Full Title: Advances in Rare Cell Isolation: An Optimization and Evaluation Study
Background: Rare nucleated CD45 negative cells in peripheral blood may be malignantsuch 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 to
investigate the problem of rare cell contamination during phlebotomy.
Methods: Firstly, 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, the 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 lowest cell spike recovery, cell viability and proliferation,
and CD45 negative cell detection. Additionally, the problem of CD45 negative cell contamination during phlebotomy was investigated.
Results: The depletion factor and recovery of the negative selection assay measuredat highest 1600-fold and 96%, respectively, leaving at
best 1.5×104 leukocytes unseparated and took 35 minutes. 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
per 2.5×107 leukocytes or 3.5ml whole blood in 89% (23/26) of the samples.
Conclusion: Our 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 hypothized that a rare cell
profile may translate into tumor marker independent screening.
Full Scientific paper is available to download from Here
Full Title: An Update of Circulating Rare Cell Types in Healthy Adult Peripheral Blood: 1 Findings of Immature Erythroid Precursors – UNDER REVIEW
Background: Circulating rare cells (CRC) are benign or malignant minuscule events in the peripheral blood or other bodily fluids.
The detection and quantification of certain CRC types is an invaluable or proposed candidate biomarker for diagnosis, prognosis and prediction of various
pathological conditions. The list of CRC types and biomarker applicability thereof continues to expand along with improvements in cell selection technology.
Past findings may suggest commonness of healthy donor peripheral blood circulating mature erythroblasts. This work suggests the occurrence of morphologically
distinct bone marrow native circulating early erythroid precursors that we intend to add to the list of CRCs.
Methods: We tested healthy individuals that varied in age and gender employing a negative cell selection assay based on magnetic bead technology
to characterize healthy adult circulating CD45 negative cell events using cell surface markers CD71 and Glycophorin-A.
Results: Positive events were detected and varied in cell and nuclear size ranging between 7.5 μm till 15.2 μm and 4.5 till 9.2 μm, respectively
with distinct appearance under bright field microscope. Cell rarity increased with cell and nuclear size. Largest EBs exceeded 12 μm in cell diameter and were found
only in 2 out of 10 donors.
Conclusion: Circulating erythroid precursors may be part of the benign CRC spectrum and are hypothized to be useful as early stage and low grade
Full Scientific paper is available to download very soon.