Publications (20)59.15 Total impact
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Article: Novel RHD alleles with weak hemagglutination and genetic Exon 9 diversity: weak D Types 45.1, 75, and 76.
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ABSTRACT: BACKGROUND: Molecular variant RHD allele analysis is best complemented by detailed characterization of the associated D phenotype. STUDY DESIGN AND METHODS: Variant D types were characterized using molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen quantification. RESULTS: We discovered three novel weak D types termed weak D Types 45.1, 75, and 76 with RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches; antigen densities of approximately 1.990, 900, and 240 D sites per red blood cell were found, respectively. Adsorption-elution technique-supported D epitope mapping of these three weak D types demonstrated the expression of all tested D epitopes. Initial molecular typing of the three investigated samples by RHD gene exon scanning polymerase chain reaction using sequence-specific priming yielded a negative reaction for A1193 located in RHD Exon 9 and could be explained by specific mutations for weak D Types 45.1 (C818T, G1195A), 75 (G1194C), and 76 (A1215C). CONCLUSION: All novel weak D types expressed all tested D epitopes. It is of interest that for weak D Types 45.1, 75, and 76, similar alleles with a maximal divergence of one amino acid only, that is, weak D Types 45, 41, and 68, respectively, have been reported so far.Transfusion 04/2013; · 3.22 Impact Factor -
Article: Modified solid-phase alloantibody detection for improved crossmatch prediction.
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ABSTRACT: Virtual crossmatching based on single-antigen bead (SAB) assays for the detection of donor-specific antibodies (DSA) has limited accuracy of predicting complement-dependent cytotoxicity crossmatch (CDCXM) results. In this study, 672 crossmatch combinations (32 allosensitized patients tested against cells from 21 high resolution-typed individuals) were analyzed to assess the potential of modified SAB tests in predicting T- or B-cell-CDCXM outcomes. Test modifications included measurement of C4d-fixation to detect complement-activating DSA ([C4d]DSA), or addition of dithiotreitol to abrogate the prozone effect ([IgG/DTT]DSA). Receiver operating characteristic (ROC) analysis revealed superior predictive accuracy of [C4d]DSA detection. Computing the mean fluorescence intensity (MFI) sum value of HLA class I [C4d]DSA in relation to T-cell-CDCXM revealed an area under the ROC curve (AUC) of 0.81. Other parameters, including DSA MFI maximum or number, were less predictive. Computing MFI sum values, AUC levels were lower for [IgG/DTT] (0.77) or [IgG]DSA detection (0.72), and did not considerably increase upon combining classifiers ([C4d] plus [IgG/DTT]: 0.82). ROC analysis revealed that [C4d]DSA detection (HLA class II) was also better at predicting B-cell-CDCXM results, even though, at very low MFI thresholds, the assay was found to provide comparably lower levels of specificity. Overall, B-cell-CDXM prediction was less precise, but could be enhanced by adjusting CDCXM thresholds to higher levels. Our data suggest particular efficiency of solid-phase complement detection as a tool for virtual crossmatching.Human immunology 10/2012; · 2.55 Impact Factor -
Article: Anti-A/B antibody depletion by semiselective versus ABO blood group-specific immunoadsorption.
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ABSTRACT: Recipient desensitization using blood group (BG)-specific immunoadsorption (ABO-IA) has proven to enable successful kidney transplantation across major ABO barriers. In this context, the efficiency of non-antigen-specific (semiselective) IA adsorbers has not yet been established. The objective of our study was to quantify anti-A/B antibody depletion by protein A-, peptide ligand- and anti-human immunoglobulin-based semiselective IA in comparison to ABO-IA. Eight ABO-IA-treated transplant candidates and 39 patients subjected to semiselective IA for a variety of different indications outside the context of ABO-incompatible transplantation were included. Antibody patterns (IgG, IgG1-4 subclasses, IgM, C4d-fixing reactivities) were analysed applying conventional agglutination testing and flow cytometry. As assessed by sensitive flow cytometric antibody detection, ABO-IA-based desensitization led to a profound even though often incomplete reduction of anti-A/B reactivities. Persistent complement- or non-complement-fixing reactivities, however, were not associated with transplant rejection or capillary C4d deposition. Single sessions of semiselective IA turned out to be more effective than ABO-IA in decreasing levels of anti-A/B IgG [median reduction to 28 versus 59% (ABO-IA) of baseline values, P < 0.001). In contrast, BG-specific IgM (74 versus 30%, P < 0.001) and IgG3 (72 versus 42%, P < 0.05) were reduced to a lesser extent, without differences between tested adsorber types. Analysis of four consecutive IA sessions revealed that inferior efficiency could not be overcome by serial treatment. Our observation of limited adsorption capacities regarding distinct BG-specific Ig (sub)classes suggests caution in applying semiselective IA techniques in ABO-incompatible kidney transplantation.Nephrology Dialysis Transplantation 11/2011; 27(5):2122-9. · 3.40 Impact Factor -
Article: Effect of the proteasome inhibitor bortezomib on humoral immunity in two presensitized renal transplant candidates.
