Ida Marie Rundgren1, Øystein Bruserud2, Anita Ryningen3, Elisabeth Ersvær4. 1. Department of Biomedical Laboratory Science and Chemical Engineering, Western Norway University of Applied Sciences, Bergen, Norway. Electronic address: ida.marie.rundgren@hvl.no. 2. Department of Clinical Science, University of Bergen, Bergen, Norway; Haukeland University Hospital, Bergen, Norway. Electronic address: oystein.bruserud@helse-bergen.no. 3. Department of Biomedical Laboratory Science and Chemical Engineering, Western Norway University of Applied Sciences, Bergen, Norway. Electronic address: anita.ryningen@hvl.no. 4. Department of Biomedical Laboratory Science and Chemical Engineering, Western Norway University of Applied Sciences, Bergen, Norway. Electronic address: elisabeth.ersver@hvl.no.
Abstract
INTRODUCTION: Monocytes are important for innate immunity and include the classical (CD14brightCD16negative), intermediate (CD14brightCD16dim) and non-classical (CD14dimCD16bright) monocyte subsets. The quantification of these functionally different subsets in peripheral blood may become useful for diagnosis and follow-up in human diseases. The aim of the present study was to investigate how different pre-analytical parameters influence analysis of monocyte subsets in peripheral blood samples. METHODS: We determined relative levels of monocytes and monocyte subsets by flow cytometry of peripheral blood samples derived from healthy individuals. A gating strategy exclusively extracting viable CD14+ monocytes and focusing on the three monocyte subsets was applied. We investigated the effects of (i) various anticoagulants (i.e. Li-Heparin, ACD-A, K2EDTA), (ii) insufficient filling of blood sampling tubes, (iii) cryopreservation. In addition, we analysed expression of the CCR2 chemokine receptor. RESULTS: The relative numbers of CD14+ monocytes depended on the anticoagulant used, whereas the fraction of the three monocyte subsets did not. Insufficient filling of blood sampling tubes altered the relative levels of monocytes out of leukocytes, but not the relative levels of the monocyte subsets. Finally, the fraction of CD14+ monocytes out of isolated peripheral blood mononuclear cells was not significantly altered by cryopreservation, but the relative percentages of monocyte subsets was altered (similar effects for ACD-A and K2EDTA samples) and this was observed in correlation to a decreased CD16 expression. CONCLUDING REMARKS: Analysis of the monocyte subsets (i.e. classical, intermediate, non-classical) in peripheral blood samples requires a careful standardization of peripheral blood sampling and pre-analytic handling of the samples with respect to the anticoagulant used, filling of sample tubes, and cryopreservation of cells prior to analysis.
INTRODUCTION: Monocytes are important for innate immunity and include the classical (CD14brightCD16negative), intermediate (CD14brightCD16dim) and non-classical (CD14dimCD16bright) monocyte subsets. The quantification of these functionally different subsets in peripheral blood may become useful for diagnosis and follow-up in human diseases. The aim of the present study was to investigate how different pre-analytical parameters influence analysis of monocyte subsets in peripheral blood samples. METHODS: We determined relative levels of monocytes and monocyte subsets by flow cytometry of peripheral blood samples derived from healthy individuals. A gating strategy exclusively extracting viable CD14+ monocytes and focusing on the three monocyte subsets was applied. We investigated the effects of (i) various anticoagulants (i.e. Li-Heparin, ACD-A, K2EDTA), (ii) insufficient filling of blood sampling tubes, (iii) cryopreservation. In addition, we analysed expression of the CCR2 chemokine receptor. RESULTS: The relative numbers of CD14+ monocytes depended on the anticoagulant used, whereas the fraction of the three monocyte subsets did not. Insufficient filling of blood sampling tubes altered the relative levels of monocytes out of leukocytes, but not the relative levels of the monocyte subsets. Finally, the fraction of CD14+ monocytes out of isolated peripheral blood mononuclear cells was not significantly altered by cryopreservation, but the relative percentages of monocyte subsets was altered (similar effects for ACD-A and K2EDTA samples) and this was observed in correlation to a decreased CD16 expression. CONCLUDING REMARKS: Analysis of the monocyte subsets (i.e. classical, intermediate, non-classical) in peripheral blood samples requires a careful standardization of peripheral blood sampling and pre-analytic handling of the samples with respect to the anticoagulant used, filling of sample tubes, and cryopreservation of cells prior to analysis.
Authors: Patrick Haider; Timothy Hoberstorfer; Manuel Salzmann; Michael B Fischer; Walter S Speidl; Johann Wojta; Philipp J Hohensinner Journal: Int J Mol Sci Date: 2022-02-07 Impact factor: 5.923
Authors: Rebecca T Veenhuis; Dionna W Williams; Erin N Shirk; Celina M Abreu; Edna A Ferreira; Jennifer M Coughlin; Todd T Brown; Pauline M Maki; Kathryn Anastos; Joan W Berman; Janice E Clements; Leah H Rubin Journal: JCI Insight Date: 2021-06-08
Authors: Ida Marie Rundgren; Anita Ryningen; Tor Henrik Anderson Tvedt; Øystein Bruserud; Elisabeth Ersvær Journal: Molecules Date: 2020-01-16 Impact factor: 4.411