The Application Of Domestic Fluorescence In Situ Hybridization Probes In Rapid Detection Of Number Of Fetal Chromosomes

OBJECTIVETo investigate the application of fluorescence in situ hybridization (FISH) to the rapid detection of fetal chromosomes, and to explore its sensitivity as well as specificity in the early diagnosis of Down syndrome (DS), from an overall perspective. Also, the domestic FISH probes used in this research were tested in various aspects like experimental requirements, stability, etc., in order to get an evaluation of its further application.METHODS(1) Both experimental group and control group were established at enrollment: With all pregnant women who had abnormal results of routinely DS included in our study, their aseptic amniotic fluid samples were collected through amniocentesis during gestational age of 17-23wks. Later on, fetal exfoliative cells were harvested from each sample and divided into 2 groups named A(FISH) and B(control). Group A went through a series of FISH procedures including preprocessing, degeneration of cells, degeneration of probes, hybridization and postprocessing, till the final step that the results were observed under the fluorescence microscopy; Whereas B group was taken into ordinary routine examine as cell incubation, screening and karyotype analysis of chromosomes. (2) Results from two groups were compared in both identity and difference. Furthermore, the strengths and limitations of each method were analyzed according to the rapidity, safety, accuracy as well as specificity. This is of key importance of guiding the clinical practice of applying most proper method to those women who are in urgent need of getting quick diagnosis before available interventions missing the window phase.(3) The fluorescence probes used in this research were simultaneously tested in several characteristics in terms of requirements of laboratory, accuracy of results and its stability.RESULTS(1) Among all the qualified amniotic fluid samples,76 out of 80 from group A were well-hybridized with respective probe in dark background, as well as distinct fluorescence signals manifestation under fluorescence microscopy; 4 out of 80 were failed (1 with bright background while other 3 were observed scattered fluorescence signals). In contrast,79 out of 80 samples from group B were successfully incubated with final karyotype analysis results. The number of fetal chromosomes were normal in 78 results and abnormal (triple 21st chromosomes) in lsample.(2) All 76 results of fetal exfoliative cells from the same sample in both groups were identical, and were verified accurate according to follow-up interview. Group A costed 2ds in average with success rate of 95%; whereas group B costed 18ds in average with 98.7%in success rate. It is relatively faster for FISH to detect the number of chromosomes, which meets the need of rapid prenatal diagnosis, thus is especially important for pregnant women who desire more time for further intervention supposing the abnormal diagnosis or so.(3) There are many disadvantages in the performance of domestic FISH probes compared with similar imported probes. Such as the over-average requirements for laboratory facilitates, instability of flurescence signals as well as the fast decay of results under microscopy. By pilot-testing, we've addressed some of the problems such as by increasing the amount of collagenase to digest the thick collagen protein surrounding the nucleus in order for the FISH probes to filter into. Also, by raising the temperature for degeneration to 77-78℃as well as elongating the time length for regeneration to 28 mins, we've greatly improved the outcomes of FISH experiments, even though the decay speed was lowered because fluorescent signals could be observed 2 days after the hybridization. In total, these domestic probes were cheaper and more easily available.CONCLUSIONS(1) Fluorescent in situ hybridization using uncultured mid-pregnancy amniotic fluid is fast and accurate in diagnosis of number of fetal chromosomes, thus has potential significance in future clinical application.(2) The process of manipulating fetal exfoliate cells from amniotic fluid is of vital importance to the success of fluorescent in situ hybridization, and is a key criteria in setting up the pregnancy week range appropriate for the test.(3) Further researches need to be conducted in laboratory quality control, and improvement of domestic FISH probes, especially in stability and reliability. In this way it can build up the foundation for future application.