Optimization of the magnetic negative selection purification technique of peripheral blood t CD4+ and b lymphocyte with analysis of cell viability, yield and purity with flow cytometry

• Grant number: 16/10083-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2016
• End date: July 31, 2017
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Nelson Zocoler Galante
• Grantee: Larissa Mitsue Okuda
• Host Institution: Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Therapeutic drug monitoring (TDM) of immunosuppressive drugs is used to improve the immunosuppressive effect while minimizing the toxicity related to exposition to high serum levels. Although TDM is widely used in clinical practice, a significant number of kidney transplant recipients have acute allograft rejection in the first year after transplantation. To improve the use of immunosuppressive drugs, new approaches of TDM have been developed. Monitoring drug concentrations at lymphocytes of peripheral blood is considering promising because it indicates the availability of the drug directly in the target sites of immunosuppression. The magnetic antibody-based cell isolation is an appropriate technology to isolate peripheral blood lymphocytes allowing high cell yields in a relatively short period of time. The efficiency of this procedure is, however, variable, being highest when cells on interest are the labeled ones (positively selected), and lower in the negative selected fraction. This study intends to optimize the magnetic antibody-based negative selection cell isolation technique to obtain single cell suspensions of peripheral blood CD4+ T and B lymphocytes with purity of at least 95%, and to systematically analyze the cell viability, yield and purity of blood samples of adult health volunteers submitted to the optimized method.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

ROMANO, PASCHOALINA; FERNANDES, MARIA DA LUZ; REZENDE EBNER, PERSIO DE ALMEIDA; DE OLIVEIRA, NAYARA DUARTE; OKUDA, LARISSA MITSUE; AGENA, FABIANA; MENDES, MARIA ELIZABETE; SUMITA, NAIRO MASSAKAZU; COELHO, VERONICA; DAVID-NETO, ELIAS; et al. UPLC-MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+and B CD19+lymphocytes. Journal of Pharmaceutical and Biomedical Analysis, v. 152, p. 306-314, APR 15 2018. (16/10083-9, 16/10043-7, 15/11389-1)