Division of Hematology/Medical Oncology, Department of Medicine, Taichung Veterans General Hospital, Taichung City, Taiwan; Ph.D. Program for Cancer Molecular Biology and Drug Discovery, and Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; Department of Post-Baccalaureate Medicine, College of Medicine, National Chung-Hsing University, Taichung, Taiwan.
Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
Pathology. 2024 Aug;56(5):671-680. doi: 10.1016/j.pathol.2024.04.001. Epub 2024 May 20.
Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.
流式细胞术可应用于荧光原位杂交（FISH）信号的检测，以有效分析染色体异常。然而，目前这种间期染色体（IC）流式-FISH 方案仅限于检测单一颜色。此外，由于 FISH 检测中的 DNA 变性步骤也会破坏细胞完整性和蛋白质结构，因此将 IC 流式-FISH 与传统的多色流式细胞术结合起来很困难，从而阻碍了随后的抗原-抗体结合，并阻碍了同时标记表面抗原和 FISH 信号。我们开发了一种用于同时检测核 IC FISH 信号和细胞表面标记物的多色流式细胞术的工作方案。该方案通过检测接受性别错配异基因造血干细胞移植（allo-HSCT）患者的血细胞性染色体含量进行了验证，这表明嵌合状态。该方法还适用于检测慢性淋巴细胞白血病（CLL）患者的 12 三体。我们首先证明了该方案在检测多种颜色以及具有高度一致性的核和表面信号方面的可行性。在临床验证实验中，使用优化的 IC 流式-FISH 方法在临床样本（n=56）中鉴定了嵌合状态；结果与传统的基于载玻片的 FISH 紧密对应（XX 细胞为 0.9649，XY 细胞为 0.9786）。在接受性别错配 allo-HSCT 的患者样本中，可以使用高灵活性的门控策略清楚地区分不同谱系中的个体嵌合状态。此外，在具有 12 三体的 CLL 样本中，该方法可以证明富含 12 三体的 FISH 信号存在于 B 细胞中，而不是 T 细胞中。最后，通过对染色体 12、X 染色体和表面标记物进行联合标记，我们可以在 allo-HSCT 后检测到具有 12 三体的罕见残留受体 CLL 细胞。这种用于多色和谱系特异性 IC 流式-FISH 的可适应方案使该技术得以发展，从而使其有可能应用于目前正在使用传统 FISH 检测的各种临床环境中。
