Cyto stat tetrachrome

Freshly isolated PBWCs were stained with Beckman Coulter CYTO-STAT tetraCHROME monoclonal antibodies: (1) CD45, CD3, CD4, and CD8 (cat no. 6607013), and (2) CD45, CD3, CD19, and CD56 (cat no. 6607073). Flow-cytometric enumeration of CD4⁺ T cells and CD8⁺ T cells was done for all enrolled patients with a NAVIOS^™ flow cytometer (BECKMAN) and analyzed using Navios Tetra software. Total white blood cell counts (WBCs) and absolute lymphocyte counts (ALCs) were determined on a Coulter XN-20 (SYSMEX) machine, and then absolute values of CD4⁺ T cells and CD8⁺ T cells were calculated by multiplying the subject’s ALC by the percentage of CD4⁺ T cells and CD8⁺ T cells subset obtained by flow cytometry, respectively.

All laboratory variables were assessed upon admission to the geriatric rehabilitation unit. Serum leptin levels were assessed using a commercially available ELISA (Human Leptin Quantikine ELISA Kit (R&D Systems, Abingdon, UK)). The serum samples were stored at −20 °C prior to analysis. The serum albumin was measured using a BNII analyser (Siemens, Saint-Denis, France). C-reactive protein (CRP) was measured by immunoturbidimetry using an Advia 1650 analyser (Siemens Healthcare, Saint-Denis, France). An ultrasensitive CRP assay was not available at the time of the study. Immunological variables were assessed using flow cytometry immunophenotyping, as described previously [25 (link)]. Absolute peripheral blood CD4 and CD8 T-cell counts were determined using a Cyto-Stat tetraCHROME device (including labelling with CD45, CD3, CD4, and CD8) and acquisition on an FC500 flow cytometer (both from Beckman Coulter, Villepinte, France). Counts of naïve, memory, and terminal effector T-cells were determined as follows: CD8 and CD4 naïve T-cells were defined as CD45RA+CD62L+, peripheral memory T-cells were defined as CD45RA−CD62L−, and terminal effector CD8 T-cells were defined as CD45RA+CD62L−CD28−. The anti-CD62L antibody was obtained from BD Pharmingen (BD Biosciences, San Jose, CA, USA) and the CD4, CD8, and CD28 antibodies (Caltag) came from Life Technologies (Carlsbad, CA, USA).