Relationship of decreased chemotaxis of technetium-99m-HMPAO-labeled lymphocytes to apoptosis

The purpose of this study was to evaluate chemotaxis and its relationship to apoptosis in 99mTc-HMPAO-labeled lymphocytes. METHODS: Peripheral lymphocytes, obtained from 12 healthy volunteers using lymphoprep, were divided in three equal fractions. One fraction was used as the control, one was labeled with cold HMPAO and one was labeled with 1.5 mCi (55.5 Mbq) 99mTc-HMPAO. Chemotaxis of T-lymphocytes was measured by the Boyden microchamber technique (BMA) (n = 8) using human monocyte chemotactic protein-3 (MCP-3) as chemoattractans. A chemotactic index was calculated as the number of HMPAO and 99mTc-HMPAO-labeled cells that migrated towards the MCP-3 solution, divided by the number of nonlabeled migrated lymphocytes. Apoptosis evaluation (n = 10) of unlabeled, HMPAO-labeled and 99mTc-HMPAO-labeled cells was performed using flowcytometry (FCM) forward light scatter analysis, 900 light scatter analysis, fluorescein-isothiocyanate (FITC)-labeled Annexin V and dye exclusion of propidium iodide. RESULTS: Chemotaxis of 99mTc-HMPAO-labeled T-lymphocytes was found to be reduced by approximately 31% (migration index of 0.69) (p = 0.01) as compared to both unlabeled and HMPAO-labeled lymphocytes, both the latter showing no difference in migration index. Whereas the mean percentages apoptotic lymphocytes in the unlabeled, 18.5%, and HMPAO-labeled fraction, 16.6%, were more or less comparable (p = 0.1), the mean percentage apoptotic cells in the 99mTc-HMPAO-labeled fraction was 51.8%, yielding a difference of 33.3% between 99mTc-HMPAO-labeled and unlabeled cells (p = 0.003). The procentual concordance between apoptotic cells (33.3%) and chemotactic impaired cells (31%) in the 99mTc-HMPAO-labeled fraction may be explained by the formation of a rigid cytoskeleton early in the apoptotic process that may theoretically limit chemotaxis. CONCLUSION: Using the BMA, chemotaxis of 99mTc-HMPAO-labeled lymphocytes was found to be reduced by approximately 31%. Furthermore, the percentage apoptotic lymphocytes induced by irradiation after labeling with 99mTc-HMPAO concurs well with the percentage of chemotaxis impaired cells.