Efficiency of Manual Scanning in Recovering Rare Cellular Events Identified by FluorescenceIn SituHybridization: Simulation of the Detection of Fetal Cells in Maternal Blood

Abstract

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Rapid Aneuploidy Detection of Chromosomes 13, 18, 21, X and Y Using Quantitative Fluorescent Polymerase Chain Reaction with Few Microdissected Fetal Cells

“…It is now well known that fetal material (cells, nucleic acids) can be detected in the blood of pregnant women. The presence of fetal cells in maternal blood has been well documented and consequently envisioned to enable NIPD [29,41,42,43,44]. In our group, Krabchi et al [45] have offered conclusive data that fetal cells are present in the maternal blood of all pregnant women and their number ranges between 2 and 6 fetal cells/ml in normal pregnancies.…”

“…This study, however, will not include characterization of single-gene disorders although this is potentially possible. We recently developed a robust detection algorithm for rare cellular events using fluorescence-based automated microscopy [29] and validated its efficiency in the detection of rare fetal cells in maternal blood (unpubl. data).…”

Validation of automatic scanning of microscope slides in recovering rare cellular events: application for detection of fetal cells in maternal blood

“…Slides were prepared from one of the stored microtubes in a modified Thermotron environmental (CDS‐5, Thermotron, Amsterdam, The Netherlands) at 25 °C and 36% humidity. The number of slides ranged from 16 to 22 with an average of 1.5–2 × 10 5 cells per slide as previously described …”

“…In this way, we were able to locate a median of four FCs per milliliter of maternal blood in euploid pregnancies by manual screening approach . The accuracy and reliability of manual screening have been validated but were dependent on operator expertise . Therefore, automation will be required before widespread application in clinical practice.…”

“…In a previous study, we developed a strategy for spreading a predefined number of rare XY cells in a pure population of XX cells on slides and validated the efficiency of manual screening . In the present study, we developed a robust custom‐made detection algorithm for detection of rare cellular events using MetaSystems' automated platform and validated its efficiency on 90 slides with a predefined number of XY cells detected by fluorescence in situ hybridization (FISH) or by primed in situ labeling (PRINS) in comparison with manual screening.…”