Recognition of low grade or asymptomatic systemic diseases suggests prevention of the worst, yet has been proven challenging ever since. Biomarker-based liquid biopsy has emerged in recent years as a practical platform for the assessment of systemic diseases yet, technical realizations were mainly focused on cancer, faced challenges in accuracy at early stage and are lacking provision of sufficient evidence of disease. In particular in cell-based cancer liquid biopsy, obstacles are rarity and heterogeneity of circulating tumor and tumor-associated rare cells. Evidence is mounting about an entire spectrum of distinct circulating rare cell types that denotes the systemic component of a certain physiological state. Therefore, circulating rare cells in combination may arise from yet, also account for systemic diseases, which we denote as multi-rare cell association and involves foremost bone marrow-derived progenitor and stem cells yet, also matured somatic cell types. One would expect immense diagnostic value in the read-out of the so called rare cell population which represents cytological evidence of abnormality. We hypothesize that comprehensive rare cell population profiling as contrasted to the biomarker screening approach may realize the premise of a biopsy as to confirm, characterize, grade, stage or predict a systemic disease. This novel approach represents the “missing link” in diagnostic care of in particular early or residual systemic disease and presumes a steady gain in knowledge about the clinical interpretation of rare cell population profiles thus, expecting the knowledge-driven transformation of cell-based liquid biopsy from suggestion to confirmation. We support our hypothesis by past findings made by others and us and provide insights how to interpret a certain rare cell population profile. The article can be found here.
Background: The circulating rare cell population is diverse and rich in diagnostic information. Its characterization and clinical exploitation by cell-based liquid biopsy is an ongoing research task. Bone marrow is one of the major contributors to the peripheral blood rare cell population and, consequently, determines individual rare cell profiles thus depending on bone marrow health status. Bone marrow damage has been associated with aggressive or late-stage systemic diseases and egress of various bone marrow cells into the blood circulation. The association of quantity and heterogeneity of circulating erythroblast with bone marrow damage is of particular interest. Methods: Circulating CD71high/CD45-/Hoechsthigh blast cells from healthy, noncancer- and cancer-afflicted donors were enriched by CD45 depletion and analyzed by immunofluorescence microscopy. Results: A new finding of aberrant and mitotic circulating erythroid-like cells that appear similar across blood donors afflicted with various systemic pathologies is reported. Further presented is a classification of said erythroblast-like cells in nine subcategories according to morphological differences between phenotypically similar cells. Conclusion: Aberrant and mitotic bone marrow-derived rare circulating erythroid-like cells can be detected in the blood of afflicted individuals but not in healthy donors, suggesting the cause of bone marrow damage. This article can be found here.
This review details state of the art knowledge of diagnostically exploitable rare cells in the blood as to introduce a complete new view on blood analysis for all kinds of serious diseases, such as cancer, myocardial infarction or pulmonary disorders. The blood circulating rare cell population is truely informative and sensitive to slightiest imbalances and microlesions in the body making it the next generation of liquid biopsy testing.
Full scientific paper is available Schreier 2020_rare cell population.
Full Title: Validation of SanoLiBio’s negative selection cell enrichment assay
Full scientific paper is available here.
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 here.

Full Title: An Update of Circulating Rare Cell Types in Healthy Adult Peripheral Blood: 1 Findings of Immature Erythroid Precursors

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 inflammation biomarker.

Full scientific paper is available here.