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Four new antigens of the second HL—A series are reported. They were established by means of lymphocytotoxic and platelet complement-fixing antisera, and they behaved in population and family studies as determined by alleles at the second HL—A sublocus. It was found that three of them were parts of a previously assumed monofactorial antigen: SL. The antigen SL thus is heterogeneous, as also seems to be true of several other HL—A antigens. If all HL—A genetic determinants presently identified in our laboratories are taken into account, 18,145 different genotypes are possible. Implications of heterogeneous HL—A antigens for histocompatibility testing and matching are discussed.

A German family showing a weak expression of the antigen HL–A2 is described. The results of the HL–A typing and of absorption experiments are mentioned, and the genetic background of this phenomenon is discussed.

H. W. Reesink, C. P. Engelfriet, G. Henn, W. R. Mayr, G. Delage, F. Bernier, T. Krusius, A. Assal, P. Gallian, C. Corbi, P. Morel, B. David, P. De Micco, H. Murokawa, H. Yugi, S. Hino, K. Tadokoro, O. Flesland, E. Brojer, M. Oe towska, G. Olim, F. Nascimento, H. Goncalves, L. Castro, M. Morais, S. L. Stezinar, M. Alvarez, S. Sauleda, R. Gonzalez, C. Niederhauser, M. Stolz, J.-P. Allain, S. Owusu-Ofori, R. Eglin, S. Stramer, M. Busch, D. M. Strong, J. Epstein, R. Biswas

SUMMARY A lymphocytotoxic antibody whose reactions are associated with the blood group A and the ABH secretor status is described.

In a mother-child pair, false exclusions in markers on chromosome 6 have been observed. The genetic incompatibilities have been caused by paternal uniparental disomy. The consequences of such cases for investigations of parentage are discussed.

Background and Objectives The aim of this study was to investigate whether in-line filtration, using a polyester filter for the preparation of red cell concentrates (RCC) and plasma (PL), leads to an altered proportion of T and B lymphocytes in the fraction of residual white blood cells (WBC). Materials and Methods The capacity of Pall WBF-2 in-line filters to reduce the numbers of T and B lymphocytes from red blood cell concentrates (RCC) and plasma (PL) of 22 donations was investigated by three-colour flow cytometry (FC) using the Tritest-Trucount kit. T and B lymphocytes were identified using monoclonal antibodies (mAbs) against CD3, CD19 and CD45, conjugated with fluorescein isothiocyanate, phycoerythrin or peridinin chlorophyll protein-A, respectively. As the number of B cells was below the detection limit of the FC method, WBC of the respective blood components of healthy donors were concentrated 25-fold by Percoll density-gradient centrifugation. In this fraction the absolute numbers of T and B cells, as well as their ratio, were determined using the Attractor software, which provides a discrimination of rare cell counts from FC in relation to debris. Results The mean numbers, as well as minima and maxima of T and B lymphocytes per unit, were as follows. T cells in RCC: 4·51 × 103 (1·68 × 102−4·09 × 104) and in PL: 1·35 × 103 (2·21–1·78 × 104); B cells in RCC: 2·33 × 103 (7·10 × 101−9·15 × 103) and in PL: 2·33 × 102 (7·5 × 102−2·8 × 103). T cells were retained, on average, at a higher level than B cells: 3·01 times higher in RCC and 1·01 times higher in PL. Conclusion After filtration, the ratio of T and B lymphocytes changed in RCC (1·95 : 1) compared with unfiltered blood, where it was 5·83 : 1. In PL the ratio did not change notably compared to unfiltered blood. The results of this research show that cell concentration (using gradient centrifugation) in combination with an appropriate FC acquisition and analysis procedure, allows both residual T and B lymphocytes (being under the detection limit without cell concentration) to be determined.

