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The preparation of periapical lesions for flow cytometry
K Fernando1, K Gulabivala, A W Barrett
1Department of Conservative Dentistry, Eastman Dental Institute for Oral Healthcare Sciences, University College London, UK.
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 proteolysis. 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 efficacy of each method was assessed by counting total cell yield and cell viability. 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 present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6-10%) CD45RB + cells.
Conclusions:
Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required.