Automation of spot counting in interphase cytogenetics using brightfield microscopy

Abstract

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5–10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction. © 1996 Wiley‐Liss, Inc.

Automated fluorescent in situ hybridization (FISH) analysis of t(9;22)(q34;q11) in interphase nuclei

“…Since intra and internuclear variation regarding shape, size, and intensity of FISH signals are significant (21), it is not possible to differentiate signals from artifacts, background noise, split, or partially overlapping signals in every case. Also, because of imperfectness of hybridization or random colocalization of signals of the same color, a signal pattern different from the expected 2R2G appears in a proportion of nuclei (7). With increasing number of signals per nucleus, the probability of random colocalization of the signals leading to false positivity in translocation negative nuclei will also increase.…”

“…Besides bone marrow karyotyping and quantitative PCR methods, FISH analysis of peripheral leukocytes has been proposed as an alternative method with several advantages (5,6). Interphase FISH is much faster, culturing of metaphases is not required, and a higher number of cells can be analyzed providing higher statistical accuracy than bone marrow karyotyping (7). The major advantage of interphase FISH is that no biased cell selection occurs, since all cells can be analyzed, not just the dividing cells.…”

“…The definition used greatly influences the false positive (FPR) and false negative rates (FNR) of the analysis, which is reflected by the heterogeneity of these values reported in the literature (Table 1). (2) Bias of the investigator might cause over or underestimation of positive events, especially at high or low concentrations of positivity (7). (3) In cases of low concentration of positivity, the number of cells analyzed must be increased to obtain statistically accurate results (15), which is very time-consuming.…”

“…The number of cells detected may be increased without significantly increasing manual workload. Most publications regarding automated FISH analysis report assessment of amplification of genes or enumeration of large centromeric probes (7,(16)(17)(18)(19). A few describe analyses of translocations applying locus specific probes (16,20).…”

“…First, cell nuclei must be correctly recognized. Nuclear debris, large clusters of overlapping or touching nuclei, or unspecific background staining must be excluded from further analysis, even if this exclusion results in the loss of a few correctly detected cells (7). Second, signals must be correctly detected.…”