaluated by using standardized test systems. RESULTS: The median WBC contamination in RBC-F was 27,000 per unit in Vienna and 50,000 in Göttingen. In FFP-F, the median WBC contamination was 13,000 (Vienna) and 31,000 (Göttingen) per unit. Coagulation factors I, V, VIII, and XI in FFP-F were not different from those in FFP-C. In addition, markers for the activation of coagulation and fibrinolysis--that is, factor XIIa, prothrombin fragments, thrombin-antithrombin complexes and fibrinogen degradation products--were not greater in FFP-F. CONCLUSION: Blood components prepared from inline-filtered whole blood meet the standards for WBC-reduced RBCs and FFP. The protein profile of FFP-F is not altered, and markers for the activation of coagulation and fibrinolysis show no increase.

Blood Coagulation Factors↗
PubMed1996

Evaluation of four monoclonal antibodies against HLA-B27 for their reliability in HLA-B27 typing with flow cytometry (FC): comparison with the classic microlymphocytotoxic test (MLCT).

Typing for HLA-B27 by serological methods is routinely performed using the microlymphocytotoxic test (MLCT). Since monoclonal antibodies (MAB) against HLA-B27 are available, flow cytometry (FC), which requires less time than the MLCT has been developed as an alternative technique. The aim of the present study was to check the accuracy and reliability of this method using different MAB against HLA-B27 in comparison to MLCT (using polyclonal antibodies against HLA-B27 and cross-reacting specificities [CRS]). FC was performed in 144 patients with HLA-B27-related rheumatic disorders (seronegative spondarthritides) using a special software package which requires corresponding calibration beads in order to achieve a standardized setup of the flow cytometer. MAB from the following producers were used: Becton & Dickinson (BD), Behringwerke (BE), One Lambda (OL), and Immunotech (IT). In addition to the critical limit of fluorescence intensity (FI) which indicates positivity if exceeded, provided by the software (but valid only for the MAB from BD), empirically twice the value of the STD calculated from the mean of the FI values of HLA-B27 positive patients was regarded a good cut off for the HLA-B27 positivity in FC measurements with the MAB used. Using a standard protocol including an incubation of whole EDTA-anticoagulated blood for 15 min with MAB against HLA-B27 (FITC-conjugated) and CD3 (PE-conjugated) and a lysis of erythrocytes, good discrimination between HLA-B27 positive and negative patients was obtained. Cross reactions with HLA-B27 positive patients occurred except when the MAB from OL was used. One false-negative result was found with OL's MAB (out of 22) and false-positive results occurred in HLA-B7+ patients when MAB from BD, BE, and IT were used. Unfortunately also 1 false-positive result (out of 57) was obtained in HLA-B7-, B27- patients with IT's MAB. Errors in the interpretation of the FC analysis might be avoided if more than one MAB (including those not cross reacting with HLA-B7) are used.

Antibodies, Monoclonal↗
PubMed1996

Sequence determination of an allele ladder for the STR polymorphism at the CD4 locus and application of the ladder in testing an Austrian Caucasian population sample.

The short tandem repeat (STR) polymorphism at the CD4 locus, designated HUMCD4, was examined by PCR, native polyacrylamide electrophoresis and subsequent silver staining using an allelic ladder of eight distinguishable alleles occurring in an Austrian Caucasian population sample as a standard size marker. The ladder was produced by pooling equal concentrations of eluted, separately amplified and sequenced alleles, which were previously identified by their different electrophoretical migration. Components of the ladder are in regular intervals of five basepairs. Alleles 4 to 8 were designated according to the number of AAAAG repeat units. The four longer alleles 8' to 11 showed a stable A to G transition in one of the repeat units and were designated counting the AAAGG unit for a AAAAG. Allele 8' was not included in the ladder because it showed the same electrophoretic mobility as allele 8. This ladder proved to be a precise and reliable tool in the analysis of 600 chromosomes of the Austrian population. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.23).

Alleles↗
PubMed1996

Risk of hepatitis C virus (HCV) transmission by anti-HCV-negative blood components in Austria and Germany.

In order to estimate the residual risk of transfusion-transmitted HCV infection, we have analyzed data from transfusion centers in Austria (Vienna) and Germany (Göttingen) from 1990 to 1995. In Vienna, the seroprevalence (RIBA-confirmed third-generation anti-HCV tests) was 0.28% in first-time donors (FTD) and the incidence of seroconversion in repeat donors (RD) was 0.049 (per 100 person years) from 1994 to 1995. In Göttingen, the prevalence of a PCR-confirmed positive third-generation anti-HCV test was 0.22% in FTDs and the incidence was 0.093 (per 100 persons years). A continuous decline of the rate of anti-HCV-positive donations and donors was observed with first- and second-generation anti-HCV tests in the years 1990-1994. The introduction of the third-generation anti-HCV test resulted in increased numbers of anti-HCV positive repeat donors, mainly due to false-positive results. Only 9% of anti-HCV-positive repeat donors were either PCR positive or RIBA positive or or indeterminate. Based on a mathematical model which takes (a) the window period, (b) the false-negative rate of anti-HCV tests, and (c) human and operational errors into consideration, we have calculated the residual risk of HCV infection. We used a window period of 74 days, a sensitivity of 98%, and an error rate of .1%. The residual risk (for third-generation anti-HCV test-negative blood components) was calculated to be 1:9000 (95% confidence interval 1:16390-1:6210) and 1:4800 (95% confidence interval 1:40000-1:1320) for Vienna and Göttingen, respectively, in 1994 and 1995. Since this conservative approach does not take the impact of ALAT screening into account, the actual risk is probably lower.

