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.

Highlights

  • Rare nucleated CD45 negative cells in peripheral blood may be malignant such as circulating tumor cells

  • We presented an in-house enrichment assay based on magnetic bead separation technology targeting rare CD45neg cells in peripheral blood by depleting normal cells

  • We proved that highly sophisticated magnetic capture technology and lengthy cell separation procedures are avoidable by enhancements in magnetic bead labelling of target cells

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Summary

Introduction

Rare nucleated CD45 negative cells in peripheral blood may be malignant such as circulating tumor cells. In oncology, nucleated CD45neg cells of epithelial or mesenchymal type are in suspicion of being malignant in the setting of confirmed cancer cases, and are believed to have originated from a primary or secondary tumour, are referred to as circulating tumour cells (CTC) [14]. Efforts to culture CTCs were purposed to investigate functional properties, pheno- and genotype, to test drug sensitivity as to support personalized therapy and/or to screen and develop new drugs [18, 19]

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