Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction

Abstract

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay), to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs. Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined by positive staining for DAPI, CD146 and von Willebrand Factor and negative staining for CD45. In addition, CECs exhibited distinct morphological features that enable differentiation from surrounding white blood cells. CECs were found both as individual cells and as aggregates. CEC numbers were higher in MI patients compared with healthy controls. VS patients had lower CEC counts when compared with MI patients but were not different from healthy controls. Both HD-CEC and CellSearch® assays could discriminate MI patients from healthy controls with comparable accuracy but the HD-CEC assay exhibited higher specificity while maintaining high sensitivity. Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

Figure 1

A typical circulating endothelial cell detected with the HD-CEC assay. The panels show a) a DAPI positive, large nucleus surrounded by numerous smaller nuclei of WBCs, b) CD45 positivity on white blood cells only, c) a CD146 positive cytoplasm, and d) von Willebrand factor positive cytoplasm with a staining pattern that is distinct from the CD146. Notice that surrounding platelets are also positive for vWF. Panel e) is the composite image of DAPI, CD45 and CD146 and panel f) is the composite image of DAPI, CD45 and vWF.

Figure 2

Gallery of representative circulating endothelial cells (CECs) found in MI patients. These cells are characterized by a large DAPI positive nucleus (blue), no CD45 staining (green), and double positive CD146 (red) and vWF (white) staining of cytoplasm. Despite heterogeneity in size and shape, CECs are morphological distinct from surrounding white blood cells.

Figure 3

CEC counts in healthy controls (C) and in patients that underwent myocardial infaction (MI) or vascular surgery (VS). Counts are reported as HD-CEC/ml blood with mean values highlighted in red.

Figure 4

CEC aggregates found in MI patients. a) and b) are representative aggregates of 2 and 3 cells, respectively, with DAPI positive nucleus (blue), no CD45 staining (green), and double positive CD146 (red) and vWF (white) staining of cytoplasm. c) Frequency histogram showing the distribution of CEC aggregates found in all MI patients. Detected CEC aggregates consisted of up to 21 CECs but aggregates containing 2–4 cells were more frequently found.

Figure 5. ROC curve for the CellSearch® assay (a) and the HC-CEC assay (b)

Figure 6

Sensitivity (a) and specificity (b) performances of each assay at different CEC/ml cut-off points. The difference in specificity correlated inversely with the CEC enumeration threshold.

Figure 7

Comparison of the overlap between CEC counts in healthy controls (C) and MI patients as determined by the HD-CEC assay and the CellSearch® assay. Y-axis is in log scale and red lines represent median values.