A comparison of the choice of monoclonal antibodies for recovery of fetal cells from maternal blood using FACS for noninvasive prenatal diagnosis of hemoglobinopathies

Abstract

Background: Isolation of adequate numbers of fetal cells circulating in the maternal circulation is the major hurdle in developing noninvasive prenatal diagnostic procedures. We used flow cytometry and a combination of different monoclonal antibodies to compare the yield and purity of the fetal nucleated red blood cells at different periods of gestation.Methods: Using a Percoll discontinuous gradient, the fetal nucleated erythrocytes were enriched from 7 ml maternal blood. In 100 samples, the enriched cells were stained with CD45, anti-fetal hemoglobin, and glycophorin A antibodies and in 30 samples they were stained with CD45, anti-fetal hemoglobin, and CD71 and then sorted and used for fetal diagnosis of hemoglobinopathies.Results: Using the first set of antibodies, although we were able to obtain a higher percentage of fetal nucleated red cells (0.07% 6 0.2%) as compared to the second set which yielded comparatively smaller numbers (0.025% 6 0.03%), there was some compromise in purity.Conclusion: Using CD45, anti-fetal hemoglobin and CD71 would be preferred as minimizing maternal contamination is more important than yield for prenatal diagnosis. q

Overview and recent developments in cell‐based noninvasive prenatal testing

“…Maternal red blood cells can be removed by bulk separation or density gradient centrifugation using Ficoll [43][44][45][46] or Percoll [47][48][49][50] in different gradient combinations, to separate the nucleated cell fraction (including fetal cells) from the maternal RBC and plasma fraction. That said, using Ficoll could result in 60%-80% fetal cell loss, 51 making the use of less aggressive methods more desirable.…”

Isolation and Enrichment of Circulating Fetal Cells for NIPD: An Overview

“…Many different positive and negative enrichment strategies have been explored, including positive enrichment by MACS with monoclonal antibodies to CD71 or glycophorin A (Gly A) or detection by FACS using monoclonal antibodies to fetal hemoglobin (HbF). Some examples are the work of Martel-Petit et al, who identified a fetus with cystic fibrosis by studying fnRBCs [ 39 ], and Souza et al, who used a combination of antibodies to increase the specificity of fnRBC selection [ 40 ].…”

Fetal Cells in Maternal Blood For Prenatal Diagnosis: A Love Story Rekindled

“…The large NIFTY trial utilized antibodies targeting either HbF, HbF and CD45, or CD71, but the study resulted in poor detection rates of aneuploidies and the correct fetal gender [4]. In 2009, a comparison between different enrichment antibodies was pursued by Souza et al who found that a combination of antibodies targeting CD45, HbF and CD71 yielded the highest specificity of fetal nRBCs but also a lower number of fetal cells, still leaving the use of nRBCs in cbNIPD with limited success [12]. Christensen and colleagues found that embryonic hemoglobin is more specific toward fetal nRBCs than HbF [13], but the group also concluded that only a small fraction of circulating fetal cells are of erythroblastic origin and therefore not the optimal cell type for cbNIPD [13,14].…”