Typing for the human lewis blood group system by quantitative fluorescence-activated flow cytometry: large differences in antigen presentation on erythrocytes between A(1), A(2), B, O phenotypes.
BACKGROUND
Lewis phenotyping by hemagglutination is an unreliable routine method for Lewis antigen designation. Now genomic typing of the Lewis gene is available. Additionally, flow cytometry has been used for typing. We wanted to compare the results of Lewis typing in healthy individuals using the three methods.


METHODS
Ninety-three randomly selected plasma donors were genotyped for inactivating Secretor (FUT2) G428A and Lewis (FUT3) T59G, T202C, C314T, G508A and T1067A point mutations. All Le(a+b-) individuals (nonsecretors) were homozygous for the FUT2 G428A mutation and all Le(a-b-) individuals had inactivating mutations on both FUT3 alleles. Fixed erythrocytes were analyzed by fluorescence-activated flow cytometry and the results were compared with hem- agglutination and genotypic data. Antigen availability was expressed as median fluorescence intensity and as percentage positive cells with fluorescence intensities > or =10(2).


RESULTS
Using an anti-Le(a) reagent a mean of 99% of erythrocytes from Le(a+b-) individuals and 1% of erythrocytes from Le(a-b-) or Le(a-b+) individuals were stained positive. Using an anti-Le(b) reagent, a mean of 71% of erythrocytes from A(1), 95% from B and 99% from O and A(2) Le(a-b+) individuals and less than 10% of erythrocytes from Le(a-b-) or Le(a+b-) individuals were stained positive. After papain treatment 100% of the erythrocytes from A(1) and A(1)B Le(a-b+) individuals stained positive without increase in background staining. The flow cytometric technique revealed large differences in staining intensities, within each ABO Le(a-b+) subgroup which was not directly correlated to plasma donation frequencies nor to Secretor or Lewis genotypes.


CONCLUSIONS
Flow cytometry may prove valuable as a Lewis blood group typing technique but also as a research tool when investigating Lewis phenotypes of human erythrocytes.
Larson
G
G
Department of Clinical Chemistry and Transfusion Medicine, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden. GL@ahlgrenska.se
Svensson
L
L
Hynsjö
L
L
Elmgren
A
A
Rydberg
L
L
D000017:ABO Blood-Group System / Q000097:blood; D000483:Alleles; D000918:Antibody Specificity; D017951:Antigen Presentation; D001788:Blood Grouping and Crossmatching; D004910:Erythrocyte Membrane / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005647:Fucosyltransferases / Q000235:genetics; D005787:Gene Frequency; D005838:Genotype; D006384:Hemagglutination; D006801:Humans; D007519:Isoantigens / Q000276:immunology; D007983:Lewis Blood Group Antigens / Q000097:blood*; D010206:Papain / Q000378:metabolism; D017354:Point Mutation; D013548:Sweden / Q000208:ethnology
D000017:ABO Blood-Group System; D007519:Isoantigens; D007983:Lewis Blood Group Antigens; D005647:Fucosyltransferases; D010206:Papain