The preparation of periapical lesions for flow cytometry

Aim To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry. Methodology PA lesions were mechanically agitated, with and without proteol ysis. This was with either 0.2% collagenase alone, or in combination with 0 .02% DNA-ase in serial incubations until all tissue was digested. The effic acy of each method was assessed by counting total cell yield and cell viabi lity. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment. Results Disaggregation of PA lesions was superior if collagenase was presen t, but: cell clumping was problematic unless the DNA-ase was also added, an d serial digestion with this combination produced optimal cell yield and vi ability. Nevertheless, the total number of cells released rarely exceeded 1 05, though viability was in excess of 80%. Mechanical agitation and proteol ysis adversely affected PBL phenotypes, but collagenase digestion limited t o 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6-10%) CD45RB + cell s. Conclusions Because of the necessity for single cell suspensions, flow cyto metry is not easily applied to the analysis of leucocytes in PA lesions, an d further refinements in tissue disaggregation and cell preparation are req uired.