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ABSTRACT: Recipient presensitization represents a major hurdle to successful renal transplantation. Previous case series have suggested that the proteasome inhibitor bortezomib directly affects the alloantibody-secreting plasma cells in rejecting allograft recipients. However, the ability of this agent to desensitize nonimmunosuppressed transplant candidates before transplantation is currently unknown. In this analysis, two sensitized hemodialysis patients were selected to receive two subsequent bortezomib cycles. Bortezomib was given at 1.3 mg/m(2) on days 1, 4, 8, and 11. Dexamethasone was added to the second cycle to enhance treatment efficiency. Serial immune monitoring included cytotoxic panel reactive antibody testing, Luminex single antigen testing for anti-human leukocyte antigen (HLA) IgG with or without C4d-fixing capability, and ABO antibody detection. During a half-year follow-up period, cytotoxic panel reactive antibody decreased from 87% to 80% (patient 1) and 37% to 13% (patient 2). Patient 1 showed a 40% reduction in binding intensities of identified Luminex HLA single antigen reactivities and, in parallel, slight reductions in ABO blood group antibody and total immunoglobulin levels. In patient 2, bortezomib did not affect circulating antibody levels in a meaningful way. Both patients showed a more than 50% reduction in the levels of anti-HLA antibody-triggered C4d deposition to Luminex beads. Our initial experience suggests that, without additional immunosuppressive measures, bortezomib has modest effects on circulating antibodies against HLA or blood group antigens. The reduced levels of antibody-triggered complement fixation, however, imply potential clinical relevance of proteasome inhibition for recipient desensitization.Transplantation 03/2010; 89(11):1385-90. · 4.00 Impact Factor -
Article: Clinical relevance of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients.
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ABSTRACT: Donor-specific alloantibodies (DSA), especially those fixing complement, may pose a particular immunologic risk to transplant recipients. To assess the clinical impact of C4d- or non-C4d-fixing (IgG) HLA sensitization, pretransplant sera obtained from 338 kidney allograft recipients prescreened by FlowPRA were retrospectively evaluated by Luminex single antigen (SA) testing using a novel fluorescent-labeled anti-C4d reagent for detection of antibody-triggered C4d deposition in addition to IgG binding. Recipients with [IgG]DSA (n = 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG] non-DSA (0%) or 283 antibody-negative patients (4%, multivariate analysis excluding retransplantation because of high co-linearity: P < 0.0001), and adversely affected 5-year death-censored graft survival (74% vs. 81% and 90%, respectively, multivariate model: P < 0.05). [C4d] DSA (n = 21) and [C4d] non-DSA (n = 25) increased rates of C4d positive rejections to a similar extent (24% and 28% vs. 4% in recipients without C4d-fixing reactivity; multivariate analysis: P <or= 0.002) with a trend towards adverse 5-year graft survival (76% and 76% vs. 90%; P <or= 0.2). In conclusion, Luminex-based characterization of HLA sensitization may be a useful strategy for risk stratification. Possibly as a result of intensified immunosuppression in presensitized recipients, identification of C4d-fixing DSA was not associated with a further increase of rejection and graft loss rates.Transplant International 08/2009; 22(10):982-9. · 2.92 Impact Factor -
Article: A novel KEL*1,3 allele with weak Kell antigen expression confirming the cis-modifier effect of KEL3.