BACKGROUND : The aim of this study was to demonstrate how ultrastructural morphology of platelets stored in different media correlate with the appearance of particular activation markers on their cell surface. STUDY DESIGN AND METHODS : Concentrates of buffy coat-derived platelets were stored in plasma or a glucose-free citrate-acetate-NaCl platelet additive solution (PAS2, Baxter Healthcare Corp.). Activation markers on platelets were measured by flow cytometry and compared with changes in the platelet cell surface as demonstrated by electron microscopy. Levels of the vasoactive cytokines vascular endothelial growth factor (VEGF) and RANTES (regulated upon activation, normal T-cell expressed and secreted) were determined in the storage medium of the platelet concentrate. RESULTS : The activation markers CD62P and CD63 and the binding of thrombospondin measured by flow cytometry were expressed to a higher extent in the PAS2 group compared with the plasma group. The difference reached significance on Day 3 (CD62P: 66.37 ± 2.44 vs. 37.83 ± 2.03, p < 0.001; CD63: 42.11 ± 3.29 vs. 34.84 ± 2.04, p < 0.05; and thrombospondin binding: 18.84 ± 3.9 vs. 13.98 ± 3.87, p < 0.001, respectively). The form factor that is related to changes of the platelet shape was determined by image analysis and correlated significantly with the cell surface expression of CD62P (p < 0.001) and with CD63 (p < 0.05) and with thrombospondin binding (p < 0.05). The chemokines VEGF and RANTES were measured at higher levels in the PAS2 group. CONCLUSIONS : With exception of baseline activation probably due to necessary handling procedures, platelets remain relatively unaltered and more stable in plasma in comparison to storage in PAS2.

Background and Objectives A molecular method for analysing whole-blood samples should be established for quality control of plasma sample logistics. Materials and Methods DNA profiles of retention samples (plasma) were compared to profiles of recent donations (whole blood). DNA extraction, amplification and detection were performed using the Qiagen DNA Blood Mini kit, the AmpFFISTR Profiler Plus Kit and capillary electrophoresis, respectively. Results Matched pairs of full profiles were obtained for all samples investigated, therefore no deviation from the standardized procedures was detected. Conclusions Modified extraction and amplification protocols enabled DNA profiling to be used for the quality control of plasma samples. Hence, DNA profiling can be used in the blood bank as a safe and easy method for quality control of sample logistics.

During the past few years the DNA commission of the International Society of Forensic Genetics has published a series of documents providing guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. This latest report addresses a relatively new area, namely Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). This report addresses nomenclature, use of allelic ladders, population genetics and reporting methods.

Background and Objectives Platelet count, thrombopoetin (TPO) level and the compartment of megakaryocyte progenitor cells (CFU-Mk) are major determinants in the regulation of thrombopoiesis. The aim of this study was to investigate the potential changes in the compartment of CFU-Mk and their correlation with serum TPO levels and platelet count after plateletpheresis. Materials and methods Twelve healthy individuals were randomly assigned to undergo single-donor plateletpheresis. A collagen-based in vitro culture system was used to determine the number of peripheral blood (PB) CFU-Mk before and after donation and on days 1, 4 and 7 thereafter. TPO levels were measured by a specific enzyme-linked immunosorbent assay and whole blood counts were performed using an automated cell counter. Results The pre-apheresis platelet count (mean ± SEM: 276 ± 13 × 109/l) decreased after plateletpheresis to a nadir of 194 ± 8 × 109/l (P

Osteopenia is common in patients with celiac disease and is believed to result from malnutrition. Osteoporosis in otherwise healthy individuals is related to genetically determined polymorphisms within the vitamin-D-receptor (VDR) gene. We hypothesized that in celiac patients particular genes of the VDR enhance the susceptibility for malnutrition-associated low-bone density.We determined allelic frequencies within the VDR gene by restriction fragment length polymorphism analysis in 92 patients with celiac disease (age, 15-83 years). Thirty-eight patients were on a gluten-free diet; 54 patients did not adhere to a diet. The determined VDR polymorphisms in 111 unrelated newborns served as controls. Osteopenia was determined by means of ultrasound measurements of the calcaneus (n = 78). Bone turnover was estimated by osteocalcin determination (n = 60).There was no difference in the frequency of the VDR gene polymorphisms in patients with celiac disease compared with controls. Adjusted ultrasound measures of the calcaneus were low in 47% of patients, but there was no difference of the VDR gene frequencies in these patients compared with those with normal ultrasound results or controls. Bone turnover was higher in patients without a gluten-free diet (P = 0.02). Again, there was no association with any particular VDR gene.Patients with celiac disease frequently have osteopenia, which is not related to any of the determined genes within the VDR.

