Intact fetal cell isolation from maternal blood: improved isolation using a simple whole blood progenitor cell enrichment approach (RosetteSep^™)

Abstract

Isolation and analysis of intact fetal cells in maternal blood is an attractive method of non-invasive prenatal diagnosis; however, detection levels are not optimal. The poor sensitivity and inconsistent recovery of fetal cells is compounded by small numbers of circulating fetal cells and loss of fetal cells during enrichment procedures. Optimizing selection criteria by utilizing less complicated methods for target cell enrichment is essential. We report here salutary results using a simple density-based depletion method that requires neither MACS (magnetic-activated cell sorting) nor flow cytometric separation for enrichment of progenitor cells. Maternal blood samples (n = 81) were obtained from women prior to invasive prenatal genetic diagnostic procedures and processed randomly within 24 h using one of two density-based enrichment methods. For progenitor cell enrichment, samples (n = 49) were labeled with a RosetteSep progenitor antibody cocktail to remove unwanted mature T-cells, B-cells, granulocytes, natural killer cells, neutrophils and myelomonocytic cells. For CD45-negative cell enrichment, samples (n = 14) were labeled with RosetteSep CD45 antibody to remove unwanted maternal white cells. The desired cellular fraction was collected and analyzed by either fluorescent in situ hybridization (FISH) or real-time PCR for the presence of intact fetal cells and to quantify Y-chromosome-specific DYS1 sequences, respectively. Overall, FISH and real-time PCR correct detection rates for the progenitor cell enrichment approach were 53% and 89% with 3% (1 out of 30 cases) and 0% false-positive detection, respectively. Fetal sequences were detected in the range from 0.067 to 1.167 genome equivalents per milliliter of blood. No fetal cells were detected using the CD45-negative enrichment method. Flow cytometric analysis of cord blood showed that a unique myeloid population of cells was recovered using RosetteSep trade mark progenitor enrichment compared with the CD45-negative enrichment method. Sensitivity of the RosetteSep progenitor enrichment approach for detection of fetal cells in this pilot study shows great promise with recovery of cells that are suitable for FISH and automated microscope scanning. This simple and rapid method may also allow expansion in culture and characterization of the fetal cell type(s) that circulate in maternal blood, hence, greatly improving reliability of non-invasive prenatal diagnosis.

Microselection - affinity selecting antibodies against a single Rare cell in a heterogeneous population

“…Likewise, foetal cells crossing the placenta barrier into the maternal peripheral blood are extremely rare -estimated to be around one per five million PBMC [2].…”

Noninvasive prenatal diagnosis of aneuploidy using cell‐free nucleic acids in maternal blood: promises and unanswered questions

“…Numerous studies have shown that both fetal cells and cff DNA circulate freely in the maternal circulation (Lo et al, , 1998Bischoff et al, 2003;Bianchi, 2004). There has been much speculation as to the source of cff DNA; one possible source is fetal nucleated red blood cells, which undergo apoptosis in the maternal circulation.…”

New trends in non-invasive prenatal diagnosis: Applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells (Review)

“…All the available information indicates that MACS technology allows the collection of cell populations significantly enriched in fetal erythroblasts or trophoblasts (19,30,(73)(74)(75). A combination of the MACS procedure with other strategies for isolation of rare cells is highly needed to obtain almost a pure cell population.…”

“…Detection of levels of fetal cells with these two methods is however difficult in relation to the small numbers of circulating fetal cells and the loss of fetal cells during the enrichment procedures. Bischoff et al (30) reported a simple and rapid-density-based progenitor cell enrichment approach. The samples were labeled with a RosetteSep™ progenitor antibody cocktail to remove unwanted maternal white cells (mature T-cells, B-cells, granulocytes, natural killer, neutrophils and myelomonocytic cells).…”

“…This method targets progenitor cells that are not necessarily of the erythroid lineage and may also allow expansion in culture and characterization of the fetal cell types that circulate in maternal blood. Some lymphocytes are long lived, and with this approach there is concern that enriched progenitors may be the vestiges of previous pregnancies and do not represent the true fetal genetic status of the current pregnancy (30). Charge flow separation (CFS) is an antibody-independent selection method of fetal cells that relies on the behaviour of cells in an electric field and a buffer counterflow gradient (31).…”