Preparation of fetal nucleated red blood cells (NRBCs) for diagnostic testing

A method of enriching for fetal nucleated red blood cells (fNRBCs) from a whole maternal blood sample from a woman, comprising:

• (a) subjecting a volume of 21 milliliters (mL) or less of the whole maternal blood sample comprising billions of cells per mL to pre-enrichment by performing a density gradient separation procedure to obtain a pre-enrichment sample comprising a volume with millions of cells comprising up to 100 fNRBCs;
• (b) subjecting the pre-enrichment sample obtained in step (a) to a further enrichment step comprising:
• (i) contacting the pre-enrichment sample with at least one fNRBC positive selection reagent immobilized to magnetic particles or beads, wherein the fNRBC positive selection reagent is monoclonal antibody (mAb) 4B9 or an antibody that competes with mAb 4B9 for binding to the same binding epitope or determinant on the surface of the fNRBCs bound by mAb 4B9 to obtain a magnetic activated cell sorting (MACS) sorted cell population, wherein the MACS-sorted cell population comprises thousands of cells comprising up to 100 fNRBCs, thereby further enriching the fNRBCs;
• or
• (ii) contacting the pre-enrichment sample with at least one fNRBC positive selection reagent conjugated to a fluorescent label, wherein the fNRBC positive selection reagent is mAb 4B9 or an antibody that competes with mAb 4B9 for binding to the same binding epitope or determinant on the surface of the fNRBCs bound by mAb 4B9 to obtain a fluorescence activated cell sorting (FACS)-sorted cell population, wherein the FACS-sorted cell population comprises thousands of cells comprising up to 100 fNRBCs, thereby further enriching the fNRBCs;
• and
• (c) performing micromanipulation on the MACS- or FACS-sorted cell population obtained in step (b) with at least one fNRBC positive selection reagent immobilized to a magnetic particle or bead, or conjugated to a fluorescent label, wherein said fNRBC positive selection reagent is mAb 4B9 or an antibody that competes with mAb 4B9 for binding to the same binding epitope or determinant on the surface of the fNRBCs bound by mAb 4B9, to prepare an enriched fNRBC cell population comprising at least 10% fNRBCs, wherein at least 1 fNRBC is present in the population,
• wherein the hybridoma producing mAb 4B9 is deposited under Deutsche Sammlung van Mikroorganismen and Zelkulturen GmbH (DSM) accession number ACC 2666.