Enrichment, identification and analysis of fetal cells from maternal blood: evaluation of a prenatal diagnosis system

• Published in: Prenatal diagnosis 1999-07, Vol.19 (7), p.648-652
• Main Authors: de Graaf, Irene M., Jakobs, Marja E., Leschot, Nico J., Ravkin, Ilya, Goldbard, Simon, Hoovers, Jan M. N.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal; Biological and medical sciences; Cell Nucleus; Cell Separation - methods; Centrifugation; Centrifugation, Density Gradient; Erythrocytes - ultrastructure; Female; Fetal Blood - cytology; Fetal Hemoglobin - analysis; Gynecology. Andrology. Obstetrics; Hemolysis; Humans; In Situ Hybridization, Fluorescence; Leukocyte Common Antigens - analysis; Male; Management. Prenatal diagnosis; Medical sciences; Pregnancy; Pregnancy. Fetus. Placenta; Prenatal Diagnosis - methods; Sensitivity and Specificity; Sex Chromosomes
• ISSN: 0197-3851; 1097-0223
• DOI: 10.1002/(SICI)1097-0223(199907)19:7<648::AID-PD600>3.0.CO;2-X

In this study we evaluated the performance of a system for the enrichment, identification and analysis of fetal cells in maternal peripheral blood. Blood samples were collected from women after chorionic villus sampling and enriched for the presence of nucleated erythrocytes using a three‐step procedure, namely: (a) centrifugation to separate nucleated red blood cells (NRBCs) from the majority of red blood cells (RBCs) and white blood cells (WBCs); (b) selective lysis of the remaining maternal RBCs; (c) separating the NRBCs from the remaining WBCs in a three‐layer density gradient. Fetal cells were identified by using a monoclonal antibody against the γ‐chain of fetal haemoglobin (anti‐HbF) and a nuclear stain (DAPI). Additionally, to further increase the specificity of the identification, and to eliminate some of the undesired staining by maternal leukocytes, a fluorescent antibody (CD45) was added. The sex chromosome complement of the cells was determined by fluorescence in situ hybridization (FISH) with X and Y‐specific probes and the results were compared with the karyotypes obtained after analysis of chorionic villi. Using the described method, in all cases where the woman was carrying a male fetus (n=18) at least one XY cell was found, while no male cells were found in women carrying a female fetus. However, in the majority of cases with a male fetus (n=11) female HbF positive cells were found indicating the presence of maternal nucleated erythrocytes. The study demonstrates that the combination of anti‐HbF and CD45 is a useful, but not fully specific, marker for fetal NRBCs and that additional markers are needed. Copyright © 1999 John Wiley & Sons, Ltd.