Posts Tagged ‘Cell counting’

Reporter: Sudipta Saha, Ph.D.

Assessment of the propensity for vascular events has been based on measurement of risk factors predisposing one to vascular injury. These assessments are based on the strong associations between risk factors such as hypertension, cholesterol levels, smoking, and diabetes which were first described almost a half century ago. The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual’s propensity for future cardiovascular events. Vascular health is now better represented as a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating endothelial progenitor cells (http://www.ncbi.nlm.nih.gov/pubmed/19124422). Accurate enumeration of circulating endothelial progenitor cells is essential for their potential application as biomarkers of angiogenesis. Different stem cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols are assessed for the purpose of circulating progenitor endothelial cell quantification (http://www.ncbi.nlm.nih.gov/pubmed/20381496). Enumeration of circulating progenitor endothelial cells are used in the assessment of various diseases and physiological states, such as: type 2 diabetes patients with peripheral vascular disease, certain phases during congestive heart failure, acute myocardial infarction, atherosclerosis, cardiovascular disease, physical training, cessation of smoking. Two modern instruments used now-a-days to measure the circulating progenitor endothelial cells are discussed below:

MACSQuant® Analyzer:

Circulating progenitor endothelial cells are defined by co-expression of the markers CD34, CD309 (VEGFR-2/KDR), and CD133, though CD133 expression is lost during maturation to endothelial cells.8-10 Since circulating progenitor endothelial cells are rare in peripheral blood, EPC enumeration protocols are rather extensive and laborious. To obtain reliable enumeration results for these rare cells, the sensitivity of flow cytometric analysis needs to be increased. This has been achieved by magnetic enrichment of circulating progenitor endothelial cells prior to flow cytometric analysis, which reduces the number of events that have to be analyzed. The circulating progenitor endothelial cell Enrichment and Enumeration Kit have been designed for enumeration of circulating progenitor endothelial cells from peripheral blood, cord blood, bone marrow, or leukapheresis products. In combination with magnetic pre-enrichment and flow cytometric analysis on the MACSQuant® Analyzer, this kit overcomes some of the limitations of circulating progenitor endothelial cell analysis and offers a simple and time effective solution for EPC enumeration. The circulating progenitor endothelial cell Enrichment and Enumeration Kit in combination with pre-enrichment and flow cytometric analysis on the MACSQuant Analyzer is an effective method to enumerate circulating progenitor endothelial cells in 10 mL of whole blood. Based on the calculated starting number of cells, the circulating progenitor endothelial cell Express Mode analysis template automatically calculates the absolute number and concentration of circulating progenitor endothelial cells in 10 mL of starting material, i.e., whole blood, bone marrow, cord blood, or leukapheresis products. The MACSQuant Analyzer has the ability to enrich cells using MACS technology. This capability makes the enumeration of circulating progenitor endothelial cells fast and easy. The entire process takes less than 2 hours to perform from blood draw to analyzed data and drastically reduces the time and difficulty of such a protocol by combining magnetic enrichment and flow cytometric analysis in one streamlined experiment (http://www.miltenyibiotec.com/downloads/6760/6764/18602/31184/MQ_ApplicationFlyer_EPC.pdf).

Attune® Acoustic Focusing Cytometer:

In cancer research, circulating progenitor endothelial cells have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis during or after chemotherapy. In healthy individuals, circulating progenitor endothelial cells are reported to be present in very low numbers: 0.01%–0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of circulating progenitor endothelial cells, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing circulating progenitor endothelial cells. The method includes a number of conventional ways to improve rare-event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1,000 μL/min with the Attune® cytometer. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic focusing cytometer would be 10–12 times longer, close to 6 hours. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method. (http://zh.invitrogen.com/etc/medialib/files/Cell-Analysis/PDFs.Par.54318.File.tmp/CO24210-Human-CEC_cancer.pdf)

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