The clinical utility of fetal cell sorting to determine prenatally fetal E/e or e/e Rh genotype from peripheral maternal blood.
OBJECTIVE
This study was undertaken to determine the fetal E/e or e/e Rh genotype prenatally from peripheral maternal blood by examining sorted fetal cells from alloimmunized and nonalloimmunized pregnancies.


METHODS
Eighteen maternal peripheral venous blood samples were obtained before amniocentesis from 15 pregnant women who were homozygous for the e allele. Five were not alloimmunized and 10 were alloimmunized. The mononuclear cell layer was isolated from the maternal blood and enriched for fetal nucleated red blood cells by flow cytometry with monoclonal antibodies to CD36 or CD71 and to glycophorin A. Eight samples were treated with CD45 monoclonal antibody-coated magnetic beads before they were sorted to deplete the maternal sample of leukocytes (CD45(+) cells). We defined the positive fetal cell fractions as the monoclonal antibody positive-sorted cells derived from the maternal samples. These included sorted cells that were CD36(+)/glycophorin A(+), CD71(+)/glycophorin A(+) and CD45(-) cells that were sorted to become CD45(-)/CD36(+)/glycophorin A(+) or CD45(-)/CD71(+)/glycophorin A(+). The negative fractions were the cells that were negative for either CD36/glycophorin A or CD71/glycophorin A or were the CD45(+) cells. Deoxyribonucleic acid was isolated from all fractions and amplified by polymerase chain reaction with allele-specific primers for the E or e Rh genes. Gel electrophoresis was performed to detect fetal E/e or e/e Rh genotype. The fetal E/e or e/e Rh genotype was confirmed by serologic and deoxyribonucleic acid testing. The accuracy of E/e or e/e Rh genotype determination from the positive cell fractions was compared with that of E/e or e/e Rh genotype determination from the negative fractions.


RESULTS
Fetal E/e or e/e Rh genotype was determined correctly in 17 of 18 of the fetal cell enriched positive fractions (94%). Fetal E/e or e/e Rh genotype was determined correctly in 11 of 14 of the maternal samples in the negative unselected cell fractions (79%). Fetal E/e or e/e Rh genotype was determined correctly in 15 of 16 sample fractions that underwent magnetic bead separation with CD45 and were subsequently sorted into positive and negative fractions (94%). Fetal E/e or e/e Rh genotype was determined correctly in 13 of 13 of the samples obtained from the alloimmunized pregnancies (100%).


CONCLUSIONS
The use of monoclonal antibodies for cell sorting or for magnetic separation predicted fetal E/e or e/e Rh genotype from peripheral maternal blood correctly in as many as 100% of alloimmunized pregnancies. Thus noninvasive fetal E/e or e/e Rh genotyping can be performed by polymerase chain reaction amplification of the rare fetal cells in maternal blood. The correct prediction of fetal E/e or e/e Rh genotype from the cell population not selected by the monoclonal antibodies suggests that there are fetal cell types other than fetal nucleated erythrocytes that can also be used as a source of fetal deoxyribonucleic acid for noninvasive genetic diagnosis. Improved technology may provide methods less laborious than cell sorting to accurately determine fetal Rh type from different fetal cell types that circulate in maternal blood.
Geifman-Holtzman
O
O
Divisions of Maternal-Fetal Medicine and Genetics and the Departments of Medicine and Pathology, State University of New York Health Science Center at Syracuse, USA. Drgeifman@aol.com
Makhlouf
F
F
Kaufman
L
L
Gonchoroff
N J
NJ
Holtzman
E J
EJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005838:Genotype; D006720:Homozygote; D006801:Humans; D017493:Leukocyte Common Antigens / Q000276:immunology; D008280:Magnetics; D008863:Microspheres; D011237:Predictive Value of Tests; D011247:Pregnancy / Q000097:blood*; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
D000911:Antibodies, Monoclonal; D012204:Rh-Hr Blood-Group System; D017493:Leukocyte Common Antigens