SubscribeIan Corless MD of Image Solutions (UK) Ltd., shows how automated interphase FISH scanning technique speeds up the slow and labour-intensive process of testing for chromosomal disorders.
In many countries, cytogenetic analysis of amniotic fluid for the diagnosis of chromosomal disorders is now a standard task and has reached an excellent level of analytical accuracy.
Traditional cytogenetic analysis requires the cultivation of foetal cells in the amniotic fluid for 1–3 weeks until analysis of metaphase chromosomes can be performed.
However, patients at high risk of chromosomal abnormalities, as indicated by sonographic abnormalities, aberrant serum screening and being of advanced maternal age, would benefit from a more rapid analysis. This is especially true in countries with laws restricting the performance of abortions between 21 and 24 weeks of gestation.
One such technique is fluorescence in situ hybridisation (FISH) on uncultured amniocytes using chromosome-specific DNA probes. Between 80 and 95% of all chromosomal disorders, expected in the second trimester, can be discovered within 24 hours if DNA probes specific for chromosomes 21, 18, 13, X, and Y are used with FISH.
Clinical trials have already shown that prenatal FISH assays have high levels of reliability, reproducibility and accuracy when compared with standard cytogenetic testing. However, one of the main factors preventing a rapid FISH test from being broadly incorporated into clinical cytogenetics laboratories to date has been lack of staff.
All that has changed with the launch of the “Duet” automated FISH scanning station by the company BioView Ltd, Rehovot, Israel. This new system uses an optimised preparation protocol and commercially available probes to screen for aneuploidy in uncultured amniotic fluid cells.
Duet, which has been approved for use by the US Food and Drug Administration (FDA), has just completed extensive testing at the Wolfson Medical Centre in Holon, Israel, where it was compared with manual FISH scoring techniques, and is detailed here.
Automated v Manual
In this study, amniotic fluid samples from 56 ongoing pregnancies were evaluated in parallel by cytogenetic analysis, using automatic FISH and manual FISH methods.
The women participating were randomly selected to represent a heterogeneous group of ages, gestational time, and indications for amniocentesis. FISH was designed to detect aneuploidies involving chromosomes 13, 18, 21, X, and Y.
Analysis of amniotic fluid sample for the presence of aneuploidy by FISH is a labour-intensive and a highly demanding task. In prenatal testing, false-positive results could lead to termination of an unaffected foetus. So samples must be evaluated carefully.
Duet’s automatic scanning locates the cells automatically and classifies them according to signal pattern into groups predefined by the user. The scanning is fast and can be performed non-stop throughout the day. The images produced by the system are high quality and realistic, so the technician can quickly verify the results and produce the report.
The automatic scanning results were found to be as accurate as the manual scoring but the analyses of samples was much less demanding in terms of technician time.
Three abnormal samples, all with trisomy 21, were detected in the study. Manual and automatic FISH scoring results were in agreement and the mean percentage of nuclei showing three signals of chromosome 21 in both methods was similar.
In normal samples, the mean frequency of the major cell population was found to be as high as 95%, whereas the mean frequency of trisomic cells found by both automatic and manual scoring was 70.9% and 72.3%, respectively.
The reported cut-off points for the proportion of cells with abnormal signal pattern needed for diagnosis by FISH were between 50 and 70% in different studies. This may reflect the nature of the test.
Theoretically, as the number of signals increases, the detection efficiency would be expected to decrease. Thus, if the probability of detecting one signal is 0.9, then two signals will be 0.81 and three 0.73. Experience from this study shows that better than 80% of cells demonstrate aneuploidy if present.
An optimised protocol
In the Wolfson study, samples were prepared according to an optimised protocol. This optimisation is crucial both for efficient automatic scanning and for reducing the number of uninformative samples, which in some studies is reported to be relatively high, especially in late gestational age.
The critical number of nuclei that need to be scored to achieve reliable results has not been established. Most groups have typically scored 50 nuclei for aneuploidy analysis. Other studies attempted to score up to 200 interphase nuclei per sample to increase accuracy.
Thus, the significant nuclei count still needs be evaluated in an expanded statistical study. Moreover, although not found in this study series, chromosomal mosaicism is a well-recognised cause of abnormal human development.
The reported incidence of mosaics found in amniocenteses ranges between 0.1 and 0.4%. In this study, up to 400 cells were scored from each sample, demonstrating the high yield of analysable nuclei from a mean sample volume of 4 ml.
High hybridisation efficiency enlarges the number of scored nuclei and may help in detecting low-level mosaicism. Scoring this high number of nuclei per sample is now practical only with the help of automation. Moreover, the images of all scanned nuclei are saved and can be revisited again in case of an inconclusive FISH result.
Although not found in this study series, chromosomal mosaicism is a well-recognised cause of abnormal human development. The reported incidence of mosaics found in amniocenteses ranges between 0.1 and 0.4%. In this study, up to 400 cells were scored from each sample, demonstrating the high yield of analysable nuclei from a mean sample volume of 4 ml.
High hybridisation efficiency enlarges the number of scored nuclei and may help in detecting low-level mosaicism. Scoring this high number of nuclei per sample is now practical only with the help of automation. Moreover, the images of all scanned nuclei are saved and can be revisited again in case of an inconclusive FISH result.
Under routine clinical conditions there is a strong demand for rapid cytogenetic diagnosis, especially in high-risk groups. However, the American College of Medical Genetics recommends that irreversible actions should not be taken on the basis of FISH alone.
Nevertheless, given the extremely small risk of false-positive FISH results and the fact that over 80% of all clinically significant chromosomal abnormalities can be detected by FISH, the number of publications supporting the integration of this technique into clinical laboratory management is now escalating. An automation capability might facilitate this process, especially in cytogenetic laboratories that analyse large numbers of samples.
This study shows that the speed, accuracy and efficiency of automatic interphase FISH scanning might be used in conjunction with conventional cytogenetics for the presence of aneuploidy in uncultured amniocytes. Automation of demanding and labour-intensive nuclei scoring will bring faster and more reliable results to ever increasing numbers of patients.