Greenwood Michael P, Newton Keith M, Pepper Kristi L, Hendrickson Heather L, Olsen Randall J, Thomas Jessica S
Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, United States.
Lab Med. 2025 Jul 11;56(4):343-350. doi: 10.1093/labmed/lmae096.
CALR mutation analysis is routinely used to diagnose BCR/ABL1-negative myeloproliferative neoplasms. The 2 most common CALR mutations are a 52-base pair (bp) deletion and a 5-bp insertion, which account for approximately 85% of cases.
To evaluate our new microfluidic chip assay, we tested CALR mutant and wild-type specimens that were previously analyzed using conventional methods at a reference laboratory. Samples included EDTA-anticoagulated peripheral blood and bone marrow specimens, air dried bone marrow aspirate smears, and formalin-fixed, paraffin-embedded bone marrow sections. CALR exon 9 was PCR amplified using 2 previously published primer pairs and a third unique primer pair designed for our new assay. Amplicons were sized using microfluidic chip analysis.
Concordance with the reference method was 100% (42/42). Intra-run and inter-run reproducibility were also 100% (3/3 and 3/3, respectively). The limit of detection was confirmed to be 6% mutant alleles.
We determined that the microfluidic chip assay to detect CALR exon 9 mutations was acceptable for clinical use. Compared with the conventional method, the microfluidic analysis assay benefits from a streamlined workflow, faster turnaround, and a smaller instrument footprint.
CALR突变分析常用于诊断BCR/ABL1阴性骨髓增殖性肿瘤。最常见的两种CALR突变是52个碱基对（bp）的缺失和5个bp的插入，约占病例的85%。
为评估我们新的微流控芯片检测方法，我们检测了CALR突变型和野生型样本，这些样本先前在参考实验室用传统方法进行过分析。样本包括乙二胺四乙酸（EDTA）抗凝的外周血和骨髓样本、空气干燥的骨髓穿刺涂片以及福尔马林固定、石蜡包埋的骨髓切片。使用2对先前发表的引物对以及为我们新检测方法设计的第三对独特引物对，对CALR第9外显子进行聚合酶链反应（PCR）扩增。使用微流控芯片分析对扩增子进行大小测定。
与参考方法的一致性为100%（42/42）。批内和批间重复性也均为100%（分别为3/3和3/3）。检测限经确认是6%的突变等位基因。
我们确定检测CALR第9外显子突变的微流控芯片检测方法可用于临床。与传统方法相比，微流控分析检测方法具有流程简化、周转更快以及仪器占地面积更小的优势。
