ABSTRACT
Objective Amniotic fluid chromosome karyotype analysis is the golden standard for prenatal diagnosis of chromosome diseases, but its success rate is low due to many factors affecting amniotic fluid culture. The research aimed to investigate the relevant factors influencing the success rate of amniotic fluid cell in situ culture and explore the condition of karyotype preparation in order to establish a stable method to prepare amniotic fluid cell chromosome with high success rate. Methods Cell culture was done in amniotic fluid obtained from 435 pregnant women with prenatal diagnosis indications by amniocentesis. Statistical analysis was done on the relationship between the amount of amniotic fluid after centrifugal precipitation, the material of operation apparatus and the culture result, as well as the relationship between ambient temperature and humidity and karyotype dispersion degree. Results Successful culture was obtain in 406 cases out of 435 cases, with the success rate being 93.3%. According to the culture result, compared with white cell mass group, the culture success rates were significantly reduced in mass brown cell group and mass fresh red cell group (P<0.01). Compared with mass fresh red cell group, the culture success rate was significantly reduced in small fresh red cell group (P<0.01). Compared with the plastic group, the culture success rate was significantly increased in glass group (P<0.01). Compared with the plastic injector and other glass group, the culture success rate was significantly improved in glass group (P<0.05). The score of the karyotype dispersion degree showed slides made at 28℃of room temperature and 50% humidity were significantly better than those made at room temperature and humidity, 28℃ of room temperature and room humidity, room temperature and 50% humidity (P<0.01). Slides made at room temperature and 50% humidity were significantly better than those at room temperature and humidity, and 28℃ of room temperature and room humidity (P<0.01). Conclusion The methods including improving the technological level of amniocentesis, handling bloody amniotic fluid timely and correctly, and the application of glass injectors and other apparatus, can effectively improve the success rate of amniotic fluid cell culture. Controlling the environmental temperature and humidity (28℃,50%) in dispersion course contributes to qualified slides for karyotype analysis, providing safeguard for prenatal diagnosis.
ABSTRACT
The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.