Detection of two different cell populations in a child is a rare event. The following case of a dispermic chimera was diagnosed before surgery due to problems in blood group determination. A 2-year-old phenotypically male child was admitted for correction of a penoscrotal hypospadia and unilateral cryptorchism. During presurgical laboratory investigation, difficulties in blood group determination occurred. Blood group typing was performed by the DiaMed-ID Micro Typing System and by FACS. Additionally, cytogenetic analysis of lymphocytes and analysis of DNA polymorphisms in different tissues were performed. Two populations of red blood cells were detected, O cells accounting for 75% and B cells for 25%. Analysis of DNA-PCR polymorphisms in lymphocytes, nails, and in cells of the oral mucous membrane demonstrated a chimerism, with two alleles inherited from the father and one from the mother. A cytogenetic analysis of cultured lymphocytes showed a mosaic 46, XY/46,XX. Surgery revealed a prostatic utricle grade III, also called pseudovagina; genitography confirmed a vagina. Bilateral gonad biopsy showed a testis on one side and an ovary on the other. This case of chimerism represents a true hermaphroditism that most probably developed by double fertilization of one or more egg nuclei by two sperms.

The STR locus D17S976 was investigated by PCR amplification and native polyacrylamide gel electrophoresis in 158 unrelated Austrian Caucasians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance was 0.792, the discriminating power was 0.980 and the observed heterozygosity rate was 0.873. Moreover two alternative denaturing electrophoretic protocols are proposed. An allelic ladder consisting of 14 sequenced alleles (236-288 bp) was constructed. Sequence analysis revealed that the locus contained three different repeat motifs: ATCA, ATCT and ACCT, all of which vary in number between alleles. The aggregate number of the three tetrameric repeat types was used for allele designation. As a repeat with a single base deletion (ATC) was found in both the smallest and the largest alleles, a ".3" was added to the allele designation in those cases. Therefore the smallest allele is designated 19.3, and the largest allele is designated 32.3. To evaluate the exact extent of sequence variation more extensive sequence studies are necessary.

BACKGROUND: Weak D phenotypes involve a quantitative variation of D. The genomic basis in weak D has been disputed, however. STUDY DESIGN AND METHODS: Five sequence-specific polymerase chain reactions (SSP- PCRs) on exons 2, 5, and 7 of the RHD gene were evaluated in 248 white and 98 Japanese blood donors and compared with the results obtained by amplification of intron 4 and serology. All methods and SSP-PCR testing on the 3′ non-coding region of the RHD gene were applied to the genotyping of 94 DNA samples derived from individuals expressing weak D phenotypes. RESULTS: Concordant results were obtained with all genotyping and phenotyping methods in testing 201 D-positive and 145 D- negative donors. Four of 94 weak D samples were typed as D-negative by amplification of intron 4 and SSP-PCR on exon 5. Phenotyping with monoclonal antibodies revealed a DVI category in one of these cases and DFR phenotype in three of these cases. One weak D sample, which reacted like normal D-positive cells with all applied monoclonal antibodies, was typed falsely negative by SSP-PCR on exon 5 because of a point mutation at nucleotide 667 (T–>G) that resulted in a Phe223Val amino acid substitution. In this individual, heterozygosity was found at two other amino acid positions (Glu233Gln and Val238Met) by restriction fragment length polymorphism analysis. CONCLUSION: Genetic diversity in weak D phenotypes is rare. Only 1 of 90 true weak D phenotypes (1.1%) had a genetic variation in testing on seven gene regions of the RHD gene.

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.

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 Gottingen). 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 Gottingen. In FFP-F, the median WBC contamination was 13,000 (Vienna) and 31,000 (Gottingen) 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.

immunization in patients whohave been previously immunized by pregnan-cy or transfusion is not prevented. Neither canimmunization against platelet-specific al-loantigens be prevented by removing leuco-cytes from platelet concentrates, but platelet-specific antibodies seem to occur mainly inpatients who develop HLA class I antibodies.Not all patients with HLA antibodies are re-fractory. Often the antibodies only react witha small percentage of panel lymphocytes andthen most random donors are compatible.Furthermore, although there are individualdifferences, some HLA class I antigens areweakly expressed on platelets. Nevertheless,refractory alloimmunized patients in need ofplatelet transfusions will continue to occur.Opinions on the optimal management of suchpatients differ. Moreover, some simple and in-teresting new crossmatch techniques have re-cently been developed. For these reasons itseemed of interest to devote an InternationalForum to the subject.There seem to be two main approaches tothe management of refractory patients: (I) theselection of platelet concentrates based on theHLA class I phenotype and, or the specificityof the HLA (and platelet-specific) antibodies,and (II) the selection of platelet concentratesby crossmatching.To obtain more information on the sub-ject, the following questions were submittedto 15 experts in the field

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. © 1996 Wiley-Liss, Inc.

