Isolation Of Fetal Nucleated Red Blood Cells In Maternal Blood Using Primed In Situ Labeling Technique

Objective To study the efficiency of primed in situ labeling(PRINS) using fetal cells isolated from peripheral blood of pregnant women for non-invasive prenatal diagnosis,and establish a rapid and precise method for isolating fetal nucleated red blood cells(NRBC)from maternal blood.Methods Maternal blood samples were obtained from 5 pregnant women,7-10 weeks of gestation.NRBCs in maternal blood were enriched by discontinuous density gradient centrifugation,Smeared and labeled by PRINS. Then the cells were collected by micromanipulaton individually,after that the remaining slides were stained with Wright-Giemsa,NRBCs were collected again. The cells collected above were performed primer extension preamplification (PEP)of the entire genome respectively,followed by short tandem repeat(STR) genotype analyses.Single NRBC was determined individually to be fetal or maternal in origin by STR genotype of NRBC and its corresponding parents.Results All of 5 pregnant women showed positive signals in the smears. A total of 14 NRBCs were labelled in all of 5 samples at a range of 1-4 per 5 ml maternal blood,13 in wich were determined to be fetal in origin by STR typing. 36 NRBCs were found with Wright-Giemsa additionally,15 cells belong to fetuses.The specificity and sensitivity of PRINS is 92.86%and 46.43% respectively. Conclusions PRINS is a reliable technique to isolate fetal NRBC from maternal blood with epsilon gene in the fist trimester.It is rapid,low cost and precise.With the optimization of conditions,the PRINS technology for prenatal diagnosis would be great potential.