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The isolation and characterization of fetal haematopoietic cells from maternal peripheral blood

University of British Columbia

Fetal cells that circulate in maternal peripheral blood during pregnancy offer a potential source of nucleated fetal material for low-risk non-invasive prenatal diagnosis. Despite some promising results and numerous investigations in this field, many of the basic questions regarding the circulation of fetal cells in the maternal blood remain unanswered. Specifically, more information on the frequency and type of fetal cells that circulate during pregnancy needs to be established, the possibility of clonally expanding fetal cells needs to be explored, and techniques that will facilitate the identification and isolation of fetal cells need to be developed before this technique can be implemented for routine prenatal screening. Progenitor cells from normal human adult bone marrow and fetal liver were sorted using multidimensional flow cytometry on the basis of expression of CD34, CD71, CD45 and Glycophorin-A and cultured in the presence of various haematopoietic cytokines. Subtle differences in phenotype and function between fetal and adult cells and a reproducible enrichment strategy were identified that could potentially be applied to identify fetal cells from maternal peripheral blood. Fluorescence activated cell sorting (FACS) was then used to target two populations of fetal cells: nucleated erythroid cells (NECs; CD71/Glycophorin-A⁺ CD45[sup lo-int] CD34⁻) and haematopoietic progenitor cells (CD34⁺ cells; CD34⁺⁺ CD71/Glycophorin-A- CD45[sup int]). Fetal cells were detected by fluorescence in situ hybridization using directly conjugated chromosome X and Y probes in 65% of the maternal peripheral bloods (fetal karyotype 46,XY). The frequency of fetal cells isolated from the NEC and CD34⁺ fractions was, respectively, 0-14 and 0-7 cells per 2x10⁷ previously frozen maternal cells (-20 mL of blood). In non-frozen samples, the yield and recovery of fetal cells was moderately improved. Culturing the CD34⁺ sorted fractions in serum-free media with cytokines improved the quality of the FISH preparations and resulted in a slight expansion in detectable fetal cells. The frequency of fetal cells isolated from cultured CD34⁺ fractions was 0-35 and 0-93 cells per 2x10⁷ previously frozen and non-frozen maternal peripheral blood cells, respectively. These results document the isolation, characterization, and enumeration of fetal cells from the maternal periphery that appear to be present in most, but not all samples analyzed.

Thesis/Dissertation

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2009-03-17

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http://hdl.handle.net/2429/6157

Experimental Medicine

University of British Columbia

UBCV

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