Target fluorescence in-situ hybridization (Target FISH) for plasma cell enrichment in myeloma.
Target fluorescence in-situ hybridization (Target FISH) for plasma cell enrichment in myeloma.
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DOI：
10.1186/s13039-016-0263-7
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发表时间：
2016
影响因子：
1.3
通讯作者：
Chan TL
中科院分区：
文献类型：
--
作者：
Ma ES;Wang CL;Wong AT;Choy G;Chan TL
细胞遗传学异常是浆细胞骨髓瘤（PCM）的重要预后标志物，在骨髓标本中富集浆细胞后，常规通过间期荧光原位杂交（FISH）与一组探针进行检测。免疫磁珠分选细胞和同时标记胞浆免疫球蛋白是常用的富集方法。我们提出了一种称为靶FISH的浆细胞富集的替代方法，这是一种自动化系统，它结合了May-Grünwald- Giemsa（MGG）染色和FISH研究对同一浆细胞的图像进行分析。本文介绍了40例PCM患者的靶FISH经验。简而言之，将浆细胞MGG染色，捕获图像，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探针组为TP 53/CEP 17、t（4;14）IGH/FGFR 3、t（14;16）IGH/MAF和CKS 1B（1 q21）/CDKN 2C（P18）。1 q21增加是18例患者（45%）中最常见的异常，其次是11例患者（27.5%）中检测到的t（4;14）IGH/FGFR 3。值得注意的是，10例患者显示t（4;14）和1 q21增益共存。2例患者出现del（17 p）/TP 53，1例与t（4;14）和1 q增加相关，另1例为独立。该患者队列均未显示t（14;16）IGH/MAF。采用临界二项函数，del（17 p）/TP 53、t（4;14）IGH/FGFR 3、t（14; 16）IGH/MAF和+1q21的FISH阳性率分别为3.4%、6.8%、5.6%和5.7%。尽管设备成本较高，但与细胞分选相比，Target FISH的总试剂成本约低10%。Target FISH的总处理时间较长，但不再需要手动荧光显微镜检查。靶FISH的主要优点是完全确定在感兴趣的细胞中检测到细胞遗传学异常，因此可以考虑更严格的分析临界值。需要预先优化细胞收集和载玻片制备过程，以在每张载玻片上积累足够的靶细胞用于分析。我们的经验表明，靶FISH适用于作为临床诊断实验室浆细胞富集的常规方法。
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.