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

Hromadníková, Ilona; Karamanov, Sultan; Houbova, Bela; Hridelova, Dana; Köfer, J.; Mrstinová, M

doi:10.1159/000059369

Cited by 9 publications

(7 citation statements)

References 25 publications

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“…We also tried to develop PNA probes specific for -hemoglobin, but with no success. We believe that some of these presumed fetal erythroblasts could be either false-positive XY cells in studies where fetal cells are identified by only one FISH procedure (Bianchi et al 2002;Hromadnikova et al 2002) or ␥ -positive fetal NRBCs where fetal cells are identified by, e.g., Kleihauer staining combined with FISH (Rodriguez de Alba et al 2001). Because of the rarity of fetal cells in maternal blood, we (Christensen et al 2003, in press), like others (cf.…”

Section: Discussionmentioning

confidence: 63%

The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis: Preliminary Results

Kølvraa

Christensen

Lykke-Hansen

et al. 2005

J Histochem Cytochem.

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Fetal cells, present in the blood of pregnant women, are potential targets for non-invasive prenatal diagnosis. The fetal erythroblast has been the favorite target cell type. We investigated four methods of enrichment for fetal erythroblasts, identifying only three fetal erythroblasts in 573 ml of maternal blood. This is much less than the expected two to six fetal cells per ml of maternal blood. Hamada and Krabchi used a cell type-independent marker, i.e., the Y chromosome in maternal blood from male pregnancies after Carnoy fixation, leaving the nuclei for hybridization with X-and Y-chromosome-specific probes. We found with a similar technique 28 fetal cells in 15 ml of maternal blood. The fetal origin of cells was confirmed by hybridizing the nuclei with X- and Y-chromosome-specific probes, using two consecutive hybridizations with the two probes in opposite colors (reverse FISH). Candidate fetal cells were inspected after each hybridization. Only cells that were found to change the color of both probe signals from first to second hybridization were diagnosed as fetal. To reduce the labor-intensive slide screening load, we used semiautomated scanning microscopy to search for candidate cells. We conclude that erythroblasts form only a small fraction of fetal cells present in maternal blood.

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Section: Discussionmentioning

confidence: 63%

The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis: Preliminary Results

Kølvraa

Christensen

Lykke-Hansen

et al. 2005

J Histochem Cytochem.

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“…Applying the described cell treatment protocol of hypotonic treatment and Carnoy's fixation after MACS enrichment (CD13 Ϫ , CD5 Ϫ , CD 71 ϩ ), Hromadnikova et al (2002) reported complications in evaluation. These were caused by lack of cells or damage to the isolated cells in seven of eight analyzed cases.…”

Section: Discussionmentioning

confidence: 99%

FISH Analysis of All Fetal Nucleated Cells in Maternal Whole Blood: Improved Specificity by the Use of Two Y-chromosome Probes

Mergenthaler¹,

Babochkina²,

Kiefer³

et al. 2005

J Histochem Cytochem.

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S U M M A R Y Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12-20 male fetal cells/ml of maternal blood were identified by XYand YY-FISH, respectively.

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“…We have applied two different enrichment methods on blood samples from women pregnant at gestational ages between 8th and 19th week: (1) A 3-step procedure consisting of a density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection on CD45/CD14-negative cells (double MACS) [9]. (2) A onestep procedure consisting of CD71 antibody selection directly from whole blood, which is simple and fast [8].…”

Section: Discussionmentioning

confidence: 99%

“…Maternal blood samples were enriched by two different methods: (1) double MACS: density gradient centrifugation followed by CD45/ CD14 depletion, which was again followed by CD71-positive selection (double MACS, Milteney Biotec, Germany) as described by Hromadnikova et al [9], and (2) IMMUNICON: CD71-positive selection performed on whole blood as described below.…”

Section: Enrichment Methodsmentioning

confidence: 99%

“…We have evaluated two methods for isolation of fetal erythroblasts from maternal blood: (1) CD71 antibody selection from whole blood using the IMMUNICON device [8] and (2) double MACS: Density gradient centrifugation followed by CD45/CD14 antibody depletion, again followed by CD71-positive selection for NRBCs from CD45/CD14-negative cells [9]. Our results indicate that CD71 antibody selection is more efficient than double MACS.…”

Section: Introductionmentioning

confidence: 99%

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Fetal Cells in Maternal Blood: A Comparison of Methods for Cell Isolation and Identification

et al. 2005

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Objective: A variety of methods have been used to select and identify fetal cells from maternal blood. In this study, a commonly used 3-step selection method is compared with selection directly from whole blood. Identification of fetal origin by XY FISH of male cells was also evaluated. Methods: Maternal blood was drawn either before invasive chorion villus sampling (pre-CVS) or after (post-CVS) from women carrying a male fetus. Fetal cells were isolated either by density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection from CD45/CD14-negative cells, or by CD71-positive selection directly from whole blood. The true origin of fetal cells recovered by the two methods was established by two rounds of XY chromosome FISH in reverse colors, in some instances combined with anti-zeta (ζ) or anti-ζ/anti-gamma (γ) antibody staining. Results: In blood samples taken post-CVS and enriched by CD71 selection directly from whole blood, fetal cells were identified with a frequency that was almost four orders of magnitude higher than in post-CVS samples enriched by the 3-step method. In blood samples taken pre-CVS and enriched by the 3-step procedure, no fetal cells were identified by reverse color FISH in 371 ml of blood. In similar samples enriched by CD71 selection on whole blood, two fetal cells were identified in 27 ml of blood. Rehybridization with X and Y chromosome probes with reverse colors was necessary to exclude false Y chromosome signals. Not all fetal cells identified by the presence of a true Y chromosome signal stained with anti-ζ antibody. Conclusions: Selection of fetal NRBCs from maternal blood by CD71-positive selection directly from whole blood is superior to density gradient centrifugation succeeded by CD45/CD14 depletion and CD71 selection of CD45/CD14-negative cells. Combining two markers for fetal origin is recommended for unambiguously identifying a cell as fetal.

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

Cited by 9 publications

References 25 publications

The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis: Preliminary Results

The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis: Preliminary Results

FISH Analysis of All Fetal Nucleated Cells in Maternal Whole Blood: Improved Specificity by the Use of Two Y-chromosome Probes

Fetal Cells in Maternal Blood: A Comparison of Methods for Cell Isolation and Identification

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