Determination of leukocyte populations, apoptosis and respiratory burst in human peripheral blood for drug evaluation.

SILVESTRINI P; ETCHEVERS L; PERALTA B; OLMOS NICOTRA, MF; STASSI A; CABAÑA E; BARAVALLE M.E; ORTEGA H.H.; RENNA, M.S.

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Congreso; II Congreso Virtual Iberoamericano de Citometría. Latinflow 2021; 2021

Red Iberoamericana de Citometría de Flujo:

In this work, the Center for Comparative Medicine (CMC) together with a clinical trial pharmaceutical company carried out a comparative study for pharmacokinetics and pharmacodynamics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) formulations. The aim of this study was to optimize the absolute neutrophil count, apoptosis and/or necrosis and respiratory burst in human peripheral blood samples from healthy volunteers by flow cytometry. Two specific antibodies were used to identify the neutrophil population: anti-CD45-FITC and anti-CD16-Alexa700 and absolute count beads were used to calculate the absolute value. For the evaluation of the respiratory burst, the dihydrorhodamine reagent (DHR) was used to measure the production of reactive oxygen species in basal and activated blood neutrophils. For cellular apoptosis and/or necrosis evaluation, blood samples were labeled with anti-CD45-FITC and subsequently with Annexin V-APC and 7AAD. The appropriate antibody and dye concentrations were determined. The results obtained in the present work have allowed optimizing the proposed methodologies for the absolute neutrophil count, apoptosis and respiratory burst. The next step will be the evaluation of rhG-CSF formulations from treated patient samples. After this development, the CMC will have a solid methodology for the evaluation of biosimilars or new drugs.