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
Keywords:
99mTc-HMPAO-WBC, flow cytometry, Media Fill, CD64, 7AAD, Annexin V.Abstract
Background and aim: Infectious processes can be diagnosed using 99mTc-HMPAO labeled autologous leukocytes. The prepared autologous leukocytes 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. For a comprehensive quality control analysis of the leukocyte preparation, the use of the cytofluorimetry technique is advisable, and, in addition, the sterility of the whole process should be assessed. This work aimed at checking the quality of the labeled leukocytes routinely prepared and used for diagnosis in our Unit, using optimized cytofluorimetry analysis with specific cell biomarkers.
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. The Media fill test demonstrated sterility throughout all the process steps.
Conclusions: Cytofluorimetry was demonstrated to be an accurate and useful method for assessing the viability of labeled autologous leukocytes. This could be used both in the process validation phase and when the scintigraphy analysis provides ambiguous results. The Media fill used in the clinical context can be run during the validation and re-validation phases of the entire process.
References
1. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection. 2012;18(3):268–81.
2. Mcafee j, Thkur M. Survey of Radioactive Agents for in Vitro Labelling of Phagocytic Leukocytes II. Particles. 1976;488–92.
3. Kumar V. Radiolabeled white blood cells and direct targeting of micro-organisms for infection imaging. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of. 2005 Dec;49(4):325–38.
4. Buscombe JR. The future of infection imaging. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of. 2006 Jun;50(2):99–103.
5. Lucignani G. The many roads to infection imaging. Eur J Nucl Med Mol Imaging [Internet]. 2007 Oct 17;34(11):1873–7. Available from: http://link.springer.com/10.1007/s00259-007-0574-3
6. Bruni C, Padovano F, Travascio L, Schillaci O, Simonetti G. Usefulness of Hybrid SPECT/CT for the 99m Tc-HMPAO-Labeled Leukocyte Scintigraphy in a Case of Cranial Osteomyelitis [Internet]. Vol. 12, The Brazilian Journal of Infectious Diseases. 2008. Available from: www.bjid.com.br
7. Ballani NS, Al-Huda FA, Khan HA, Al-Mohannadi S, Mahmood H, Al-Enezi F. The value of quantitative uptake of99mTc-MDP and 99mTc-HMPAO white blood cells in detecting osteomyelitis in violated peripheral bones. J Nucl Med Technol. 2007 Jun;35(2):91–5.
8. Peters AM. The utility of [99mTc]HMPAO-leukocytes for imaging infection. Semin Nucl Med. 1994 Apr;24(2):110–27.
9. Ballinger JR, Gulenchyn KY, Reid RH. Radiopharmaceutical Factors in the Variable Quality of [99mTc]HM-PAO Images of the Brain. J nucl Med. 1990;31:118–22.
10. Capriotti G, D’Alessandria C, Garin E, Weber E, Devillers A, Lehmann K, et al. An in vitro study to compare 99mTc-stannous colloids and 99mTc-HMPAO for labelling human leukocytes. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of. 2004 Sep;48(3):229–36.
11. Hung JC, Iverson BC, Toulouse KA, Mahoney DW. Effects of Methylene Blue Stabilizer on In Vitro Viability and Chemotaxis of 99m Tc-Exametazime-Labeled Leukocytes. Vol. 43, THE JOURNAL OF NUCLEAR MEDICINE •. 2002.
12. Strober W. Tripan blu exclusion test for cell viability. Curr Protoc Immunol. 2001;Appendix 3:A.3B.1-A.3B.2.
13. Norme di buona preparazione dei radiofarmaci per la Medicina Nucleare. D.M. 30-3-2005 . G.U. anno 146° Numero 168.
14. Auletta S, Riolo D, Varani M, Lauri C, Galli F, Signore A. Labelling and Clinical Performance of Human Leukocytes Labelled with 99m Tc-HMPAO Using Leukokit® with Gelofusine versus Leukokit® with HES as Sedimentation Agent. Contrast Media Mol Imaging. 2019;2019.
15. Stocks SC, Ruchaud-Sparagano MH, Kerr MA, Grunert F, Haslett C, Dransfield I. CD66: role in the regulation of neutrophil effector function. Eur J Immunol. 1996 Dec;26(12):2924–32.
16. Ruchaud-Sparagano MH, Stocks SC, Turley H, Dransfield I. Activation of neutrophil function via CD66: Differential effects upon β2 integrin mediated adhesion. Br J Haematol. 1997;98(3):612–20.
17. van de Winkel JG, Capel PJ. Human IgG Fc receptor heterogeneity: molecular aspect and clinical implications. Immunol Today. 1993;14(5):215–21.
18. Hoffmann JJML. Neutrophil CD64: A diagnostic marker for infection and sepsis. Vol. 47, Clinical Chemistry and Laboratory Medicine. 2009. p. 903–16.
19. Okumura M, Thomas ML. Regulation of immune function by protein tyrosine phosphatases. Vol. 7, Current Opinion in Immunology. 1995.
20. Crowley LC, Marfell BJ, Scott AP, Waterhouse NJ. Quantitation of apoptosis and necrosis by Annexin V, Propidium Iodide Uptake, and Flow Cytometry. Cold Springs Harb Protoc. 2016 Nov 1;11.
21. Schmid I, Krall WJ, Uittenbogaart CH, Braun J, Giorgi J V. Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry. 1992;13(2):204–8.
22. De Vries EFJ, Roca M, Jamar F, Israel O, Signore A. Guidelines for the labelling of leucocytes with 99mTc-HMPAO. Vol. 37, European Journal of Nuclear Medicine and Molecular Imaging. 2010. p. 842–8.
23. Prakash Srinivasan Timiri Shanmugam TSIJ. Biocompatibility Protocols for Medical Devices and Materials. 1st ed. Elsevier; 2023.
24. Dragar Č, Kralj S, Kocbek P. Bioevaluation methods for iron-oxide-based magnetic nanoparticles. Int J Pharm. 2021 Mar;597:120348.
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Copyright (c) 2025 Rita Paroni, Claudio Maioli, Giulia Marchetti, Giacomo Crosta, Giuseppina Vismara, Mahrail Nan, Aldijana Sadikovic, Ilaria Goggi, Anna Petroni, Antonella Capozza
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