Validation of a 12-color flow cytometry assay for acute myeloid leukemia minimal/measurable residual disease detection

IF 2.3 3区医学 Q3 MEDICAL LABORATORY TECHNOLOGY

Cytometry Part B: Clinical Cytometry Pub Date : 2023-08-22 DOI:10.1002/cyto.b.22140

Sa A. Wang, Jeffrey L. Jorgensen, Shimin Hu, Fuli Jia, Shaoying Li, Sanam Loghavi, Chi Young Ok, Beenu Thakral, Jie Xu, L. Jeffrey Medeiros, Wei Wang

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引用次数: 0

Abstract

Background

Acute myeloid leukemia (AML) minimal/measurable residual disease (MRD) by multicolor flow cytometry is a complex laboratory developed test (LDT), challenging for implementation. We share our experience in the validation of a 12-color AML MRD flow cytometry assay to meet stringent regulatory requirements.

Methods

We worked under the guidelines of the CLSI HL62 publication, illustrated the details of the validation process that was tailored to uniqueness of AML MRD, and tested its clinical validity in 61 patients. The “trueness” was determined by correlating with concurrent molecular genetic testing and follow-up bone marrow examinations.

Results

Under assay specificity, we shared the details of panel design, analysis, and criteria for interpretation and reporting. The assay accuracy was assessed by testing known positive and negative samples and correlating with molecular genetic testing and follow-up bone marrow examination. The limit of detection (LOD) and limit of quantification (LOQ) were validated to a level between 0.01% and 0.1%, varied from the leukemia-associated immunophenotypes (LAIP) and the numbers of events obtained for analysis. Assay linearity, precision and carry over studies all met acceptable criteria. In the clinical validity test, the concordance was 93%, specificity 98% and sensitivity 83%. The most challenging aspects of the assay were the discrimination of pre-leukemic cells (persistent clonal hematopoiesis) or underlying myelodysplastic clones from AML MRD with immunophenotypic switch or subclone selection.

Conclusion

The validation met all criteria and obtained FDA IDE (investigational device exemption) approval. This study provides ample technical and professional details in setting up the AML MRD flow cytometry assay and illustrates through the example of the “fit for purpose” validation process. We also highlight the need for further characterization of abnormal blasts bearing the potential for AML relapse.

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12色流式细胞术检测急性髓系白血病最小/可测量残留疾病的验证。

背景：通过多色流式细胞术检测急性髓细胞白血病（AML）最小/可测量残留病（MRD）是一种复杂的实验室开发的检测方法，实施起来具有挑战性。我们分享了我们在验证12色AML MRD流式细胞术检测以满足严格监管要求方面的经验。方法：我们在CLSI HL62出版物的指导下工作，说明了根据AML MRD的独特性定制的验证过程的细节，并在61名患者中测试了其临床有效性。“真实性”是通过同时进行分子遗传学检测和后续骨髓检查来确定的。结果：在分析特异性下，我们分享了面板设计、分析以及解释和报告标准的细节。通过检测已知的阳性和阴性样本并与分子遗传学检测和后续骨髓检查相关联来评估检测的准确性。检测限（LOD）和定量限（LOQ）被验证为0.01%至0.1%之间的水平，不同于白血病相关免疫表型（LAIP）和用于分析的事件数量。测定线性、精密度和结转研究均符合可接受的标准。在临床有效性测试中，一致性为93%，特异性为98%，敏感性为83%。该测定最具挑战性的方面是通过免疫表型转换或亚克隆选择从AML MRD中区分白血病前期细胞（持续性克隆造血）或潜在的骨髓增生异常克隆。结论：该验证符合所有标准，并获得了美国食品药品监督管理局IDE（试验器械豁免）批准。本研究提供了建立AML MRD流式细胞术测定的充分技术和专业细节，并通过“适用”验证过程的例子进行了说明。我们还强调了对具有AML复发潜力的异常母细胞进行进一步表征的必要性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Cytometry Part B: Clinical Cytometry 医学-病理学

CiteScore

6.80

自引率

32.40%

发文量

审稿时长

>12 weeks

期刊介绍： Cytometry Part B: Clinical Cytometry features original research reports, in-depth reviews and special issues that directly relate to and palpably impact clinical flow, mass and image-based cytometry. These may include clinical and translational investigations important in the diagnostic, prognostic and therapeutic management of patients. Thus, we welcome research papers from various disciplines related [but not limited to] hematopathologists, hematologists, immunologists and cell biologists with clinically relevant and innovative studies investigating individual-cell analytics and/or separations. In addition to the types of papers indicated above, we also welcome Letters to the Editor, describing case reports or important medical or technical topics relevant to our readership without the length and depth of a full original report.