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.

In order to estimate the residual risk of transfusion-transmitted HCV infection, we have analyzed data from two transfusion centers in Austria (Vienna) and Germany (G6ttingen) 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 Gottingen, 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 person 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 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 0.1%. 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 Gottingen, 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.

The tetranucleotide repeat loci D6S389 and D6S1051 situated nearby the HLA class II region (6p) were investigated in an Austrian Caucasoid population sample. Typing of the amplification products and cycle sequencing were carried out using denaturing capillary electrophoresis. For D6S389 and D6S1051, 19 and 7 different alleles were observed. Sequencing of D6S389 revealed a (GAAA)n repeat pattern in the common alleles. Additionally, infrequent “.1” alleles were seen. Alleles of D6S1051 showed a simple (GATA)n repeat structure and an A/G-SNP next to the repeat region as well as a T/C-SNP in the 3′-flanking region. D6S389 proved to be a highly polymorphic marker. D6S1051 is less polymorphic, but interesting because of the existing SNPs.

On the occasion of a study on mutation rates of human STR loci, numerous alleles have been sequenced. Several new sequence or length variant alleles were found.

In this study, we investigated the short tandem repeat loci C1_4_4, C2_4_4 and C3_3_6 situated in the HLA class I region in a sample of 153 unrelated Austrian Caucasoid individuals by multiplex PCR. The sequence structure of C1_4_4 and C3_3_6 alleles is described, as well as the sequence of a new C2_4_4 allele, allele 23.

Unusual peak height ratios of the two amplified fragments in the X–Y homologous amelogenin gene in two multiplex STR kits (16 loci) required further investigations in three male Caucasoid individuals. Mosaicism is discussed as a possible reason for these findings.

ZusammenfassungDie Übertragung von HlV-lnfektionen durch Blutkomponenten, die sich bislang noch nicht virusinaktivieren lassen, ist eine extrem seltene, aber typische unerwünschte Arzneimittelwirkung der Transfusion. Um dieses Restrisiko für HIV zu ermitteln, haben wir die Daten von 1985-1993 aus Wien und Göttingen ausgewertet. Im Jahr 1993 war die Prävalenzrate der HIV-lnfektion bei Erstspendern 1:7000 in Wien und 1:7900 in Göttingen. Die Inzidenz einer Neuinfektion unter den Dauerspendem war 1:42000 in Wien und 1:67 000 in Göttingen. Es wurde ein statistisches Modell zur Berechnung des Restrisikos entwickelt, das sowohl (1) das diagnostische Fenster der HIV-Teste, (2) die Sensitivität der Anti-HIV-Teste, als auch (3) globale Testfehler (bzw. Nichtberücksichtigung eines Testergebnisses) berücksichtigt. Wir ermittelten ein Restrisiko (für 3. Generations-anti-HIV-Tests mit einem diagnostischen Fenster von 22 Tagen) in Wien von 1:520000 und in Göttingen von 1:900000 für das Jahr 1993. Die gleichzeitig durchgeführten Look-back-Untersuchungen erbrachten HlV-lnfektionen bei 3 Empfängern (auf 1900000 Blutspenden) in Wien und 1 Empfänger in Göttingen (für 160000 Blutspenden).Mit Hilfe unseres Modells ist nicht nur eine Risikoabschätzung möglich, sondern es läßt sich auch der Effekt weiterer Maßnahmen zur Reduktion des Restrisikos errechnen.

