Ma Edmond S K, Wang Candy L N, Wong Anthony T C, Choy Gigi, Chan Tsun Leung
Division of Molecular Pathology, Department of Pathology, Hong Kong Sanatorium & Hospital, Clinical Pathology Laboratory, 1/F Li Shu Fan Block, 2 Village Road, Happy Valley, Hong Kong.
Mol Cytogenet. 2016 Aug 16;9:63. doi: 10.1186/s13039-016-0263-7. eCollection 2016.
Cytogenetic abnormalities are important prognostic markers in plasma cell myeloma (PCM) and detection is routinely performed by interphase fluorescence in-situ hybridization (FISH) with a panel of probes after enrichment of the plasma cells in the bone marrow specimen. Cell sorting by immunomagnetic beads and concurrent labeling of the cytoplasmic immunoglobulin are the usual enrichment methods. We present an alternative method of plasma cell enrichment termed Target FISH, which is an automated system that combines the images of May-Grünwald- Giemsa (MGG) staining and FISH study on the same plasma cell for analysis.
Our experience of Target FISH on 40 PCM patients was described. Briefly, plasma cells were MGG stained, image captured, de-stained, FISH probe hybridized and finally relocated for simultaneous analysis of morphology and FISH signal pattern. The FISH probe panel was TP53/CEP17, t(4;14) IGH/FGFR3, t(14;16) IGH/MAF and CKS1B(1q21)/CDKN2C(P18). Gain of 1q21 was the most common abnormality detected in 18 patients (45 %), to be followed by t(4;14) IGH/FGFR3 detected in 11 patients (27.5 %). Of note, 10 patients showed coexistence of both t(4;14) and 1q21 gain. Two patients showed del(17p)/TP53, one in association with t(4;14) and 1q gain while the other was stand alone. None of this patient cohort showed t(14;16) IGH/MAF. Using the critical binomial function, the normal cutoff FISH positive value for del(17p)/TP53 was 3.4 %, t(4;14) IGH/FGFR3 was 6.8 %, t(14;16) IGH/MAF was 5.6 % and +1q21 was 5.7 %.
The equipment cost notwithstanding, when compared with cell sorting, the total reagent cost was around 10 % lower in Target FISH. The total processing time was longer for Target FISH but manual fluorescence microscopy was no longer necessary. The main advantage of Target FISH was the complete certainty that the cytogenetic abnormality was detected in the cells of interest, and hence a more stringent analytical cutoff value might be considered. Optimization of the cell collection and slide preparation process upfront was required to accrue adequate target cells on each slide for analysis. Our experience suggested that Target FISH was applicable as a routine method of plasma cell enrichment in clinical diagnostic laboratories.
细胞遗传学异常是浆细胞骨髓瘤（PCM）重要的预后标志物，常规检测方法是在骨髓标本中富集浆细胞后，通过间期荧光原位杂交（FISH）技术使用一组探针进行检测。通过免疫磁珠进行细胞分选并同时标记细胞质免疫球蛋白是常用的富集方法。我们提出了一种称为靶向FISH的浆细胞富集替代方法，这是一种自动化系统，它将同一浆细胞的May-Grünwald-Giemsa（MGG）染色图像和FISH研究图像相结合进行分析。
描述了我们对40例PCM患者进行靶向FISH检测的经验。简要来说，浆细胞经MGG染色、图像采集、脱色、FISH探针杂交，最后重新定位以同时分析形态和FISH信号模式。FISH探针组合包括TP53/CEP17、t(4;14)IGH/FGFR3、t(14;16)IGH/MAF和CKS1B(1q21)/CDKN2C(P18)。1q21增益是最常见的异常，在18例患者（45%）中检测到，其次是在11例患者（27.5%）中检测到的t(4;14)IGH/FGFR3。值得注意的是，10例患者同时存在t(4;14)和1q21增益。2例患者显示del(17p)/TP53，其中1例与t(4;14)和1q增益相关，另1例为单独出现。该患者队列中均未显示t(14;16)IGH/MAF。使用临界二项式函数，del(17p)/TP53的正常FISH阳性截断值为3.4%，t(4;14)IGH/FGFR3为6.8%，t(14;16)IGH/MAF为5.6%，+1q21为5.7%。
尽管设备成本较高，但与细胞分选相比，靶向FISH的总试剂成本低约10%。靶向FISH的总处理时间较长，但不再需要人工荧光显微镜检查。靶向FISH的主要优点是能够完全确定在感兴趣的细胞中检测到细胞遗传学异常，因此可以考虑采用更严格的分析截断值。需要预先优化细胞收集和玻片制备过程，以便在每张玻片上积累足够的目标细胞进行分析。我们的经验表明，靶向FISH可作为临床诊断实验室中浆细胞富集的常规方法。
