High gradient magnetic cell sorting and internal standardisation substantially improve the assay for somatic mutations at the glycophorin A (GPA) locus.
In the MACS-BR6 version of the GPA assay [Int. J. Radiat. Res. 70 (1996) 131] variant red blood cells (RBC) are isolated from 5 x 10(8) normal RBC by magnetic cell separation (MACS) before detection and quantification by immunolabelling and flow cytometry as in the classical BR6 assay. In the present work it is described how the MACS-BR6 assay is improved by internal standardisation with FITC-labelled RBC. This modification of the assay has the advantage that (i) the analysis of variants is not disturbed by the overwhelming number of normal RBC that (ii) the precision of the assay is improved and finally that (iii) a sufficient number of variants is available for further investigations. Tn positive RBC behave in MACS like variants. It is demonstrated that in normal individuals Tn cells (frequency: approximately 2 x 10(-8)) do not disturb the assay.
Hempel
Klaus
K
Klinik und Poliklinik für Nuklearmedizin der Universität, Würzburg, Josef-Schneider-Str 2, D-97080 Würzburg, Germany. hempel@nuklearmedizin.uni-wuerzburg.de
Deubel
Werner
W
Lorenz
Reinhard
R
Reiners
Christoph
C
D000328:Adult; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism; D002469:Cell Separation / Q000379:methods* / Q000592:standards*; D004912:Erythrocytes / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D014644:Genetic Variation; D006021:Glycophorins / Q000235:genetics*; D006801:Humans; D019656:Loss of Heterozygosity; D008951:MNSs Blood-Group System / Q000235:genetics; D008280:Magnetics; D009154:Mutation; D018401:Sample Size
D015295:Antigens, Tumor-Associated, Carbohydrate; D005456:Fluorescent Dyes; D006021:Glycophorins; D008951:MNSs Blood-Group System; C040286:Tn antigen; D016650:Fluorescein-5-isothiocyanate