We report on flow cytometric IgG subclass determinations of red cell antibodies using polyclonal FITC-labeled antibodies. The limit of detection of this method was 1 ng anti-D per 1 x 10(7) red cells. The inter- and intra-assay coefficients of variance were 8.2 and 2.3%, respectively. In 8 newborns with a positive direct antiglobulin test (DAT) in the gel centrifugation test (GCT), due to ABO antibodies, IgG1 was detected in all and IgG2 additionally in 4 of these cases. In 5 severe cases of hemolytic disease of the newborn (HDN) due to anti-D, large amounts of IgG1 were found, and in 3 of these 5, IgG3 in combination with IgG1. In 8 mild or moderate HDN cases (4 anti-D, 2 anti-E, 1 anti-Fya, 1 anti-Jka), phototherapy sufficed, and IgG1 was the only antibody. In 7 adult patients with malignant lymphoma and a positive DAT (GCT), only small amounts of IgG1 red cell autoantibodies could be demonstrated by flow cytometry. In 5 further patients with malignant lymphoma, a positive DAT, and severe hemolytic anemia, large amounts of IgG1 autoantibodies were found and IgG3 was also present in 3 of these cases. Flow-cytometric determination of IgG subclasses may be a useful tool in immunohematology, since subclass determinations were possible in all of these cases. This method is suited for clinical routine and offers the possibility of sufficient standardization.

Die Sicherheit von Blut- und Blutkomponentenkonserven ist ein wichtiges Anliegen der Transfusionsmedizin. Auch wenn es bei der Anwendung transfusionsmedizinischer Praparationen keine absolute Sicherheit fur den Therapieempfanger geben kann, so wird durch die Einbeziehung wesentlicher Sicherheitsverbesserungen grose Annaherung an das nicht erreichbare Idealbild moglich gemacht. Zur Sicherheitsverbesserung zahlen 1.) eine genau gefuhrte Spendererfassung, 2.) die Herstellung der Blutkomponenten nach den Prinzipien des Arzneimittelgesetzes bzw. der GMP (Gute Herstellungsordnung)-Richtlinien, 3.) die diagnostische Aufarbeitung transfusionsmedizinisch relevanter Parameter und dabei die Beachtung von GMP-/GLP-Richtlinien, 4.) die adaquate Produktlagerung und -versorgung und – als leider zu wenig beachteter Sicherheitsgarant – 5.) die Beachtung strenger Indikationsrichtlinien fur diesen Therapiezweig. In der vorliegenden Arbeit werden die Teilergebnisse der 1. Grazer Konsensustagung „Transfusionsmedizin”, welche die Indikationsrichtlinien betreffen, mitgeteilt.

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10(7) and 10(8)/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll-Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll-Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/l resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the alpha-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the beta 2-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.

In a collaborative study, we calculated mutation rates at 23 different STR loci after investigation of nearly 24,000 meioses including all Profiler™, Profiler Plus™, COfiler™, SGM Plus™ and Powerplex™ 16 loci except Penta D and Penta E and further loci, of which D12S391, D17S976 and SE33 are the most informative: CD4, CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1132, D8S1179, D12S391, D13S317, D16S539, D17S976, D18S51, D19S433, D21S11, F13A1, F13B, FES, FGA, SE33, TH01, TPOX and VWA. The alleles presumed to be taking part in mutations were sequenced. The possible origin and the possible structural variations of mutational events in microsatellites are discussed.

The STR locus C 2_4_4 situated in the HLA class I region (6p21.3) was investigated in an Austrian Causasoid population sample of 247 unrelated individuals. Beside length polymorphism, the alleles at this locus also revealed sequence polymorphism.

In a follow up study, DNA typing of short tandem repeat polymorphisms, HLA and ABO antigen typing was carried out on a pair of chimeric twins, which had already been investigated by serological and cytogenetical methods two decades ago. The aim of the study was to find out whether or not DNA typing of short tandem repeat polymorphisms is an appropriate method to characterize twin chimeras.

The performance of the new STR typing kit Powerplex™ 16 was investigated on degraded DNA samples. After optimization of the reaction conditions, comparative typings on six archived whole blood samples and the corresponding plasma retention samples as well as on whole blood DNA, which had been stored at 4 °C and room temperature for 20 days, were performed with Powerplex™ 16 and AmpFlSTR® SGM Plus™. Concerning the loci common in both kits, no differences of performance could be calculated. As expected, allele dropout of loci was observed with increasing fragment length, which unfortunately included some of the most informative marker systems. Inclusion of further loci, which show amplification products smaller than 250 bp, into STR multiplex kits or combination of two different kits might help to increase the information obtainable on degraded DNA samples.