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ABSTRACT: KEL1 (K) is the most immunogenic red blood cell antigen of the Kell blood group system. The frequently occurring anti-KEL1 alloantibodies may cause hemolytic transfusion reactions as well as severe hemolytic disease of the fetus and newborn. So far, reports on weak KEL phenotypes are scarce. Blood samples of two unrelated Central European propositi with faint reactions in routine KEL1 typing were analyzed by extended serologic phenotyping, flow cytometry, genotyping by polymerase chain reaction with sequence-specific priming, and genomic DNA sequencing of separated parental KEL alleles. Both propositi exhibited an unusual KEL:1,2,3,4 phenotype: markedly weakened and negative reactions were observed in serologic KEL1 typing in gel and tube technique, respectively. No KEL1 epitope loss was detected using five different monoclonal anti-KEL1 reagents. KEL genotyping confirmed KEL*1/2 and KEL*3/4 (Kpa/Kpb) heterozygosity of both individuals. Importantly, DNA sequencing of the two separated parental alleles of both propositi revealed a KEL*1-specific coding nucleotide T578 and a KEL*3-specific T841 on the same allele. This novel KEL*1,3 (KKpa)allele was associated with an approximately 80 percent reduction in KEL1 expression, compared to KEL:1,2,-3,4 controls. The low KEL1 density was attributed to acis-modifier effect of KEL3, so far only reported in association with weakened expression of KEL2 (k). This is the first description of the KEL*1,3 allele encoding KEL1 and KEL3 on the same molecule. In individuals with weakened KEL1 because of KEL3 in cis, very sensitive serologic or molecular genetic techniques may be required for reliable Kell typing.Transfusion 05/2009; 49(4):733-9. · 3.22 Impact Factor -
Article: Application of a Multivariant, Caucasian-Specific, Genotyped Donor Panel for Performance Validation of MDmulticard®, ID-System®, and Scangel® RhD/ABO Serotyping.
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ABSTRACT: BACKGROUND: Validations of routinely used serological typing methods require intense performance evaluations typically including large numbers of samples before routine application. However, such evaluations could be improved considering information about the frequency of standard blood groups and their variants. METHODS: Using RHD and ABO population genetic data, a Caucasian-specific donor panel was compiled for a performance comparison of the three RhD and ABO serological typing methods MDmulticard (Medion Diagnostics), ID-System (DiaMed) and ScanGel (Bio-Rad). The final test panel included standard and variant RHD and ABO genotypes, e.g. RhD categories, partial and weak RhDs, RhD DELs, and ABO samples, mainly to interpret weak serological reactivity for blood group A specificity. All samples were from individuals recorded in our local DNA blood group typing database. RESULTS: For 'standard' blood groups, results of performance were clearly interpretable for all three serological methods compared. However, when focusing on specific variant phenotypes, pronounced differences in reaction strengths and specificities were observed between them. CONCLUSIONS: A genetically and ethnically predefined donor test panel consisting of 93 individual samples only, delivered highly significant results for serological performance comparisons. Such small panels offer impressive representative powers, higher as such based on statistical chances and large numbers only.Transfusion Medicine and Hemotherapy 01/2009; 36(3):219-225. · 1.16 Impact Factor -
Article: Mosaicism due to myeloid lineage restricted loss of heterozygosity as cause of spontaneous Rh phenotype splitting.
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ABSTRACT: Spontaneous Rh phenotype alteration interferes with pretransfusion and prenatal blood group examinations and may potentially indicate hematologic disease. In this study, the molecular background of this biologic phenomenon was investigated. In 9 patients (3 with hematologic disease), routine RhD typing showed a mixture of D-positive and D-negative red cells not attributable to transfusion or hematopoietic stem-cell transplantation. In all patients, congenital and acquired chimerism was excluded by microsatellite analysis. In contrast to D-positive red cells, D-negative subpopulations were also negative for C or E in patients genotyped CcDdee or ccDdEe, respectively, which suggested the presence of erythrocyte precursors with an apparent homozygous cde/cde or hemizygous cde/- genotype. Except for one patient with additional Fy(b) antigen anomaly, no other blood group systems were affected. RH genotyping of single erythropoietic burst-forming units, combined with microsatellite analysis of blood, different tissues, sorted blood cell subsets, and erythropoietic burst-forming units, indicated myeloid lineage-restricted loss of heterozygosity (LOH) of variable chromosome 1 stretches encompassing the RHD/RHCE gene loci. Fluorescent in situ hybridization studies indicated that LOH was caused by either somatic recombination or deletion. Therefore, most cases of spontaneous Rh phenotype splitting appear to be due to hematopoietic mosaicism based on LOH on chromosome 1.Blood 10/2007; 110(6):2148-57. · 9.90 Impact Factor -
Article: Genetic diversity of KELnull and KELel: a nationwide Austrian survey.
