Martinez Ryan J, Kang Qing, Nennig Davis, Bailey Nathanael G, Brown Noah A, Betz Bryan L, Tewari Muneesh, Thyagarajan Bharat, Bachanova Veronika, Mroz Pawel
From the Department of Laboratory Medicine and Pathology (Martinez, Nennig, Thyagarajan, Mroz).
The Division of Hematology and Oncology, Department of Internal Medicine (Kang, Tewari), University of Michigan, Ann Arbor.
Arch Pathol Lab Med. 2022 Jan 1;146(1):92-100. doi: 10.5858/arpa.2020-0454-OA.
CONTEXT.—: Quantification and detection of the t(9;22) (BCR-ABL1) translocation in chronic myelogenous leukemia and B-lymphoblastic leukemia are important for directing treatment protocols and monitoring disease relapse. However, quantification using traditional reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is dependent on a calibration curve and is prone to laboratory-to-laboratory variation. Droplet digital polymerase chain reaction (ddPCR) is a novel method that allows for highly sensitive absolute quantification of transcript copy number. As such, ddPCR is a good candidate for disease monitoring, an assay requiring reproducible measurements with high specificity and sensitivity.
OBJECTIVE.—: To compare results of ddPCR and RT-qPCR BCR-ABL1 fusion transcript measurements of patient samples and determine if either method is superior.
DESIGN.—: We optimized and standardized a 1-step multiplexed ddPCR assay to detect BCR-ABL1 p190 and ABL1 e10 transcripts. The ddPCR optimization included varying cycle number and primer concentration with standardization of droplet generation and droplet number and analyses to improve data sensitivity. Following optimization, ddPCR measurements were performed on clinical samples and compared with traditional RT-qPCR results.
RESULTS.—: Droplet digital polymerase chain reaction was able to detect the BCR-ABL1 p190 transcript to 0.001% (1:10-5) with a calculated limit of detection and limit of quantitation of 4.1 and 5.3 transcripts, respectively. When tested on patient samples, ddPCR was able to identify 20% more positives than a laboratory-developed 2-step RT-qPCR assay.
CONCLUSIONS.—: Droplet digital polymerase chain reaction demonstrated increased detection of BCR-ABL1 compared with RT-qPCR. Improved detection of BCR-ABL1 p190 and the potential for improved standardization across multiple laboratories makes ddPCR a suitable method for disease monitoring in patients with acute B-lymphoblastic leukemia.
在慢性粒细胞白血病和B淋巴细胞白血病中，t(9;22)(BCR-ABL1)易位的定量和检测对于指导治疗方案及监测疾病复发至关重要。然而，使用传统逆转录定量聚合酶链反应（RT-qPCR）进行定量依赖于校准曲线，且易于出现实验室间差异。微滴式数字聚合酶链反应（ddPCR）是一种能够对转录本拷贝数进行高灵敏度绝对定量的新方法。因此，ddPCR是疾病监测的良好候选方法，这是一种需要具有高特异性和灵敏度的可重复测量的检测方法。
比较患者样本中ddPCR和RT-qPCR检测BCR-ABL1融合转录本的结果，并确定哪种方法更具优势。
我们优化并标准化了一步多重ddPCR检测方法，以检测BCR-ABL1 p190和ABL1 e10转录本。ddPCR的优化包括改变循环数和引物浓度，同时对微滴生成、微滴数量进行标准化，并进行分析以提高数据灵敏度。优化后，对临床样本进行ddPCR检测，并与传统RT-qPCR结果进行比较。
微滴式数字聚合酶链反应能够检测到低至0.001%（1:10-5）的BCR-ABL1 p190转录本，计算得出的检测限和定量限分别为4.1和5.3个转录本。在患者样本检测中，与实验室开发的两步RT-qPCR检测方法相比，ddPCR能够多识别出20%的阳性样本。
与RT-qPCR相比，微滴式数字聚合酶链反应对BCR-ABL1的检测能力有所提高。对BCR-ABL1 p190检测能力的提高以及在多个实验室实现更好标准化的潜力，使ddPCR成为急性B淋巴细胞白血病患者疾病监测的合适方法。