DNA profiles were generated from blood and a tissue sample (buccal swab) of a healthy female showing irregular agglutinations in serological blood grouping and from whole blood samples of her parents in order to prove the presumption of chimerism and differentiate between its different forms. Genetic patterns of different cell types, including double maternal and paternal contribution of alleles at some loci, could be detected in whole blood and the buccal swab sample. Thus, the chimerism detected in the proposita was not restricted to the blood.

The aim of the study was to compare results obtained by electrophoresis under denaturing conditions with results of native horizontal gel electrophoresis and to investigate additional population data on the STR locus SE33 (HumACTBP2) in an Austrian Caucasoid population sample. The results of the comparative study showed that a conversion of results between both typing strategies is not feasible for the entire size range of this locus.

The genetics of the HLA system are described as well as the phenotypic and genomic HLA polymorphisms. The influence of the HLA system in matching for organ transplantation and the tolerance induced by blood transfusion are discussed.

One to two per cent of patients in need of red cell transfusion carry irregular antibodies to red blood cell (RBC) antigens and have to be supplied with specially selected blood units. To be able to respond to those requests, blood centres have to screen a significant number of donors for a variety of antigens serologically, which is a costly and through the shortage of reagents, also limited procedure. To make this procedure more efficient, the Austrian Red Cross has developed a genotyping assay as an alternative approach for high throughput RBC typing.A multiplex polymerase chain reaction (PCR) assay was designed for typing 35 RBC antigens in six reaction mixes. The assay includes both common as well as high-frequency-alleles: MNS1, MNS2, MNS3 and MNS4; LU1, LU2, LU8 and LU14; KEL1, KEL2, KEL3, KEL4, KEL6, KEL7, KEL11, KEL17 and KEL21; FY1, FY2, FYB(WK) and FY0 (FYB(ES)); JK1 and JK2; DI1, DI2, DI3 and DI4; YT1 and YT2; DO1 and DO2; CO1 and CO2; IN1 and IN2. The assay was validated using 370 selected serologically typed samples. Subsequently 6000 individuals were screened to identify high frequency antigen (HFA)-negative donors and to facilitate the search for compatible blood for alloimmunized patients.All controls showed complete concordance for the tested markers. The screening of 6000 donors revealed 57 new HFA-negative donors and the blood group database was extended by approximately 210,000 results.The study shows that in practice, this high-throughput genotyping assay is feasible, fast and provides reliable results. Compared to serological testing, this molecular approach is also very cost-efficient.

The influence of treatment of platelets with citrate buffer (pH 7.2), chloroquine, or citric acid at pH 3 on the expression of HLA class I antigens and 'thrombocyte-specific' glycoproteins was investigated by means of flow cytometry. After treatment with citric acid at pH 3 and chloroquine, the expression of HLA class I was significantly reduced, while the density of the molecules GPIa/IIa, GPIIb, and GPIIb/IIIa (GP = glycoprotein) carrying 'thrombocyte-specific' antigens was not or only weakly decreased on the surface of the platelets. The use of two monoclonal antibodies (HC-10 and HC-A2) against the native heavy chain of the HLA class I molecule revealed that 'antigen stripping' with chloroquine or citric acid does not affect the entire molecule: only the beta 2-microglobulin is cleaved, or only some epitopes on the heavy chain are altered by this procedure. The treatment with citric acid yielded better results with respect to the removal of HLA class I activity and the preservation of 'thrombocyte-specific' glycoproteins. The presence of the heavy chain of HLA class I molecules on the surface of platelets after treatment with citric acid and chloroquine confirms the hypothesis that platelets--like nucleated cells--bear HLA class I antigens inserted in the cell by a cytoplasmic and a transmembrane domain.