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ABSTRACT: Besides ABO and RH, the KEL blood group system, including the two antithetical antigens KEL1 and KEL2, is the most important owing to the frequent appearance of anti-KEL alloantibodies and their considerable clinical significance. So far, only limited information was available on KEL variant alleles determining the rare silent KELnull and KELel phenotypes with absent or diminished KEL antigen expression detected only by adsorption-elution techniques, respectively. For a systematic investigation of the KELnull and KELel phenotypes, 401 KEL:1,-2 samples (representing 2.6% of all Austrian KEL:1,-2 samples) and 811 KEL:1,2 samples were genotyped for the KEL*1/KEL*2-specific single-nucleotide polymorphism. All heterozygous KEL*1/KEL*2 and 4 additional KELnull samples were subjected to detailed immunohematologic examination and allele-specific sequencing. In 14 KEL:1,-2 samples, discrepant KEL*1/KEL*2 heterozygosity was observed, indicating the presence of silent or barely expressed KEL*2 alleles, whereas all KEL:1,2 individuals were homozygous for KEL*2. In the course of further molecular analysis, 8 novel KEL*2null and 2 KEL*2el alleles were discovered, representing 67 and 33 percent of previously known KEL*2null- and KEL*2el-encoding alleles, respectively. In addition, two different known KEL*2null and KEL*2el alleles each were confirmed. The immunohematologic properties of KEL variant red blood cells were defined by extended KEL phenotyping and flow cytometric KEL1, KEL2, KEL4, and KEL7 antigen as well as total Kell protein quantification. For the first time, exact KELnull and KELel population frequencies could be established in this population.Transfusion 05/2007; 47(4):703-14. · 3.22 Impact Factor -
Article: Platelet autoantibodies are common in hepatitis C infection, irrespective of the presence of thrombocytopenia.
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ABSTRACT: To investigate the generation of platelet antibodies in hepatitis C virus (HCV)-infected individuals and their relation to the development to thrombocytopenia with the aim of using their detection as a diagnostic aid of immune thrombocytopenia in these patients. We tested by the monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA) for the presence of platelet antibodies against specific glycoprotein (GP) targets (GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIIIb, GPV, and FcRgammaIIa) in 48 HCV-infected individuals of various stages of disease and compared the results with those from 35 patients with alcoholic liver cirrhosis. Thirty-two HCV-infected individuals (66%) had detectable platelet antibodies. The most common target was GPIIb/IIIa, but all other GP were also targets. Results were not different from patients with alcoholic liver cirrhosis. There was no correlation between antibodies and platelet counts, or the stage of disease, or the viral genotype, or a discernible influence of treatment with alpha-interferon. While platelet autoantibodies are common in individuals with HCV infection, their detection does not assist in the diagnosis of immune thrombocytopenia.European Journal Of Haematology 01/2007; 77(6):513-7. · 2.61 Impact Factor -
Article: Surface disinfection of packed red blood cells with 70% ethanol.
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ABSTRACT: No data or recommendations are available on feasibility of surface disinfection of blood bags, but some circumstances can make such procedures inevitable. Impact of immersion of blood bags in 70% ethanol for 30min was investigated with respect to alcohol penetration and changes of hemolysis parameters in the product, and bag material changes influencing material stability and composition. After immersion ethanol concentration in blood bags was below detection limit. Hemolysis parameters did not differ between blood products that had been exposed to ethanol and a control group. Inner surface of the bag material was unchanged according to our infrared spectrometry results. Also endurance testing showed no altered results. We conclude that immersion of blood bags in 70% ethanol for surface disinfection is a safe procedure for the quality of the blood product and the bag material.International journal of surgery (London, England) 02/2006; 4(2):118-21. -
Article: Novel weak D types 31 and 32: adsorption-elution-supported D antigen analysis and comparison to prevalent weak D types.
