Discussion On Optimization Of Experimental Conditions For Karyotype Analysis And FISH Detection In Multiple Myeloma

Objective: By combining the application of the multiple myeloma(MM)cell culture systems were optimized through adding cytokines and extending the culture time,the experimental procedures of fluorescence in situ hybridization(FISH)were optimized and stable laboratory conditions for fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms(FICTION)were established,to explore the application value of the combined three methods in improving the detection rate of abnormal genetics in MM,it is expected to provide more reliable laboratory basis for the prognosis judgment,clinical diagnosis and treatment of MM.Methods:1.The bone marrow specimens from 22 MM patients were subjected to conventional 24 h short-term culture method and improved method,that is,IL-6(10ng/m L)+GM-CSF(40ng/m L)was added and extended to culture for 6d,To analyze whether the improved method can effectively improve the detection rate of abnormal genetics in MM patients by conventional cytogenetic analysis(CCA).2.The bone marrow specimens from 20 MM patients were pretreated with Triton X-100/4%PFA before FISH detection to observe whether the fluorescence signal quality of the probe could be improved or not.3.The bone marrow specimens from 19 MM patients were detected by CCA,FISH and FICTION simultaneously to explore the effection of FICTION technology on the abnormal genetic detection rate.Results:1.After improving the culture conditions,the number and quality of karyotypes available for analysis were significantly improved,the success rate of culture was increased from 77.3% to 86.4%,and the detection rate of abnormality was increased from 9.1% to 40.9%.2.After the pretreatment with Triton X-100/4%PFA,the signal of FISH probe was enhanced,the clutter was decreased,the fluorescence of background was significantly decreased,and the number of non-signal cells detected by 1q21,RB1,D13S319,P53 and Ig H probes were obviously decreased,the differences were statistically significant which P-value of the five probes were 0.008,0.002,0.001,0.002 and 0.001 respectively.3.The positive rate of FICTION(89.5%)was significantly higher than that of FISH(60.0%)(P=0.030).The percentage of abnormal cells was higher when FICTION was detected in 15 MM specimens with abnormal chromosomal clone or critical value by FISH.There were significant differences among 1q21,RB1,D13S319 and Ig H probes except P53,which P-value were 0.000,0.038,0.004 and 0.001 respectively.4.The combination of improved CCA and FICTION can improve the detection rate of genetic abnormalities in MM patients.Conclusion: The success rate of MM bone marrow culture and the abnormal detection rate of CCA can be significantly improved by adding IL-6+GM-CSF and prolonging the culture time;The quality of the fluorescence signal of the probe can be significantly improved during FISH detection by using Triton X-100/4%PFA;The abnormal detection rate of FICTION is higher than that of ordinary FISH;The detection rate of genetic abnormality in MM patients can be improved through the combination of improved CCA and FICTION.