Plasmodium falciparum malaria has long been a killer of the young, and has selected for polymorphisms affecting not only erythrocytes, but the immunogenetics of three histocompatibility systems: ABO, human leukocyte antigen (HLA), and CD36. The ABO system is important because the original allele, encoding glycosylation with the A sugar, acts as an adhesion ligand with infected red blood cells (iRBC), thereby promoting vasoocclusion. The prevalence of blood group O, which reduces this cytoadhesion, has increased in endemic areas. Other adaptations which could mitigate A-mediated rosetting include weaker A expression and increased soluble A secretion. The role of the HLA system in malaria has been harder to verify. Although HLA-B53 and DRB1*04 may be associated with clinical outcome, HLA studies are challenged by numerous comparisons in this most polymorphic of systems, and confounded by increasingly heterogeneous populations. Certain HLA markers may also reflect linkage artefact with other malaria-relevant polymorphisms. HLA may be less important because the parasite predominantly invades a compartment which does not express HLA. Adhesion of iRBCs is also mediated by CD36, expressed on platelets, monocytes, and microvascular endothelium. CD36 on monocytes is involved in clearing iRBC, while CD36 on platelets and the endothelium may play a role in tissue sequestration. The genetics of CD36 expression are complex, and recent research is fraught with inconsistent results. The solution may lie in examining genotype-phenotype correlations, zygosity effects on differential tissue expression, or other mechanisms altering CD36 tissue expression. Carefully designed prospective studies should bridge the gap between in-vitro observations and clinical outcomes.

Das humane Leukozytenantigen- (HLA-)System umfasst Zellproteine, welche die Gewebevertraglichkeit masgeblich bestimmen und in ihrer Gesamtheit auch als Hauptgewebsvertraglichkeitskomplex (»major histocompatibility complex«, MHC) des Menschen bezeichnet werden. Das HLA-System weist einen auserordentlichen Polymorphismus auf, derzeit sind mehr als 4000 Allele fur die Gene der 5 klassischen Vertreter – HLA-A, -B, -C, -DR und -DQ – beschrieben. Die dreidimensionale Struktur der HLA-Determinanten wurde weitgehend charakterisiert; dies ermoglichte ein eingehendes Verstandnis ihrer Funktion im Rahmen der Immunantwort. Die Bestimmung der Typen des HLA-Systems besitzt grose klinische Bedeutung fur die Spenderauswahl bei Organ- und Stammzelltransplantationen, bei Thrombozytentransfusionen sowie fur die Diagnostik einiger Krankheiten.

The etiology of autoimmune diseases is multifactorial with genetic factors being an important prerequisite. There are two clinical manifestations of autoimmune thyroiditis: the goitrous form (Hashimoto's thyroiditis) and the atrophic variant, which is characterized by hypothyroidism (primary myxoedema). Different genetic markers were assumed to be predisposing factors for the distinct clinical presentation. In the present study, we determined HLA A,B,C,DR,DQ alloantigens serologically and HLA-DQ by gene analysis in patients with nongoitrous autoimmune thyroiditis and randomly chosen controls. To verify the exact classifications, thyroid volume (median 5.85 ml) was measured by ultrasonography. HLA-DR5 was found in 16 of 36 (44%) patients with nongoitrous autoimmune thyroiditis and in only 26 of 175 controls (15%) (Pc = 0.0018). There was a tendency towards a lower frequency of HLA-DR7 with 6% positivity in patients vs. 29% in controls (Pc = 0.052). Regarding HLA-DQ, DQ7 was found in 17 of 35 patients (48%) vs. 21 of 98 controls (21%) (Pc = 0.028) (relative risk 3.5). No other association was found with HLA-A,B,C and HLA-DR and -DQ. Our data indicate that the genetic susceptibility to autoimmune nongoitrous thyroiditis is closely associated to HLA-DR5 and DQ7 and not distinct from goitrous disease. We conclude that factors other than genetic ones explain the different immunological and clinical manifestation of chronic lymphocytic thyroiditis.

Twenty-three bilateral testicular germ cell tumors (four synchronous and 19 sequential tumors) were investigated for potential risk factors. The incidence of maldescensus testis was not found to be higher than in patients with unilateral disease. The histologic findings of the first tumor did not have any effect on the incidence of the second tumor. In 21 patients (four synchronous and 17 sequential tumors), histocompatibility antigens (HLA) were determined; HLA-B14 was increased significantly in the sequential tumor group. Tendencies toward an increase of HLA-DR5 and HLA-DR7 also were found. The HLA-DR1, HLA-DR3, and HLA-DR4 showed a tendency toward a decreased frequency. Therefore genetic factors might be important in the development of sequential bilateral testicular cancers.