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ABSTRACT: Weak D types are thought to express rather quantitative than qualitative D antigen variants. Distinct type-specific phenotypes and weak D cases with anti-D alloimmunization, however, suggest a variable degree of D antigen alteration. Variant D types were investigated by use of molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen density determination. Two novel weak D types were discovered, termed weak D type 31 and 32 with single RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches, with antigen densities of approximately 130 and 50 D sites per red blood cell, respectively. Adsorption-elution technique-supported D epitope mapping of these two weak D types, the recently described weak D type 26, and of the most common Central European weak D types (weak D types 1, 2, 3, 4.0, and 4.1) demonstrated the expression of all tested D epitopes. In contrast, a distinct D epitope loss was detected in weak D type 15 and partial D control samples. All novel and prevalent weak D types expressed all tested D epitopes. Our results indicate that adsorption-elution techniques may be of advantage whenever D epitope loss is suspected in extremely weak D variants.Transfusion 11/2005; 45(10):1574-80. · 3.22 Impact Factor -
Article: A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization.
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ABSTRACT: The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion- and pregnancy-induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption-elution techniques, with so far only poorly defined antigenic properties. A comprehensive immunohematologic analysis of five of the six currently known DEL genotypes was performed. DEL phenotypes associated with the RHD(M295I), RHD(IVS3+1g>a), RHD(K409K), RHD(X418L), or RHD(IVS5-38del4) allele were characterized with extended serology and flow cytometry. Epitope mapping with adsorption-elution revealed a prominent D epitope loss in the RHD(IVS3+1g>a)-associated DEL phenotype, whereas in the other four DEL types no signs of qualitative D antigen alteration were detected. The observation of alloanti-D in two RHD(IVS3+1g>a) cases confirmed the partial nature of this DEL phenotype. The RHD(M295I) phenotype exhibited the highest D antigen expression among all investigated DEL types, as determined by a semiquantitative adsorption-elution approach and flow cytometry. In conclusion, evidence is provided that different DEL genotypes code either for partial or complete D antigen expression and that this finding is clinically relevant.Transfusion 10/2005; 45(10):1561-7. · 3.22 Impact Factor -
Article: Milestones in immunohematology.
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ABSTRACT: The milestones in immunohematology, as seen by immunogeneticists, are described.Transplant Immunology 09/2005; 14(3-4):155-7. · 1.46 Impact Factor -
Article: Anti-D immunization by DEL red blood cells.
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ABSTRACT: No data are available on the immunogenicity of extremely weak D variants called DEL. Evaluation of alloanti-D formation in a D- female patient after transfusion of apparently D- blood from an Austrian donor led to discovery of a so far unknown DEL type. Standard blood group serologic methods were applied. Molecular typing, RHD sequencing, and D epitope mapping was performed and the absolute D antigen density determined. After transfusion of RBCs typed D- by routine serology, the recipient developed alloanti-D. Further evaluation with an indirect antiglobulin test confirmed donor RBCs to be D-. Molecular typing, however, demonstrated the presence of the RHD gene in one donor, and RHD sequencing revealed a deletion of four nucleotides in RHD intron 5 (RHD IVS5-38del4) as the only difference compared to the normal RHD gene. Adsorption-elution techniques demonstrated a DEL phenotype without apparent loss of D epitopes. This study documents the clinical significance of the DEL phenotype in blood units that was capable of inducing anti-D in a recipient. Qualitative data are provided on D epitope expression in DEL RBCs.Transfusion 05/2005; 45(4):520-6. · 3.22 Impact Factor -
Article: Presence of RHD in serologically D-, C/E+ individuals: a European multicenter study.
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ABSTRACT: RHD blood group alleles with reduced or absent antigen expression are a clinically significant and heterogeneous group. Study design and methods: To detail population genetics data on apparently D- individuals in central Europe, a six-center study was performed with participants from Austria, Germany, Slovenia, Switzerland, and Russia. A total of 1700 serologically D- samples, positive for C and/or E, were investigated. Observed unexpressed RHD alleles were 59 RHD-CE-D+ hybrid alleles, 9 apparently regular RHD, 1 new RHD(Y401X); DELs were 8 RHD(M295I), 6 RHD(IVS3+1G>A), and 1 new RHD(X418L); and weakly expressed RHDs were 2 weak D type 5, 1 weak D type 1, 1 RHD category VI type 1, and 1 novel weak D type 26. Although weak D type 26 was shown to have one of the lowest D antigen densities ever observed, it gave rise to anti-D immunization in a transfused D- individual. The relative occurrence of RHD among serologically D- samples, positive for C and/or E, differed significantly in the investigated central European regions. Considering the growing use of molecular typing techniques, correct identification of blood group alleles with scarce or missing antigen expression is of utmost clinical importance and requires reliable population-based frequency data.Transfusion 04/2005; 45(4):527-38. · 3.22 Impact Factor -
Article: Quality of drainage blood: survival of red cells after re-transfusion and content of free hemoglobin and potassium.
