Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Zheng, Yun‐Ling; Carter, N. P.; Price, CJ; Colman, Sue; Milton, P. J. D.; Hackett, GeraldA; Greaves, MF; Ferguson‐Smith, Malcolm A.

doi:10.1136/jmg.30.12.1051

Cited by 121 publications

(82 citation statements)

References 21 publications

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“…The information regarding specific cell types and fetal genotype will not be obtained. These drawbacks of PCR can be overcome by the approach proposed by Zheng et al [14], in which fetal cells could be analyzed by simultaneous immunotyping and FISH. They noted, however, that the FISH probes did not hybridize 100% of the cells, and that with a small number of fetal cells to analyze, errors could still occur.…”

Section: Discussionmentioning

confidence: 99%

Frequencies of Fetal Nucleated Red Blood Cells in Maternal Blood during Different Stages of Gestation

Kuo

1998

Fetal Diagn Ther

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Nucleated red blood cells (NRBCs) isolated by a triple density gradient from 100 ml peripheral blood samples of 100 pregnant women and 30 women postpartum were subjected to morphological analysis and PCR quantitation. The number of NRBCs steadily increased from 5.3 (frequency: 2.4 × 10^–7) in early gestation to 98.2 (frequency: 4.2 × 10^–6) near term. The number of male cells increased from 6 (frequency: 2.7 × 10^–7) in early gestation to a peak of 31 (frequency: 1.48 × 10^–6) in the second trimester, and slightly decreased to 27 (frequency: 1.31 × 10^–6) near term. Both NRBCs and male fetal cells rapidly disappeared after delivery. The result implies that a significant proportion of NRBCs in maternal blood are of fetal origin before 24 weeks of gestation while in late gestation the majority of NRBCs may be of maternal origin.

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Section: Discussionmentioning

confidence: 99%

Frequencies of Fetal Nucleated Red Blood Cells in Maternal Blood during Different Stages of Gestation

Kuo

1998

Fetal Diagn Ther

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“…Staining procedures were similar to those followed for immunophenotyping with UCHÁ [1,5]. Slides were fixed for 10 min in 2% formaldehyde in phosphate-buffered saline (PBS) at room temperature, rinsed in Tris-buffered saline (TBS) for 2 min twice, and washed for 10 min in TBS.…”

Section: Methodsmentioning

confidence: 99%

Improved Specificity of NRBC Detection in Chorionic Villus Sample Supernatant Fluids Using Anti-Zeta and Anti-Epsilon Monoclonal Antibodies

Mavrou

Kοlialexi²,

Zheng³

et al. 1999

Fetal Diagn Ther

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Objective: Fetal erythrocytes leak from fetal capillaries at the time of chorionic villus sampling (CVS). It has been reported that in approximately 60% of CVS cases fetal nucleated red blood cells (NRBC) can be isolated from the supernatant fluid by immunophenotyping with monoclonal antibody (Ab) against the γ-chain of fetal hemoglobin and used as an additional source for confirmation of the fetal karyotype. However, the increased prevalence of β-thalassemia mutations in countries such as Greece results in many pregnant women who produce γ-positive cells. This makes it difficult to distinguish between the fetal and maternal origin of the NRBC. Use of Abs against embryonic hemoglobin chains ζ and ε may increase specificity for fetal NRBC detection. Methods: Mouse monoclonal Abs against Hb-ζ and Hb-ε were used in order to examine if specificity for fetal NRBC detection in CVS supernatant fluids could be improved. 41 samples were studied using anti-ζ and 20 using anti-ε monoclonal Abs. Results: Anti-ζ or anti-ε positive erythrocytes were, respectively, identified in 52 of 61 CVS samples and anti-ζ or anti-ε positive NRBC were present in all cases. The mean number of Hb-positive erythrocytes identified with the anti-ζ Ab was 58 and the mean number of NRBC 29. The mean number of anti-ε positive erythrocytes was 30 and of NRBC 23. FISH with X and Y chromosome specific probes was performed in 26 cases and the results were concordant with the CVS karyotype. Statistical analysis using the correlation test showed that anti-ζ and anti-ε were more specific for the detection of embryonic NRBCs. Conclusions: Since embryonic monoclonal Abs show increased specificity, they should be preferentially used for NRBC detection in CVS supernatant fluids. Furthermore, the increased specificity of anti-ζ and anti-ε Abs may considerably improve prenatal diagnosis from fetal cells isolated from maternal circulation.

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“…PCR detected paternal DNA sequences from the purified fetal cell fraction but not from the unsorted maternal blood. 43 The above cases of multiple rounds of cell isolation were possible, as several methods of sorting were already available for each cell type. For example, a lot of cell types from blood have multiple cell surface markers identified, or they have unique cell sizes and densities that separate them from other cell types.…”

Section: Discussionmentioning

confidence: 99%

Development of the “Three-step MACS”: a Novel Strategy for Isolating Rare Cell Populations in the Absence of Known Cell Surface Markers from Complex Animal Tissue

Lee¹,

Lufkin²

2012

J Biomol Tech

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To circumvent the difficulty of isolating specific cell populations by MACS from dissociated complex animal tissue, when their proportions reached levels similar to that of the background, we developed the "Three-step MACS" strategy. Cells of interest are defined by their expression of a particular gene(s) of interest rather by than their natural cell surface markers or size. A two-component transgenic cell surface protein, for two sequential rounds of MACS, is expressed under the promoter control of the endogenous gene of interest by means of gene targeting and the generation of transgenic tissue. An initial step to remove dead cells is also used. Here, we describe proof-of-concept experiments, using the biotin acceptor peptide (BAP)-low-affinity nerve growth factor receptor as the two-component protein. The first component, the BAP, can be biotinylated in specific subsets of cells expressing a particular gene by expressing the biotinylating enzyme, hBirA ϭ humanized BirA (hBirA), under the promoter control of another gene defining the specific subpopulation. We showed that a rare population of cells (1.1% of the 13.5 days postcoital mouse embryo) could be enriched to a sufficiently high purity (84.4%). From another sample with 0.1% of our cells of interest, we achieved a 40.3% pure sample. The low cost, speed, and technical ease of the Three-step MACS also make it scalable and hence, an ideal method for preparing sufficient quantities of biological samples for sensitive, high-throughput assays.

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Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Cited by 121 publications

References 21 publications

Frequencies of Fetal Nucleated Red Blood Cells in Maternal Blood during Different Stages of Gestation

Frequencies of Fetal Nucleated Red Blood Cells in Maternal Blood during Different Stages of Gestation

Improved Specificity of NRBC Detection in Chorionic Villus Sample Supernatant Fluids Using Anti-Zeta and Anti-Epsilon Monoclonal Antibodies

Development of the “Three-step MACS”: a Novel Strategy for Isolating Rare Cell Populations in the Absence of Known Cell Surface Markers from Complex Animal Tissue

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