Isolation and Analysis of Rare Cells in the Blood of Cancer Patients Using a Negative Depletion Methodology.
ABSTRACT A variety of enrichment/isolation technologies exist for the characterization of rare cells in the blood of cancer patients. In this article, a negative depletion process is presented and discussed which consists of red blood cell (RBC) lysis and the subsequent removal of CD45 expressing cells through immunomagnetic depletion. Using this optimized assembly on 120 whole blood specimens, from 71 metastatic breast cancer patients, after RBC lysis, the average nucleated cell log depletion of 2.56 with a 77 percent recovery of the nucleated cells. The necessity of exploring different anti-CD45 antibody clones to label CD45 expressing cells in this enrichment scheme is also presented and discussed. An optimized, four-color immunofluorescence staining is conducted on the cells retained after the CD45-based immunomagnetic depletion process. Different types of rare non-hematopoietic cells are found in these enriched peripheral blood samples and a wide range of external and internal markers have been characterized, which demonstrates the range and heterogeneity of the rare cells.
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ABSTRACT: There is not yet a consensus on the reliability of the methods that should be used for the detection of rare disseminated tumor cells from non-hematological malignancies. In this review, we will discuss the advantage and drawbacks of the classical approach of immunocytochemistry and the molecular detection by reverse transcriptase polymerase chain reaction (RT-PCR). The interpretation of the biological significance of circulating tumor cells and the pitfalls of the detection techniques are the main causes of discrepancy between the conclusions of different tumor-cell detection (TCD) studies.Annals of Oncology 08/2000; 11(7):785-92. · 6.58 Impact Factor
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ABSTRACT: In stem-cell research a major difficulty is caused by the lack of distinctive features that allow the identification of human mesenchymal stem cells (hMSC). Until now, there has been no specific marker and the most common way to identify hMSC is by their characteristic stem-cell properties: self-replication and differentiation potential. However, these findings can only be revealed retrospectively, and, once differentiated, hMSC lose their stem-cell character. The aim of this study was to establish four-colour immunofluorescence of several markers simultaneously in order to address the problem of how to identify hMSC on the single-cell level. The four markers collagen-I, collagen-IV, fibronectin and CD44 are known to be expressed by hMSC. Antibody binding was detected using secondary antibodies conjugated to FITC, Alexa546, TexasRed and AMCA. Because the distinction between Alexa546 and TexasRed was not possible on conventional digital images using standard filter sets, we performed spectral image acquisition. The image was subsequently decomposed into its pure spectral components, which permitted linear unmixing. Using this procedure we were able to demonstrate four-colour immunofluorescence on hMSC. With the possibility of using more sophisticated marker profiles and/or additional markers, four-colour immunofluorescence offers the opportunity of identifying hMSC on the single-cell level without performing differentiation assays.Journal of Anatomy 03/2004; 204(2):133-9. · 2.23 Impact Factor
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ABSTRACT: To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes. A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 10(5) total leukocytes in buffy coat or 1 cancer cell to 10(8) total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis. The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log(10) and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used). To the authors' knowledge, no examples in the literature exist of a 5.17 log(10) enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log(10) enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.Experimental Hematology 11/2004; 32(10):891-904. · 2.81 Impact Factor