Zahedipour Fatemeh, Ranjbaran Reza, Behzad Behbahani Abbas, Afshari Khalil Tavakol, Okhovat Mohammad Ali, Tamadon Gholamhossein, Sharifzadeh Sedigheh
Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran.
Avicenna J Med Biotechnol. 2017 Apr-Jun;9(2):104-108.
Acute Promyelocytic Leukemia (APL) is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t(15; 17) (q22; q21), which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform (L) of PML-RARa fusion transcript in NB4 cell line.
To achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR.
In the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 at 42°. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript.
The concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring.
急性早幼粒细胞白血病（APL）是急性髓系白血病的一个亚类。95%的APL病例存在染色体畸变t(15; 17) (q22; q21)，这阻碍了细胞分化。确定潜在分子病变的特征对于确定最佳治疗策略具有重要价值。本研究的目的是开发一种新的强大的流式荧光原位杂交（Flow-FISH）技术，以检测NB4细胞系中PML-RARα融合转录本的长异构体（L）。
为实现最佳固定条件，使用了两种不同的固定剂，包括2%多聚甲醛和75%乙醇。通透步骤使用0.2% Triton X-100和0.2%皂苷。在杂交过程中，采用了广泛的时间和温度范围，并在荧光共振能量转移（FRET）系统中设计了探针。结果通过荧光显微镜检测和逆转录聚合酶链反应（RT-PCR）进行确认。
在本研究中，成功优化了一种将原位杂交与流式细胞术相结合的新技术，以检测PML-RARα转录本的存在。使用2%多聚甲醛和0.2%皂苷的标准固定和通透方案在流式细胞术中获得了最佳荧光结果。此外，结果表明杂交的最佳时间和温度为42℃下2小时。逆转录聚合酶链反应和荧光显微镜的结果证实了PML-RARα转录本的存在。
Flow-FISH结果与逆转录聚合酶链反应和荧光原位杂交这两种其他技术的结果之间的一致性表明，该方法可作为临床样本中APL的诊断测试和微小残留病（MRD）监测。
