High-throughput multiplex PCR genotyping for 35 red blood cell antigens in blood donors.

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Influence of clinical factors on the haemolysis marker haptoglobin.

BACKGROUND: Plasma haptoglobin determination is clinically used as parameter for haemolysis. To date, however, the influence of the mode of haemolysis (extravascular vs. intravascular) and of nonhaemolytic conditions on haptoglobin concentration and its reliability as a haemolysis marker remain poorly defined. MATERIALS AND METHODS: In a total of 479 individuals, the influence of haemolytic and nonhaemolytic conditions on plasma haptoglobin levels was investigated. RESULTS: All studied types of haemolytic disease (n = 16) were associated with markedly decreased plasma haptoglobin levels, without significant differences between intravascular vs. predominantly extravascular haemolysis. Diminished haptoglobin values were also observed in patients with liver cirrhosis, which normalized after liver transplantation. In contrast, markedly increased haptoglobin levels were found in patients with inflammation. In patients with haemolysis and a concomitant acute-phase response, however, haemolysis-dependent haptoglobin depletion was not attenuated. Interestingly, patients with a strongly positive direct antiglobulin test or high cold agglutinin titre but no further evidence for haemolysis had normal haptoglobin values. Likewise, anaemia owing to bone marrow failure, acute gastrointestinal or chronic diffuse blood loss, and end-stage kidney disease were associated with normal haptoglobin levels. CONCLUSIONS: Plasma haptoglobin depletion is a reliable marker for the instant diagnosis of accelerated red cell destruction irrespective of the site of haemolysis or the presence of inflammation. The capacity of this parameter to predict haemolysis appears to be limited in patients with liver cirrhosis and decreased haptoglobin production only.

ACTBP2 (alias ACTBP8) is localized on chromosome 6 (band 6q14).

The locus ACTBP2 (SE33) is localized on chromosome 6 (band 6q14). This has been demonstrated by typing a large Caucasoid three-generation kindred of Austrian origin for SE33 and several chromosome 6 markers.

Removal of T and B lymphocytes by in-line filtration: evaluation of the efficiency of a polyester filter type (Pall WBF-2) by flow cytometric counting.

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 x 10(3) (1.68 x 10(2)-4.09 x 10(4)) and in PL: 1.35 x 10(3) (2.21-1.78 x 10(4)); B cells in RCC: 2.33 x 10(3) (7.10 x 10(1)-9.15 x 10(3)) and in PL: 2.33 x 102 (7.5 x 10(2)-2.8 x 10(3)). 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.

Kinetics of CFU-Mk after automated plateletpheresis.

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 x 10(9)/l) decreased after plateletpheresis to a nadir of 194 +/- 8 x 10(9)/l (P < 0.001), showed a gradual increase on days 1 and 4, and reached pre-apheresis values by day 7 (280 +/- 11). The serum TPO levels were found to be significantly increased on days 1 and 7 as compared to baseline levels (baseline value 103.3 +/- 18.5 pg/ml versus day-1 value 135.8 +/- 25.8 pg/ml and day-7 value 132 +/- 30.19 pg/ml; P < 0.03 and P < 0.03, respectively). The numbers of CFU-Mk in PB were significantly elevated on day 4 only (125 +/- 21 colonies/ml of PB versus pre-apheresis values of 68 +/- 18 colonies/ml of PB, P < 0.005). CONCLUSIONS: These findings suggest that platelet loss during plateletpheresis affects thrombopoiesis at the progenitor cell level, probably through alterations in TPO plasma concentrations.

Further sequence and length variation at the STR loci HumFES/FPS, HumVWA, HumFGA and D12S391.

This paper reports population data and statistics for the HumFES/FPS, HumVWA, HumFGA and D12S391 loci in Austria. The sequences of some rare and new variant alleles which have been identified in the course of the present population study and other investigations are described. Sequence variation occurred in a HumFES/FPS allele revealing an (ATTT)9 structure and an A to C transversion in the 5' flanking region. At the HumVWA locus the structural type of the common allele 14 has been found in one allele 13 and in three examples of allele 15. Additionally the TCTA (TCTG)3(TCTA)n structure has been observed in three examples of allele 13 and one allele 14, which is very uncommon. Another allele 14 showed a C to T transition in the third of nine TCTA repeats. The sequences of three length variations at the HumFGA locus, namely the alleles 16, 19.2 and 21.2 are reported. At the D12S391 locus a novel 19.1 allele was found in this study. An extended nomenclature is proposed for the HumVWA locus to denominate sequence variants in a precise but simple way.

RHD genotyping in weak D phenotypes by multiple polymerase chain reactions.

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.

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.

Population study of the STRs HUMCD4 and HUMF13A1 in Catalonia (Northeast Spain).

Allele and genotype frequencies were determined in a population sample from Catalonia (northeast Spain) for two short tandem repeat loci (HUMCD4 and HUMF13A1), using the polymerase chain reaction (PCR). After denaturing PAG electrophoresis, 6 alleles were identified for HUMCD4 in a sample of 157 unrelated individuals, and 11 alleles for HUMF13A1 in a sample of 141 individuals. No deviation from Hardy-Weinberg equilibrium was found. The HUMCD4 and HUMF13A1 loci demonstrated a heterozygosity of 0.6815 and 0.7305 respectively.

A new allele at the short tandem repeat locus HumF13A01.

We investigated the (AAAG)n short tandem repeat (STR) polymorphism HumF13A01 an Austrian Caucasoid population sample (n = 674). PCR amplified fragments were detected on an automatic A.L.F. DNA sequencer using laser-induced fluorescence. A total of 14 alleles could be identified including a new 179 bp allele which was designated allele 3. Sequence determination of allele 3 confirmed the typing results by revealing three continuous copies of the core repeat, whereas in sequencing of 54 additional alleles no further variants or microheterogeneities could be observed. The population data showed no significant deviation from Hardy-Weinberg equilibrium.

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.

Additional variability at the D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution.

The highly polymorphic STR locus D12S391 was investigated in an Austrian population sample (N = 150) by PCR-amplification, comparative detection on native and denaturing polyacrylamide gels and solid phase single stranded sequencing of three size variant alleles and several additional alleles. A total of 15 alleles, distinguishable by size under denaturing conditions, could be detected. No deviations from Hardy-Weinberg equilibrium were observed in the population investigated (P = 0.52). Sequencing of size variants designated 17.3 and 18.3 showed an incomplete (GAT) repeat unit at position two of the tandem region. Additional new sequence variants due to varying compositions of the number of (AGAT) and (AGAC) repeats could be identified. Due to distinct electrophoretical mobilities of alleles of the same size but different sequence structures, denaturing detection conditions should be employed when the aim is standardization.

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.

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).

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).