Adult↗
PubMed1996

HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay.

Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.

Adult↗
PubMed1996

Genotypes of the vitamin-D-receptor gene and bone mineral density in Caucasoid postmenopausal females.

OBJECTIVE: To evaluate the relation of bone mineral density (BMD) to inherited polymorphisms within the gene coding for the vitamin D receptor (VDR) in postmenopausal women. METHODS: DNA samples from 163 post-menopausal women and from 112 controls were genotyped for common allelic determinants within the VDR gene as defined by the restriction enzyme sites for BsmI, ApaI, and TaqI. BMD was evaluated at the lumbar spine (L2-L4) by dual energy X-ray absorptiometry. Single restriction enzyme site polymorphisms as well as their joint genotypes were evaluated for their association with BMD in postmenopausal women. RESULTS: The three restriction site polymorphisms showed a close association indicating linkage disequilibrium (P < 0.0001). There was no relationship between any of the single restriction site polymorphisms and BMD. The distribution of the five most common joint genotypes among postmenopausal women and controls was similar. There was no relation of BMD and any of the five most common VDR genotype groups. Females more than 5 years after menopause had a significantly lower BMD than those less than 5 years after menopause (P < 0.001). This age-related decrease of BMD was also decernible within genotype groups. CONCLUSION: In our population, the postmenopausal period has a major influence on BMD, but BMD is not controlled significantly by any of the common allelic variations within the VDR gene.

Absorptiometry, Photon↗
PubMed1996

Specificities of anti-platelet antibodies in multitransfused patients with haemato-oncological disorders.

The clinical condition and the formation of platelet-reactive antibodies influence the post-transfusion platelet increment. We analysed the specificities of platelet-reactive antibodies in 81 multitransfused patients with haemato-oncological diseases refractory to platelet transfusions, or prior to a scheduled stem cell transplantation. In 17 additional patients we prospectively determined the development of platelet-reactive antibodies at the time of chemotherapy in weekly intervals. Sera were tested by the monoclonal antibody-specific immobilization of platelet antigens (MAIPA)-technique for antiplatelet antibodies against HLA class I antigens, the human platelet-specific alloantigens (HPA)-1, -2, -3, -5, and the platelet membrane glycoproteins (GP) Ia/IIa, Ib/IX, IIb/IIIa (panreactive). Platelet reactive antibodies were found in 54% of blood samples. They were frequent in patients with a history of possible previous immunization, particularly if patients were refractory to platelet transfusions. Platelet-reactive HLA antibodies were the most common antibodies. Even patients without a known risk of primary immunization who received exclusively leucocyte-depleted blood products formed antibodies. By the MAIPA technique, even LCT-negative sera were found to contain platelet-reactive antibodies.

Adolescent↗
PubMed1996

Flow cytometric detection of platelet-reactive antibodies and application in platelet crossmatching.

BACKGROUND: Alloimmunization against HLA or platelet antigens can cause refractoriness to platelet transfusions in multiply transfused patients. Crossmatching of platelet concentrates is effective in overcoming this problem. STUDY DESIGN AND METHODS: A flow cytometric assay was used for simultaneous detection of lymphocyte-reactive and platelet-reactive antibodies in a single sample using fluorescein isothiocyanate-labeled anti-IgG. This assay was compared with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay in selected sera containing HLA and platelet antibodies. In a further study, this assay was compared with lymphocytotoxicity test results from thrombocytopenic patients, for whom platelet concentrates were ordered. The results of both assays were then correlated with the 1-hour corrected count increment, with a corrected count increment greater then 7500 considered as an adequate transfusion response. RESULTS: The results of the MAIPA and flow cytometric assay in detecting platelet-reactive antibodies correlated well (p<0.0001, r = 0.84). The sensitivity and specificity of the flow cytometric assay in detecting platelet-reactive antibodies were 94.7 and 96.3 percent, when the MAIPA assay was taken as a reference. In unselected sera from patients, the sensitivity and specificity of the flow cytometric assays were, respectively, 72.7 and 91.7 percent in detecting lymphocyte-reactive antibodies and 70.6 and 77.7 percent in detecting platelet-reactive antibodies, when the lymphocytotoxicity test was used as a reference. With regard to an adequate transfusion response, the sensitivities and efficiencies were 20.0 and 82.1 percent, 33.3 and 84.3 percent, and 70.0 and 88.6 percent for the lymphocytotoxicity test and the lymphocyte-reactive and platelet-reactive flow cytometric assays, respectively. CONCLUSION: Flow cytometric crossmatching appears to be an effective method of detecting platelet-reactive antibodies that may affect the success of platelet transfusions. This procedure is well-suited for routine conditions and can be performed within 2 hours.

