Quality control by Flow Cytometry and Media Fill Test of autologous leukocytes marked with 99mTc-HMPAO to be used for the “In Vivo” infection diagnosis: A pilot study

Background and aim: Infectious processes can be diagnosed using99mTc-HMPAO labeled autologous leukocytes, that once separated and labeled should be neither activated nor apoptotic, so capable of reaching the infectious site. Commonly, only cell vitality by Trypan Blue test is assessed. This work aimed at checking the quality of the labeled leukocytes used for diagnosis, using an optimized cytofluorimetry analysis. Methods: Leukocytes from 11 patients were separated, labeled, and controlled by cytofluorimeter after labeling with Annexin V, CD 66, CD 64, CD 45, and 7AAD, markers of activation, apoptosis, and lysis, respectively. In addition, the sterility of the entire process was assessed using “Media Fill test”. Results: In all the patients tested, about 80% of the cells remained inactivated and suitable for diagnostic use “in vivo.” The small portion of activated leukocytes did not affect the preparation’s diagnostic use. Only one patient showed a total apoptosis of 76%, and another one of 30% vs the mean of the other 9 patients of 1±0.8%. The Media fill test demonstrated sterility throughout all the process steps. Conclusions: Cytofluorimetry was demonstrated to be an accurate and useful method. This could be used both in the process validation phase and when the scintigraphy analysis provides ambiguous results. With a major number of cases, this methodology could provide a good correlation between leukocytes vitality and scintigraphy result, more accurate than Trypan blue test. The Media fill used in the clinical context can be run during the validation and re-validation phases of the entire process.