Translational Cancer Pathology Laboratory, School of Biomedical Sciences, The University of Western Australia, Crawley, Western Australia, Australia.
Haemato-Oncology Diagnostic Service, Department of Haematology, Addenbrooke's Hospital, Cambridge University Hospital, NHS Foundation Trust, Cambridge, UK.
Cytometry A. 2019 May;95(5):521-533. doi: 10.1002/cyto.a.23769. Epub 2019 Apr 24.
Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.
成像流式细胞术正在成为评估白血病的一种诊断工具。它具有标准流式细胞术的功能，并为每个细胞生成具有可量化数值数据的高分辨率数字图像。我们展示了一种自动化高通量方法在悬浮免疫表型全细胞上进行荧光原位杂交（FISH）的应用，该方法称为“免疫流式 FISH”。本研究的目的是证明免疫流式 FISH 可用于检测 CLL 中的染色体异常，特别是 12 三体和 17p 缺失。分离单核细胞并用荧光标记的 CD3、CD5 和 CD19 单克隆抗体进行免疫表型分析。固定后，细胞进行透化处理，dsDNA 变性，并与染色体 12 或 17 计数（CEP12 和 CEP17）和 17p12 位点特异性 FISH 探针杂交。使用 Amnis ImageStream®X Mark II 对细胞进行分析，以评估 FISH 阳性 CLL 细胞的数量和百分比以及 FISH 斑点计数的 CD5/CD19 阳性 CLL 细胞与 CD3/CD5 阳性 T 细胞的比值（FISH“平均斑点比”）。迄今为止，已检测到约 8%的病例存在 17p 缺失，del(17p)范围为 3.5-22.8%，FISH“平均斑点比”为 0.86-0.96。免疫流式 FISH 还检测到一个带有 +12 的微小残留病病例，检测限为 0.13%，并检测到一个具有非典型表型和细胞遗传学的罕见病例。免疫流式 FISH 可以检测到表型鉴定为 CD5/CD19 阳性 B 细胞中的 del(17p)。与当前的临床 FISH 检测相比，分析细胞数量增加了 100 倍，并且增加了细胞表型，提高了敏感性和特异性。此外，免疫流式 FISH 分析在独特的临床情况下具有特定的实用性，例如残留疾病和非典型生物学病例，这对于预后和 MRD 分析可能具有重要意义。该方法将有助于许多血液恶性肿瘤的治疗决策和疾病监测。
