Ultra-rare-event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs

Abstract

Background The performance of a fully automated scanning cytometer incorporating previously reported high‐precision autofocus and accurate image segmentation was evaluated for the detection of “ultra‐rare” cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. Methods Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate–conjugated anti‐fetal hemoglobin and 4,6‐diamidino‐2‐phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 107 nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare‐event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. Results Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false‐positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false‐positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. Conclusion This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high‐speed autofocus and accurate real‐time image segmentation for ultra‐rare event detection. Cytometry 39:285–294, 2000 © 2000 Wiley‐Liss, Inc.

Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations

“…Until now the automation was done using the traditional epifluorescent (25) or inverted microscopy stages (1,26), which were designed for manual use, not for high-throughput use.…”

“…Recently, enhanced focus has been set on the rare-cells of the human organism (e.g., physiologic and pathologic stem cells, fetal cells, circulating tumor cells) (1)(2)(3).…”

Toward complete laser ablation of melanoma contaminant cells in a co‐culture outgrowth model via image cytometry

“…Other flow sorters like the microfabricated fluorescence-activated cell sorter claim to have higher sensitivity of 99% (1) but still cannot work practically in the ultrarare (less than 1:10,000) cell regime. Microscope-based automated image cytometers, although slower thus far than flow cytometers, have demonstrated higher sensitivity and specificity (2). The information content and thereby the effective signal-to-noise ratios are greater in high-resolution images (where hundreds of pixels record information about each cell over tens of milliseconds or longer dwell times) than in flow cytometers (where photomultiplier tubes record linear data streams over $1-10 ls dwell times).…”

“…A laser on a microscope can create short radiation pulses focused at the submicron scale for quickly and effectively killing target cells with minimal collateral damage when properly controlled (8). We combined laser ablation with image cytometry instrumentation that previously demonstrated the ability to locate as rare as 1:20 million fetal cells in a model population (2). These model ultrarare fetal cell detection experiments demonstrated false positive rates of 12.6 cells per million and one false negative in 15 experiments of 1 million cells each.…”

Clinical applications of laser scanning cytometry

“…Mercury arc lamps are relatively inexpensive and provide multiple emission bands for excitation but may generate uneven illumination and unstable excitation power; this hampers quantitative analysis. New developments might help to solve this problem (8). In contrast, laser light provides a highly stable light source but multiple line excitation generally cannot be performed by a single laser.…”