Isolation of fetal nucleated red blood cells from maternal blood

Abstract

To find a simple, effective method of isolating fetal cells from maternal peripheral blood for prenatal diagnosis, 45 women were studied with their gestation being 6–14 weeks and age 21–30 years. The fetal cells were isolated from maternal blood by using discontinuous density gradient centrifugation. Some of the isolated cells were made smear and counted under the microscope; others were used for predicting fetal sex by PCR amplification of Y chromosome specific DYZ1 gene. The major cells in the upper separation interface were lymphocytes and monocytes, with occasionally seen nucleated red blood cells (NRBC); while those in the middle separation interface were neutrocytes, with NRBC scattering. The ratio of NRBC/nucleated cells was 1. 98±0.28×10⁻⁵. There was no significant difference between the first and second trimester (P>0.05). The amount of isolated fetal cells was sufficient for prenatal genetic diagnosis. Male pregnancy was correctly predicted in 10 out of 13 cases. It is concluded that the method of discontinuous density gradient centrifugation was of considerable importance in the development of non-invasive prenatal genetic diagnosis.

Keywords

maternal peripheral blood / nucleated red blood cell / sex identification / polymerase chain reaction

References

[1] WalknowskaJ, ConteF A, GrumbachM M, et al.. Practical and theoretical implications of fetal/maternal lymphocyte transfer. Lancet, 1969, 1: 1119-1119

[2] 1995,13(2):114

[3] TakabayashiH, KuwabaraS, UkitaT, et al.. Development of non-invasive fetal DNA diagnosis from maternal blood. Prenat Diagn, 1995, 15: 74-74

[4] SekizawaA, WatanaboA, KimuraT, et al.. Prenatal diagnosis of the fetal RhD blood type using a single fetal nucleated erythrocyte from maternal blood. Obstet Gynecol, 1996, 87(4): 501-501

[5] ThomasM R, TutschekB, FrostA, et al.. The time of appearance and disappearance of fetal DNA from the maternal circulation. Prenat Diagn, 1995, 15(7): 641-641

[6] ThilaganathanB, Meker-HomjiN J, NicolaidesK H, et al.. Blood transferrin receptor expression in chromosomally abnormal fetuses. Prenat Diagn, 1995, 15(3): 282-282

[7] KorstL M, PhelanJ P, AhnM O, et al.. Nucleated red blood cells: An update on the marker for fetal asphyxia. Am J Obstet Gynecol, 1996, 175: 843-843

[8] HumeR, PazoukiS, HallasA, et al.. The ontogeny of the glucose-6-phosphatase enzyme in human embryonic and fetal red blood cells. Early Hum Dev, 1995, 42(2): 85-85

[9] LoY M, CorbettaN, ChamberlainP F, et al.. Presence of fetal DNA in maternal plasma and serum. Lancet, 1997, 350(9076): 485-485