D. W. M. Schwartz

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 μl FRCC of FP using 50 μl 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/μl. 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/μl for the CD45/CD14 staining and 0.1 WBC/μl 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 × 90° light scatter signal dot plot. PLT counting was as described for WBC. The DL value was about 2 PLT/μl. 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.

Strategy to Detect Chimerism in Allogeneic Bone Marrow Transplant Recipients by PCR‐Amplification Fragment Length Polymorphism Analysis of Microsatellite Polymorphisms

Monitoring chimerism in recipients of bone marrow transplants (BMT) is important for diagnosing engraftment and residual disease and thus for selecting the optimal therapy in these patients. Unfortunately, traditional polymorphic marker systems, such as red blood cell alloantigens, polymorphic isoenzymes or chromosome markers and methods to type for these markers (hemagglutination, various electrophoretic methods, karyotyping [ 1,2]) have a limited sensitivity and are often not applicable because there is either no informative constellation or interference from transfused red cells or plasma. The HLA polymorphism cannot be used either, since usually allogeneic BMT is carried out only in HLA-identical donor-recipient constellations. The significant progress in molecular biology techniques in the last years has not only opened the opportunity for typing polymorphic markers at the genomic level but has also enormously increased the number of marker systems available. The majority of these new markers belong to the microsatellite polymorphisms [3], which are simple repetitive sequences in intronic regions of the genome, with the number of repeats being highly variable among individuals (variable number of tandem repeats, VNTR). Dispersed throughout the whole genome of humans and other higher organisms, they provide excellent markers for genetic linkage mapping, identifying disease susceptibility genes and individual identification. The development of rapid and reliable typing techniques has led to a broad range of applications [see ref. 4, for a recent review] in human and animal

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 OLs 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 ITs 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.

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.

Simple and Rapid Typing for VNTR Polymorphisms Using High Resolution Electrophoresis of PCR Products on Rehydratable Polyacrylamide Gels

The invention of the Polymerase Chain Reaction (PCR, Saiki et al 1988) has opened a wide new field of applications in molecular biology. Many new PCR-based techniques to type for polymorphic markers are now available and thus forensic haemogenetics is certainly moving to a new age. The highly polymorphic Variable Number of Tandem Repeat (VNTR) -loci offer a system of markers with high informativeness and have already been employed for paternity and identity testing for a few years. So far, most work has been done by analysis of enzyme-digested amplification products (RFLP) which is a cumbersome method with limited sensitivity. Therefore, typing of VNTR’s and STR’s (Short Tandem Repeats, a subset of VNTR’s with very short repeat sequences: 2–4bp) by Amplification Fragment Length Polymorphism (AMPFLP) is about to become the method of choice, since DNA-sequencing, of course the most precise way to type for a genetic polymorphism, will presumably not be available to most workers in the forensic field in the near future. We have undertaken studies to optimize conditions for high resolution electrophoresis on rehydratable polyacrylamide gels for the VNTR marker systems ApoB, COL2A1, D1S80, YNZ22 and for the STR loci SE33 and TC11 in order to obtain a simple protocol that leads to reproducible and reliable results and to further employ this protocol for local population and family studies and for the forensic casework in the future.

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).

Typing for STR-loci by Electrophoresis on Rehydratable Polyacrylamide Gels: Phenotype and Allele Frequencies of SE33 and TC11 in an Austrian Population Sample

Short tandem repeat (STR) microsatellite loci (Edwards et al 1991) are becoming increasingly important in forensic haemogenetics. Like the VNTR-AMPFLP microsatellite loci they represent highly polymorphic markers for genetic linkage studies, human identification and paternity testing and can be typed by direct high resolution polyacrylamide gel electrophoresis (PAGE). Because of their short overall size (100-500bp) STR loci may still be successfully amplified by PCR from highly degraded material (stains) when other methods fail to produce a typeable result. In order to establish procedures and references for reliable typing of the STR-loci SE33 (Polymeropoulos et al 1992) and TC11 (Edwards et al 1992) we optimized conditions for high resolution PAGE with subsequent silver staining of the amplification products and employed this method for typing a local population sample.

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).

Kinetics of CFU-Mk after automated plateletpheresis

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.

AMPFLP-Typing of the D21S11 Microsatellite Polymorphism: Allele Frequencies and Sequencing Data in the Austrian Population

Human DNA microsatellite polymorphisms (Variable Number of Tandem Repeats, VNTR) with short repeat sizes (2–5bp) have become very useful markers in forensic haemogenetics in the last few years. For a number of reasons, typing for these markers is more and more preferred to conventional techniques in forensic casework: The large number, high polymorphism and thus the high information content of microsatellite markers The use of just one technique (PCR-Amplification Fragment Length Polymorphism, AMPFLP) for all markers The possibility to successfully employ these markers even for very low amounts of highly degraded stains However, it must be stated that there are still some problems and restrictions with these systems. Typing by AMPFLP on polyacrylamide gels is not always very clear-cut, especially with certain markers (e.g. APOB, SE33) because of interalleles (variants with incomplete repeats or sequence variants with distinct electrophoretic mobility) and/or the limited resolution capability of electrophoresis. The allele frequency distribution in different populations has shown significant differences for certain systems, whereas other loci showed a more uniform distribution, at least in the populations studied so far. Since the amount of data is limited, especially for the microsatellites with short core repeat length (Short Tandem Repeats, STR) population studies are still required. Furthermore, detailed sequencing studies should also be performed for all markers in forensic use since only with this technique the polymorphism can be determined to the ultimate level. For routine use the AMPFLP technique will still stay the standard, but it is required that a marker consisting of alleles with known sequence is used with this technique [DNA comission of the ISFH, 1994].