Simple and Fast “Double‐MACS” Sorting of Fetal Erythroblasts from Maternal Blood for PCR‐Based Paternity Analysis

Abstract

Our procedure for prenatal paternity testing combines depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell sorting (MACS)' with PCRbased analysis of DNA.Venous blood samples (20 ml) were collected in a 5% EDTA solution from healthy women with uncomplicated pregnancies. The PBMCs were stained after ficolation (Pharmacia, Upsalla) with CD45 PE (murine IgG1, KC56, Coulter) and anti-IgG1 MACS beads and CD14 MACS beads (both from Miltenyi Biotec). After depletion of CD45' and CD14+ cells (leukocytes and monocytes) on a MACS (A2) column, the negative cells were stained with CD71 FTTC (murine IgG2a, Becton Dickinson) and anti-IgG2 MACS beads. CD71' cells (erythroblasts) were enriched by a second MACS separation on a miniMACS. The enrichment of the CD71' cells could be improved up to 80% by using haptenated CD45 and haptenated CD7 1 (both Miltenyi Biotec) and corresponding MACS beads.The highly polymorphic loci of the gene system HLA-DQA l2 and variable number of tandem repeats (VNTR) of the polymorphic gene system D1S803 were analyzed by PCR with and without preamplification with unspecific primers (PEP): After DNA extraction from the double-MACS sorted cells with phenol-chloroform, the concentration of DNA was quantified photometrically. HLA-DQA1 typing was carried out according to the manufacturer's instructions for the "Ampli Type HLA-DQa" system (Cetus, Emmeryville, CA). Amplification of the DlS80 locus was carried out by using the primers described by Kasai et al.5 The PCR cycles had the following phases: denaturation at 95 "C for 1 min; primer annealing at 65 "C for 1 min; primer extension at 70 "C for 2 min. A total of 30 cycles was carried out in a Perkin-Elmer thermocycler. Electrophoretical separation of amplified D 1 S80 alleles was performed 144

Non-invasive prenatal diagnosis of aneuploidies: new technologies and clinical applications

“…The first monoclonal antibody used for enrichment of fetal cells was directed against CD71, the transferrin receptor [30]; however, purity after FACS was low. An improvement was later made by using MACS depletion of anti-CD45 prior to selection of CD71-positive cells [43]. However, the methods used so far have had low recovery rates.…”

Recent advances in non‐invasive prenatal DNA diagnosis through analysis of maternal blood

“…Purity can be further enhanced by MACS depletion of maternal cells and fetal lymphocytes with anti‐CD45 prior to positive selection for CD71‐positive cells. Using this method, approximately 20 fetal cells are obtained from a 20 mL maternal blood sample 153 . However, even following double MACS separation, the purity of NRBC remains low, and a prolonged interval is required for NRBC detection.…”

“…Using this method, approximately 20 fetal cells are obtained from a 20 mL maternal blood sample. 153 However, even following double MACS separation, the purity of NRBC remains low, and a prolonged interval is required for NRBC detection. The advantages of using MACS include the short time required for the procedure itself and its relatively low cost.…”

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis

“…Despite the lack of functional characterization, these cells are recognized as ideal candidates for noninvasive prenatal diagnosis (NIPD) . Presence of both primitive and definitive fetal EryPs has been demonstrated in maternal blood, and the hEryP has been shown to be the most abundant cell type during the first trimester …”

“…Isolation of hEryPs from maternal blood remains a challenge due to their extreme rarity and lack of specific surface markers . Only a single fetal cell per mL of blood may be retrieved following enrichment . Attempts to expand the numbers of fetal cells by culture have been mostly unsuccessful due to contamination with maternal cells .…”