Advances in rare cell isolation: an optimization and evaluation study

Abstract

BackgroundRare nucleated CD45 negative cells in peripheral blood may be malignant such as circulating tumor cells. Untouched isolation thereof by depletion of normal is favored yet still technological challenging. We optimized and evaluated a novel magnetic bead-based negative selection approach for enhanced enrichment of rare peripheral blood nucleated CD45 negative cells and investigated the problem of rare cell contamination during phlebotomy.MethodsFirstly, the performance of the magnetic cell separation system was assessed using leukocytes and cultivated fibroblast cells in regard to depletion efficiency and the loss of cells of interest. Secondly, a negative selection assay was optimized for high performance, simplicity and cost efficiency. The negative selection assay consisted of; a RBC lysis step, two depletion cycles comprising direct magnetically labelling of leukocytes using anti-CD45 magnetic beads followed by magnetic capture of leukocytes using a duopole permanent magnet. Thirdly, assay evaluation was aligned to conditions of rare cell frequencies and comprised cell spike recovery, cell viability and proliferation, and CD45 negative cell detection. Additionally, the problem of CD45 negative cell contamination during phlebotomy was investigated.ResultsThe depletion factor and recovery of the negative selection assay measured at most 1600-fold and 96%, respectively, leaving at best 1.5 × 104 leukocytes unseparated and took 35 min. The cell viability was negatively affected by chemical RBC lysis. Proliferation of 100 spiked ovarian cancer cells in culture measured 37% against a positive control. Healthy donor testing revealed findings of nucleated CD45 negative cells ranging from 1 to 22 cells /2.5 × 107 leukocytes or 3.5 mL whole blood in 89% (23/26) of the samples.ConclusionOur assay facilitates high performance at shortest assay time. The enrichment assay itself causes minor harm to cells and allows proliferation. Our findings suggest that rare cell contamination is unavoidable. An unexpected high variety of CD45 negative cells have been detected. It is hypothesized that a rare cell profile may translate into tumor marker independent screening.

Neuroendocrine Neoplasms of the Small Bowel and Pancreas

“…This reflects the inability to capture all tumour cells, the heterogeneity of captured cells and the limitations in the molecular assessment of a single cell. Currently, technological inadequacies in the "capture and count" strategy, as well as the complexity of single cell analysis, remain limitations [89,94]. As a diagnostic test, CTC measurement is only accurate in ∼50% of NEN [93] (that is, CTC can be detected in only half of the cases of confirmed metastatic NEN).…”

The Blood Circulating Rare Cell Population. What Is It and What Is It Good for?

“…Furthermore, mature cells may be discerned between platelet carrying cells with cytoplasm and those having released their content appearing as cytoplasm-free or naked MKC that may still measure 20 µm in size [11,25,26]. Our investigations on CRC relying on negative selection [27] suggest findings of naked MKC in healthy donor peripheral blood, when compared with published imagery and descriptions of morphology (Figure 1). the CD41a marker.…”

“…The authors reported the detection of DRAQ5+/CD41+/concanavalinA−/glycophorin A−/CD45−/CD16− cells and suggested those to be circulating MKC. Similar in principle and suited for MKC isolation are other magnetic cell separation platforms relying on negative selection [27,34]. Moreover, circulating cytoplasmic MKC are best isolated from the right atrium or inferior vena cava using a pressure fixation method [21].…”

Novel method for DNA methylation analysis using high‐performance liquid chromatography and its clinical application

“…However, methods for efficient capture of circulating tumor cells (CTC) from blood samples have been recently developed for liquid biopsy. [34][35][36] Adams et al 34 report that pathologically definable, apoptotic and epithelial-mesenchymal transition-like CTC accounted for 17%, 16% and 58% of cells isolated by one such capture method, respectively. Because our HPLC method is able to detect 5% DNA methylation ( Figure S5), 16%-58% of cancer cells in liquid biopsy samples would be detectable using our system.…”