Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood

Gänshirt‐Ahlert, Dorothee; Burschyk, Monika; Garritsen, Henk; Helmer, L.; Miny, Peter; Horst, Jürgen; Schneider, H. P. G.; Holzgreve, Wolfgang

doi:10.1016/0002-9378(92)91603-8

Cited by 123 publications

(67 citation statements)

References 9 publications

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“…3,10 After sorting, separate fetal cell identification and genetic diagnosis procedures promote loss of rare cells and compromise specificity. Thus, an accurate slide-based identification system, which allows genetic analysis of only fetal cells among what is typically a 100-to 1000-fold higher postenrichment background of contaminating maternal erythroblasts, is desirable.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

Choolani

O'Donnell

Campagnoli

et al. 2001

Blood

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Isolating fetal erythroblasts from maternal blood offers a promising noninvasive alternative for prenatal diagnosis. The current immunoenzymatic methods of identifying fetal cells from background maternal cells postenrichment by labeling ␥-globin are problematic. They are nonspecific because maternal cells may produce ␥-globin, give poor hybridization efficiencies with chromosomal fluorescence in situ hybridization (FISH), and do not permit simultaneous visualization of the fetal cell identifier and the FISH signal. We describe a novel technique that allows simultaneous visualization of fetal erythroblast morphology, chromosomal FISH, and ⑀-globin labeled with AMCA (7-amino-4-methylcoumarin-3-acetic acid).AMCA was chosen as the fluorescent label to circumvent the problem of heme autofluorescence because the mean difference in relative fluorescence intensity between fetal erythroblasts stained positive for antiglobin antibody and autofluorescence of unstained cells was greater with AMCA (mean 43.2; 95% confidence interval [CI], 34.6-51.9; SD ‫؍‬ 14.0) as the reporting label compared with fluorescein isothiocyanate (mean 24.2; 95% CI, 16.4-31.9; SD ‫؍‬ 12.4) or phycoerythrin (mean 9.8; 95% CI, 4.8-14.8; SD ‫؍‬ 8.0). Median FISH hybridization efficiency was 97%, comparable to the 98% (n ‫؍‬ 5 paired samples) using Carnoy fixative. One ⑀-positive fetal erythroblast was identified among 10 5 maternal nucleated cells in 6 paired mixture experiments of fetal erythroblasts in maternal blood (P < .001). Male ⑀-positive fetal erythroblasts were clearly distinguishable from adult female ⑀-negative erythroblasts, with no false positives (n ‫؍‬ 1000). The frequency of fetal erythroblasts expressing ⑀-globin declines linearly from 7 to 14 weeks' gestation (y ‫؍‬ ؊15.8 ؋ ؉ 230.8; R 2 ‫؍‬ 0.8; P < .001). We describe a rapid and accurate method to detect simultaneously fetal erythroblast morphology, intracytoplasmic ⑀-globin, and nuclear FISH. IntroductionIsolating fetal nucleated red blood cells (NRBCs) from maternal blood should allow first trimester noninvasive prenatal diagnosis of aneuploidy and monogenic disorders. 1 There are currently 3 steps: enrichment of fetal cells in maternal blood, identification of fetal cells among background maternal cells, and diagnosis using fluorescence in situ hybridization (FISH) or single-cell techniques. Antibody directed against the ␥ chain of fetal hemoglobin is commonly used for both the fetal cell enrichment 2,3 and identification 4 steps. There may be a case for using ␥-globin for sorting, favoring yield over purity, but it is not nearly specific enough for accurate fetal erythroblast identification because of increased maternal fetal hemoglobin production in pregnancy 5 and ␤ thalassemia. 6,7 In contrast, ⑀-or -globin chains appear specific for fetal cells in chorionic villus supernatants 7 but have not been tested in maternal blood. Whereas -globin is occasionally produced in adults with ␣ thalassemia, 8 ⑀-globin has not been found in adult peripheral blood, 9 making it the prefer...

