Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei 100226, Taiwan.
Institute of Applied Mechanics, National Taiwan University, Taipei 10617, Taiwan.
Anal Chem. 2023 Feb 14;95(6):3274-3282. doi: 10.1021/acs.analchem.2c04260. Epub 2023 Feb 3.
Rare cells in the blood often have rich clinical significance. Although their isolation is highly desirable, this goal remains elusive due to their rarity. This paper presents a systemic approach to isolate and characterize trophoblasts from the maternal circulation. A microfluidic rare cell disc assay (RaCDA) was designed to process an extremely large volume of up to 15 mL of blood in 30 min, depleting red blood cells (RBCs) and RBC-bound white blood cells (WBC) while isolating trophoblasts in the collection chip. To minimize cell loss, on-disc labeling of cells with fluorescent immuno-staining identified the trophoblasts. Retrieval of trophoblasts utilized an optimized strategy in which multiple single cells were retrieved within the same micropipette column, with each cell encapsulated in a fluid volume (50 nL) separated by an air pocket (10 nL). Further, whole-genome amplification (WGA) amplified contents from a few retrieved cells, followed by quality control (QC) on the success of WGA via housekeeping genes. For definitive confirmation of trophoblasts, short-tandem repeat (STR) of the WGA-amplified content was compared against STR from maternal WBC and amniocytes from amniocentesis. Results showed a mean recovery rate (capture efficiency) of 91.0% for spiked cells with a WBC depletion rate of 99.91%. The retrieval efficiency of single target cells of 100% was achieved for up to four single cells retrieved per micropipette column. Comparison of STR signatures revealed that the RaCDA can retrieve trophoblasts from the maternal circulation.
血液中的稀有细胞通常具有丰富的临床意义。尽管人们非常希望能够分离这些细胞，但由于其稀有性，这一目标仍然难以实现。本文提出了一种从母体循环中分离和鉴定滋养层细胞的系统方法。设计了一种微流控稀有细胞盘检测（RaCDA），可在 30 分钟内处理多达 15 毫升的血液，耗尽红细胞（RBC）和 RBC 结合的白细胞（WBC），同时在收集芯片中分离滋养层细胞。为了最大限度地减少细胞损失，使用荧光免疫染色对细胞进行盘上标记，以识别滋养层细胞。回收滋养层细胞采用了一种优化策略，即在同一微吸管柱内回收多个单细胞，每个细胞封装在 50 nL 的流体体积（50 nL）中，由 10 nL 的气隙隔开。此外，全基因组扩增（WGA）从几个回收的细胞中扩增内容物，然后通过管家基因对 WGA 的成功进行质量控制（QC）。为了明确确认滋养层细胞，将 WGA 扩增内容物的短串联重复（STR）与来自母体白细胞和羊膜穿刺术的羊水细胞的 STR 进行比较。结果显示，对于含有 99.91%WBC 耗竭率的标记细胞，平均回收率（捕获效率）为 91.0%。每个微吸管柱最多可回收四个单细胞，实现了 100%的单个靶细胞回收效率。STR 特征的比较表明，RaCDA 可以从母体循环中回收滋养层细胞。
