Development, Characterization, and Use of Monoclonal Antibodies Made to Antigens Expressed on the Surface of Fetal Nucleated Red Blood Cells

Abstract Background: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5–1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity. Methods: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens. Results: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0–419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples. Conclusion: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.

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

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6404.2005 ◽

2005 ◽

Vol 53 (3) ◽

pp. 319-322 ◽

Cited By ~ 17

Author(s):

Susanne Mergenthaler ◽

Tatiana Babochkina ◽

Vivian Kiefer ◽

Olaf Lapaire ◽

Wolfgang Holzgreve ◽

...

Keyword(s):

In Situ Hybridization ◽

Y Chromosome ◽

Blood Cells ◽

Maternal Blood ◽

Fish Analysis ◽

Blood Samples ◽

Fetal Cells ◽

Nucleated Red Blood Cells ◽

Noninvasive Prenatal Diagnosis

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 XY- and YY-FISH, respectively.

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A Silicon-based Coral-like Nanostructured Microfluidics to Isolate Rare Cells in Human Circulation: Validation by SK-BR-3 Cancer Cell Line and Its Utility in Circulating Fetal Nucleated Red Blood Cells

Micromachines ◽

10.3390/mi10020132 ◽

2019 ◽

Vol 10 (2) ◽

pp. 132 ◽

Cited By ~ 3

Author(s):

Gwo-Chin Ma ◽

Wen-Hsiang Lin ◽

Chung-Er Huang ◽

Ting-Yu Chang ◽

Jia-Yun Liu ◽

...

Keyword(s):

Red Blood Cells ◽

Pregnant Women ◽

Blood Cells ◽

De Novo ◽

Capture Rate ◽

Genetic Diagnosis ◽

Maternal Blood ◽

Capture Efficiency ◽

Nucleated Red Blood Cells ◽

Silicon Based

Circulating fetal cells (CFCs) in maternal blood are rare but have a strong potential to be the target for noninvasive prenatal diagnosis (NIPD). “Cell RevealTM system” is a silicon-based microfluidic platform capable to capture rare cell populations in human circulation. The platform is recently optimized to enhance the capture efficiency and system automation. In this study, spiking tests of SK-BR-3 breast cancer cells were used for the evaluation of capture efficiency. Then, peripheral bloods from 14 pregnant women whose fetuses have evidenced non-maternal genomic markers (e.g., de novo pathogenic copy number changes) were tested for the capture of circulating fetal nucleated red blood cells (fnRBCs). Captured cells were subjected to fluorescent in situ hybridization (FISH) on chip or recovered by an automated cell picker for molecular genetic analyses. The capture rate for the spiking tests is estimated as 88.1%. For the prenatal study, 2–71 fnRBCs were successfully captured from 2 mL of maternal blood in all pregnant women. The captured fnRBCs were verified to be from fetal origin. Our results demonstrated that the Cell RevealTM system has a high capture efficiency and can be used for fnRBC capture that is feasible for the genetic diagnosis of fetuses without invasive procedures.

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