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Section: Introductionmentioning

confidence: 99%

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

Choolani

O'Donnell

Campagnoli

et al. 2001

Blood

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“…In previous reports, the density gradient solutions of 1.077 g/ml or sometimes 1.083 g/ml were generally used to eliminate non-NRBCs and to enrich fetal cells [1,2,[8][9][10]13]. We compared 1.083 to 1.090 g/ml Percoll solution.…”

Section: Discussionmentioning

confidence: 99%

Improvement of Fetal Cell Recovery from Maternal Blood: Suitable Density Gradient for FACS Separation

Sekizawa¹,

Farina²,

Zhen³

et al. 1999

Fetal Diagn Ther

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Objective: To improve the recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for noninvasive prenatal genetic diagnosis. Methods: Blood samples were obtained from 10 women at 8–22 weeks of gestation. Samples were split and mononuclear cells were isolated using 1.083 and 1.090 g/ml of Percoll solution. Flow sorting with antibody to fetal hemoglobin and fluorescence in situ hybridization (FISH) analysis were used to evaluate the number of fetal cells recovered. Results: In samples separated with the 1.090 density gradient, the yield of true γ-hemoglobin-positive cells (median 21.0, range 2.2–303.8) was 1.9 times higher than that in the 1.083 density (median 11.1, range 1.1–87.5), although it took 2.1-fold longer time to flow sort the γ-hemoglobin-positive cells. In 7 out of 10 cases, the number of γ-hemoglobin-positive cells recovered from the 1.090 density gradient was 3 times or greater than that from 1.083 gradient. After FISH analysis, we detected a median of 13.3 (range 2.2–98.8) fetal NRBCs per 10-ml maternal blood in the 1.090 density gradient, whereas a median of 11.0 fetal NRBCs were detected in the 1.083 gradient (range 1.1–35.0). The number of fetal NRBCs in the 1.090 density was significantly higher than that in the 1.083. Conclusion: Increased Percoll density results in improved fetal cell recovery in fresh posttermination maternal samples. The increased yield of fetal cells using this gradient may permit better noninvasive detection of fetal chromosome as well as DNA abnormalities in maternal blood.

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“…No problem with persistence into the next pregnancy was observed due to their short life and limited replicating capacity [5,6]. They express several unique antigens [13], such as transferrin receptor (CD71) [8,14,15], erythropoietin receptor [16], thrombospondin receptor (CD36), glycophorin A [1], blood type and fetal liver surface antigens [17,18] and HLA antigens [19,20], that allow their enrichment from the maternal circulation. However, even after enrichment, the majority of NRBCs are of maternal origin [12,[21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Non-Invasive Fetal Sex Determination on Fetal Erythroblasts from the Maternal Circulation Using Fluorescence in situ Hybridisation

Hromadníková

Karamanov²,

Houbova³

et al. 2002

Fetal Diagn Ther

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Objective: The main purpose of this study was to evaluate the potential of a non-invasive method for fetal sex determination in clinical practice using dual-colour fluorescence in situ hybridisation (FISH) analysis. We evaluated the differences in nucleated red blood cell (NRBC) recovery from the maternal circulation using various slide preparation procedures following high-gradient magnetic cell separation (double MACS). Methods: NRBCs were enriched from peripheral blood mononuclear cells of 63 pregnant women between 12 and 37 weeks of gestation by double MACS involving simultaneous CD14+ and CD45+ maternal cell depletion and CD71+ fetal cell enrichment. Isolated cells were analysed by dual-colour FISH with X- and Y-specific probes. Before applying the FISH technique, cells were treated using three different protocols. Cells were either fixed in methanol:acetic acid (3:1) and dropped immediately onto glass slides (protocol 1) or treated with 75 mM KCl before resuspension in fixative (protocol 2). Alternatively, isolated cells were transferred onto glass slides and then treated using a method described in the literature (protocol 3). Results: Using various slide preparation procedures, fixed cell numbers as well as the quality of slides differed significantly. Using protocol 1, fetal sex was well determined in 30 cases, in 15 out of 17 male fetuses (1–13, mean 3 fetal cells were found among 5–164, mean 50 maternal cells) and in 15 female fetuses (7–178, mean 56 fixed cells). On the other hand, interpretation difficulties occurred in 7 out of 8 studied cases using protocol 2 due to a lack of cells or damage to the isolated cells. The highest recovery of fixed cells was achieved using protocol 3 (27–411, mean 186); fetal cells positive for the Y signal (2–12, mean 6) were detectable in all studied cases (n = 16). In 7 of the samples from women carrying female fetuses, we could only detect cells with two X signals (51–182, mean 103). All of the experiments were interpretable due to the presence of compact cells with well-visible red and green signals. Conclusion: Our study revealed that using protocol 3 as the post-MACS treatment results in improved NRBC recovery and enables a reliable prospective non-invasive fetal sex determination.

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Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood

Cited by 123 publications

References 9 publications

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

Improvement of Fetal Cell Recovery from Maternal Blood: Suitable Density Gradient for FACS Separation

Non-Invasive Fetal Sex Determination on Fetal Erythroblasts from the Maternal Circulation Using Fluorescence in situ Hybridisation

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