Flow Cytometry Unit, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney, Australia.
Cytometry B Clin Cytom. 2013 Mar;84(2):96-103. doi: 10.1002/cyto.b.21067. Epub 2013 Jan 2.
Levels of residual disease (RD) are an independent predictor of progression-free survival (PFS) and overall survival (OS) in patients treated for chronic lymphocytic leukemia (CLL). We modified the international standardized approach (ISA) to RD detection using flow cytometry by developing a single tube 10 color antibody assay.
A single tube incorporated the following monoclonal antibodies: CD81FITC, CD22PE, CD3ECD, CD5PercP5.5, CD20PECY7, CD79bAPC, CD38A700, CD43APC Alexa750, CD19eFluor 450, and CD45KO. A modified ISA gating strategy was developed that removed contaminating events. Sensitivity assays were performed using dilution with normal peripheral blood and bone marrow. Clinical samples were compared using the ISA and the single tube assay.
Dilution studies showed that sensitivity of 0.001% was achievable when a minimum of 1.8 × 10(6) total events were acquired. One hundred twenty-nine samples were analyzed and showed RD levels from 0.003 to 22%. In 80 samples analyzed with both assays, there was an excellent correlation between the two methods (slope = 1.0, intercept = 0.07 and R2 = 0.992) and results from Bland-Altman analysis showed a bias of 0.04 ± 0.38 with 95% confidence interval of -0.71 to 0.79. Removal of contaminating events in the single tube assay led to a significant reduction in RD values (P = 0.0014).
The single tube 10-color assay for the detection of RD in CLL provides equivalent results to the ISA but requires fewer cells, uses fewer reagents, and allows for simpler analysis. By directly removing contaminating events, it improves the accuracy of CLL RD detection and may reclassify the status of some patients following chemotherapy.
在接受慢性淋巴细胞白血病 (CLL) 治疗的患者中，残留疾病 (RD) 水平是无进展生存期 (PFS) 和总生存期 (OS) 的独立预测因子。我们通过开发一种单管 10 色抗体检测方法，对 RD 检测的国际标准化方法 (ISA) 进行了修改。
单管中包含以下单克隆抗体：CD81FITC、CD22PE、CD3ECD、CD5PercP5.5、CD20PECY7、CD79bAPC、CD38A700、CD43APC Alexa750、CD19eFluor 450 和 CD45KO。开发了一种改良的 ISA 门控策略，可去除污染事件。通过用正常外周血和骨髓稀释进行了敏感性检测。使用 ISA 和单管检测对临床样本进行了比较。
稀释研究表明，当获得至少 1.8×10^6 个总事件时，可实现 0.001%的灵敏度。分析了 129 个样本，RD 水平为 0.003 至 22%。在 80 个用两种方法分析的样本中，两种方法之间存在极好的相关性（斜率=1.0，截距=0.07，R^2=0.992），Bland-Altman 分析结果显示偏差为 0.04±0.38，95%置信区间为-0.71 至 0.79。在单管检测中去除污染事件会导致 RD 值显著降低（P=0.0014）。
用于检测 CLL 中 RD 的单管 10 色检测法与 ISA 提供等效的结果，但需要更少的细胞、更少的试剂，并允许更简单的分析。通过直接去除污染事件，提高了 CLL RD 检测的准确性，并且可能重新分类一些化疗后的患者状态。