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ABSTRACT: Re-transfusion of drainage blood is widely used in orthopedic surgery, but objective evidence of the efficacy of re-transfusion of drainage blood in view of post-transfusion survival of RBC has not been given so far. With this study we wanted to evaluate the efficacy and safety of transfusion of drainage blood collected with HandyVac autotransfusion system. In 7 patients red cells in drainage blood were labeled with biotin and percentage of labeled red cells in circulation were determined immediately after re-transfusion, and during 10 days after surgery. To assess further unwanted side-effects of re-transfusion of drainage blood potassium and free hemoglobin were determined in the collected blood. Ten days after re-transfusion at mean 78.9% of drainage-blood derived RBC were found in circulation. Free hemoglobin in drainage blood ranged from 16.8 to 59.2 mg/dL; potassium in drainage blood ranged from 3.84 to 4.52 mmol/L. Our results suggest that re-transfusion of drainage blood collected with HandyVac autotransfusion system is an efficient procedure that seems to be safe in view of free hemoglobin and potassium in the product.International journal of surgery (London, England) 02/2005; 3(4):250-3. -
Article: Molecular and serologic characterization of DWI, a novel "high-grade" partial D.
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ABSTRACT: Accurate D antigen identification is essential for pretransfusion and prenatal evaluation to prevent anti-D alloimmunization. Quantitative and qualitative D variants may pose typing problems and require particular consideration because of differing potential for anti-D induction. A novel partial D, DWI, was discovered in an anti-D-alloimmunized D+ Austrian woman. This D variant was investigated by RHD genotyping and nucleotide sequencing, as well as characterization of its serologic properties. The proposita exhibited a single-nucleotide exchange in RHD Exon 7 (1073T>C) predicting a Met358Thr substitution in the sixth extracellular loop of the RhD polypeptide. All DWI individuals identified (the proposita and two relatives) were genotyped DWIdCcee, which, together with the family tree, was highly suggestive of a DWICe haplotype association. Epitope mapping studies revealed only minor D antigen modification with weakening but not loss of epitopes D1.1, D9.1, and D16.1. Antigen density varied individually between 8000 and 8600 D sites per erythrocyte. No known low-frequency Rh antigen was detected. Despite the highly retained D epitope composition, the DWI proposita's serum sample contained alloanti-D from an immunization event many years earlier. The findings of this investigation emphasize the possible clinical significance of "high-grade" partial D variants that are likely to be missed by routine serology.Transfusion 05/2004; 44(4):575-80. · 3.22 Impact Factor -
Article: Flow cytometry based detection of HLA alloantibody mediated classical complement activation.
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ABSTRACT: Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.Journal of Immunological Methods 04/2003; 275(1-2):149-60. · 2.20 Impact Factor -
Article: The tetranucleotide repeat polymorphism C2_4_4: population data and linkage disequilibria with HLA class I.
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ABSTRACT: The tetranucleotide repeat locus C2_4_4 situated in the HLA class I region (6p21.3) and the HLA-ABC specificities were investigated in an Austrian population sample of 240 unrelated Caucasoid individuals. The analysis of the linkage disequilibrium between C2_4_4 and HLA class I showed several significant values, especially when factors coded for by so-called "superhaplotypes" were considered; such linkage disequilibria are of importance for the practical use of HLA coded short tandem repeats.Immunobiology 02/2003; 207(2):137-40. · 3.20 Impact Factor
Top co-authors
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Institutions
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2007–2012
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Medical University of Vienna
- Universitätsklinik für Innere Medizin II
Vienna, Vienna, Austria
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2003–2004
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University of Vienna
Vienna, Vienna, Austria
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