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Talk:MitoEAGLE blood cells 1

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Phase 1.3: Lund retreat May 2018

ยป MitoEAGLE Lund 2018


Phase 1.2: Poznan retreat Jan 2018

ยป MitoEAGLE Poznan 2018


Phase 1.1: Innsbruck retreat Jan 2018

ยป MitoEAGLE Innsbruck 2018

Abstract Version beta 01
  1. The evaluation of mitochondrial function remains crucial for the diagnosis of a spectrum of human pathologies. Peripheral blood provides an easily accessible source of biological material like cells. However, mitochondrial respiratory studies in blood cells still require standardization. The procedures applied in bioenergetic assessments involve multiple steps including pre-analytical analytical and normalization phase. Therefore, the aim of this report is to review and summarize methods using peripheral blood mononuclear cells and platelets for mitochondrial respiratory studies. For this purpose we have used original data from multiple laboratories combined with literature data.
  2. This methodological review is not primarily to present results on patients, but to characterize control groups for pathophysiological studies. During pre-analytical phase smoking, alcohol intake, BMI threshold, lifestyle intervention and medications should be considered for inclusion / exclusion of controls. Moreover, all subjects should be matched for sex, age and ethnicity. Conditions like time of sampling, fasting and resting state are terms of standardization.
  3. Anticoagulants are used for whole blood sampling /e.g. K-EDTA, lithium heparin, sodium citrate/ and discussed for their effect on cells isolation, yield, cells fraction purity and respiration.
  4. The time and temperature during transport and storage of whole blood before specific cells separation influences next analytical phases and affect final outcome. Characterization of whole blood provides the basis for inclusion / exclusion for further processing of the sample.
  5. Separation procedures depend on the type of cells required for respirometric studies / PLT, PBMC or PLT and PBMC/. Media used, centrifugation conditions, cell counting and cell viability methods are under discussion. Purity of preparation / e.g. PLT contamination in PBMCs fraction, purity of PLT fraction/, recovery and yield of cells from the whole blood are also presented in this report.
  6. There are limited data on the effect of storage and conditions during processing of isolated cell types before respirometry. Temperature, media, antibiotics, proteinase inhibitors cocktail, density of cells during storage, tilting of cell fraction might significantly affect the measurements.
  7. Specific protocols are used for respiration assessments in intact and permeabilized. The type of medium used for respirometry /MiR 05, RPMI, plasma/ need further evaluations.
  8. Finally, we focus on the normalization, which is important to interpret correctly and compare the results of respiratory studies. It should include data on contamination of PBMCs with PLTs, cell viability /e.g. succinate test/, flux control ratios, total protein concentration, citrate synthase activity. Some other markers, like clusters of differentiation specific for leukocytes and platelets can be taken into consideration.
  9. To conclude, we summarize emerging recommendations for mitochondrial respiratory studies in intact and permeabilized PBMC and PLT towards building a data base of healthy subjects that can be used as controls in clinical studies.
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