Antibodies, Monoclonal↗
PubMed1996

Genotyping of the human platelet antigen systems 1 through 5 by multiplex polymerase chain reaction and ligation-based typing.

BACKGROUND: Platelet-specific antibodies may be involved in refractoriness to platelet transfusions, disorders such as neonatal alloimmune thrombocytopenia, and post-transfusion purpura. Genotyping for the major human platelet antigen (HPA) systems HPA-1 through HPA-5 is of considerable help in establishing the diagnoses of these diseases. STUDY DESIGN AND METHODS: A new genotyping method is described. Alleles of all five systems are amplified in a multiplex polymerase chain reaction. Subsequently, aliquots of the amplification products are thermocycled in the presence of a pair of allele-specific oligonucleotide probes and a heat-stable ligase. After heat denaturation, the probes hybridize adjacent to complementary sequences of the amplification product. In a perfect match, the two probes become covalently joined. Detection of the ligation product is performed with an enzyme-linked immunosorbent assay. RESULTS: Complete concordance of genotypes between the ligation-based typing and established genotyping methods was determined in 54 Austrian (HPA-1, -2, -3, and -5) and 56 Japanese (HPA-4) individuals. Ligation-based genotyping of HPA-1 polymorphism using platelet-derived RNA as starting material gave concordant results in all 15 cases tested. CONCLUSION: Multiplex polymerase chain reaction in combination with ligation-based typing allows fast typing of large numbers of platelet donors and screening for critical antigens in pregnant women.

Base Sequence↗
PubMed1996

Ligation based HLA-B*27 typing.

We have established a ligation based typing method to detect HLA-B*27 alleles at the DNA level. The method requires amplification of exon 2 of the HLA-B locus from genomic DNA by the polymerase catalyzed chain reaction (PCR) using group specific primers. An aliquot of the PCR amplification product, heat stable ligase and a pair of oligonucleotide probes, designed to hybridize adjacently to HLA-B*27 specific sequences of the amplified DNA are subsequently thermocycled. If the probes are perfectly complementary they become ligated otherwise they stay separated. The ligation of probes can be detected through their different labels by an enzyme linked immunosorbent assay (ELISA). Ligation based detection of beta-actin sequences which have been co-amplified serves as positive control for each PCR reaction. We observed complete concordance when typing 76 HLA-B*27 positive and 107 HLA-B*27 negative individuals either by serology or by the ligation based approach. We conclude that ligation based typing is a reliable tool for the DNA based detection of HLA-B*27 alleles. The procedure allows automation to a large extent and should be easily applicable to the typing of other HLA-class I alleles.

Base Sequence↗
PubMed1996

Allelic ladder characterization of the short tandem repeat polymorphism in intron 6 of the lipoprotein lipase gene and its application in an Austrian Caucasian population study.

The short tandem repeat (STR) polymorphism HumLPL (TTTA)n, which is located in intron 6 of the lipoprotein lipase gene, was investigated by AMPFLP (amplification fragment length polymorphism)-technique using an allelic ladder consisting of amplified alleles of this locus as a standard size marker. The allelic ladder was prepared by pooling equal concentrations of six separate alleles, which were identified by their different electrophoretic mobility in native polyacrylamide gel, eluted and subsequently amplified. Sequence analysis of the ladder alleles and allele 7, which is not included in the ladder, showed a regular repeat structure with 7 and 9 to 14 repetitions of the core repeat. The allelic ladder was employed in the analysis of the genotypes of 550 unrelated Caucasoids of Austria. No new alleles were found. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.195).

Alleles↗
PubMed1995

Tumor necrosis factor-alpha induction of major histocompatibility complex class II antigen expression is inhibited by interferon-gamma in a monocytic cell line.

Regulation of major histocompatibility complex (MHC) class II antigen expression by cytokines has been suggested to play a major role in the initiation and propagation of immune and autoimmune processes. The analysis of class II gene regulation benefits greatly from the existence of mutants with defects in regulatory factors. We report the establishment of a subclone of the human monocytic cell line U937, termed C119/9, with unusual cytokine regulation of MHC class II expression. In contrast to the parental U937 cell line, only tumor necrosis factor (TNF)-alpha, and not interferon (IFN)-gamma induces the expression of MHC class II antigens on C119/9 cells, and paradoxically, this induction was inhibited almost completely by IFN-gamma. The HLA-DR induction is controlled at the transcriptional level by the first 150 bp of the class II promoter which contains all the class II consensus elements. Both HLA-DR and -DQ mRNA are induced by TNF-alpha treatment, and both are diminished upon co-treatment with TNF-alpha and IFN-gamma. This antagonism between TNF-alpha and IFN-gamma seem to be restricted to MHC class II genes. This subline of U937 cells may be useful in further studies of MHC class II regulation.

Blotting, Northern↗