Proposed strategy for circulating progenitor stem cells assessment by multiparameter flow cytometry analysis

Objective: To optimize the gating strategy for circula-ting progenitor stem cells (cPCs) assessment based on our old protocol and latest literature data.
Methods: Blood samples from 10 STEMI patients were collected aseptically by venipuncture in sterile EDTA vacutainers. Collection tubes were stored at room tem-perature and processed within 4 hours after sampling, using a lyse-no wash protocol. For counting the ab-solute number of cells, TruCOUNT Tubes have been used. The following mouse anti-human monoclonal antibodies were employed: FITC-labelled anti-CD45, PE-labelled anti-CD309, APC-labelled anti-CD34, BV421-labelled anti-CD133, and PE-Vio770-labelled anti-CD184. Isotype control antibodies were also uti-lized. Optimal working concentration of each antibody was determined by serial titration. A LSR Fortessa 5 la-sers analyzer and BD FACSDiva analysis software were used to enumerate and analyze cPCs. Roughly 100 000 CD45+ cells were acquired.
Results: Gating with isotype controls decreased the frequency of false positives events. The following cell subpopulations have been identified: CD34+CD45+, CD133+CD34+CD45+, KDR+CD34+CD45+, KDR+ CD133+CD34+CD45+ and CD184+CD34+CD45+.
Conclusions: Herein we presented a five-color flow cytometric approach for cPCs assessment. This gating strategy could be employed for cPCs accurate enume-ration in various medical conditions. Acknowledge-ments: This work was supported by a grant of the Ro-manian National Authority for Scientific Research and Innovation, CNCS/CCCDI UEFISCDI, project num-ber PN-III-P2-2.1-PED-2016-1333, within PNCDI III.

ISSN – online: 2734 – 6382
ISSN-L 1220-658X
ISSN – print: 1220-658X
The Romanian Journal of Cardiology is indexed by:
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CODE: 379
CME Credits: 10 (Romanian College of Physicians)