Difference between revisions of "Sumbalova 2017 MiP2017 WG4"

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{{Abstract
{{Abstract
|title=[[Image:MiPsocietyLOGO.JPG|left|90px|Mitochondrial Physiology Society|MiPsociety]]
|title=[[Image:SumbalovaZ.JPG|left|90px|Zuzana Sumbalova]] Purity of blood cells fractions in respirometric studies: peripheral blood mononuclear cells and platelets.
|info=[[MiP2017]]
|info=[[MiP2017]]
|authors=Sumbalova Z, Garcia-Souza LF, Velika B, Menz V, Gatterer H, Burtscher M, Gnaiger E
|year=2017
|year=2017
|event=MiP2017
|event=MiP2017
|abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]
|abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]  
Respiration of peripheral blood mononuclear cells (PBMC) and platelets (PLT) can be a valuable tool for functional diagnostics of mitochondrial disorders. The isolation of blood cells from whole blood is relatively easy, but a common problem is the impurity of the target cell fraction. PLT and neutrophils are the main contaminants in the PBMC fraction; isolated PLT fraction can contain traces of lymphocytes and monocytes. Metabolically active contaminating cells may significantly contribute to the respiration typically expressed per cell count of the target cells.
 
In our study, we aimed to 1) increase purity of the PBMC fraction by improving the isolation method; 2) establish a contamination threshold for the necessitiy of O<sub>2</sub> fluxes correction; 3) develop a method for the correction of O<sub>2</sub> fluxes to contribution by contaminating cells.
 
The PBMC and PLT were isolated from the same blood samples [1]. The purity of each preparation was determined by counting on hematology analyzer Sysmex XN-350 (Sysmex Corporation). The same respirometric protocols were applied for both cell types in O2k-FluoRespirometer (Oroboros Instruments, Austria) at 37°C. The contribution of PLT respiration to respiration of PBMC fraction was calculated for each individual from the ratio of PLT to PBMC in PBMC fraction (N<sub>PLT</sub>/N<sub>PBMC</sub>)<sub>PBMC</sub> and O<sub>2</sub> fluxes in PLT fraction measured in parallel. The contribution of PBMC to respiration of PLT fraction was calculated from the ratio of PBMC to PLT in PLT fraction (N<sub>PBMC</sub>/N<sub>PLT</sub>)<sub>PLT</sub> and corrected O<sub>2</sub> fluxes of PBMC.
 
The ratio of PLT to PBMC in whole blood, (N<sub>PLT</sub>/N<sub>PBMC</sub>)<sub>blood</sub>, varied significantly between individuals in the range of 60 – 212 (average 120.6 ± 35 sd, n=77). The PBMC fraction isolated with our standard method [1] contained about 5.8% of the whole blood ratio, being (<sub>NPLT</sub>/<sub>NPBMC</sub>)<sub>PBMC</sub> in the range of 1.8 – 18.3, with the average 7.1 ± 3.5 sd. Relative contribution of PLT respiration to respiration of PBMC fraction was in each respiratory state slightly different and accounted in average to ~ 2% per unit of (N<sub>PLT</sub>/N<sub>PBMC</sub>)<sub>PBMC</sub>. As the average (N<sub>PLT</sub>/N<sub>PBMC</sub>)<sub>PBMC</sub> was around 7, the average contribution of PLT to total O<sub>2</sub> flux in PBMC fraction was around 14%, but varied between individuals from ~4 to 50% [2].
 
The ratio of PBMC to PLT in PLT fraction (N<sub>PBMC</sub>/N<sub>PLT</sub>)<sub>PLT</sub> varied between 0.00021 – 0.0021 with an average 0.0008 ± 0.0005 sd. (n=23). Relative contribution of PBMC to respiration of PLT fraction differed in each respiratory state and accounted in average to ~ 4.3% per 0.001 unit of (N<sub>PBMC</sub>/N<sub>PLT</sub>)<sub>PLT</sub>.
 
Minor alterations on the isolation method, such as higher volume of washing medium (with a concomitantly higher g) or one extra washing step did not significantly improve the purity of PBMC fraction. If a threshold for the correction of O<sub>2</sub> fluxes from contaminating cells is set to 5% of total O<sub>2</sub> flux, the correction of PBMC respiration is necessary if (N<sub>PLT</sub>/N<sub>PBMC</sub>)<sub>PBMC</sub> > 2.5 in PBMC fraction. The correction of PLT respiration is necessary if (NPBMC/NPLT)PLT >0.0012 in PLT fraction. Normalization of O<sub>2</sub> fluxes per cell count of target cells gives the most consistent results, as contaminating cells contribute also to citrate synthase activity or protein concentration.
 
The purity of target cell preparation is an important parameter in respirometric studies, that should be determined in every preparation for quality control and data comparison between laboratories. A device counting all cell types should be used for that and O<sub>2</sub> fluxes should be corrected if contamination exceeds certain threshold. The purity of PBMC fraction could be improved by addition of more washing steps, the effect of which on PBMC respiration should be tested.
|editor=[[Kandolf G]]
|editor=[[Kandolf G]]
|mipnetlab=AT Innsbruck Oroboros, SK Bratislava Sumbalova Z, AT Innsbruck Burtscher M, AT Innsbruck Gnaiger E
}}
{{Labeling
|area=Respiration
|tissues=Blood cells, Platelet
|instruments=O2k-FluoRespirometer
}}
}}
{{Labeling}}
== Affiliations ==
== Affiliations ==
::::Sumbalova Z(1,2), Garcia-Souza LF(1,3), Velika B(1,4), Menz V(3), Gatterer H(3), Burtscher M(3), Gnaiger E(1,5)
::::#Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
::::#Pharmacobiochemical Lab, 3rd Dept Internal Medicine, Fac Medicine, Comenius Univ, Bratislava, Slovakia
::::#Inst Sport Science, Univ Innsbruck, Austria
::::#Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik Univ Košice, Slovakia
::::#Oroboros Instruments, Innsbruck, Austria. – [email protected]
== References and acknowledgement ==
::::#Sumbalova Z, Hiller E, Chang S, Garcia L, Droescher S, Calabria E, Volani C, Krumschnabel G, Gnaiger E (2016) Isolation of blood cells for HRR. Mitochondr Physiol Network 21.17:1-15.  [[MiPNet21.17_BloodCellsIsolation|»»Bioblast Link]]
::::#Sumbalova Z, Garcia-Souza LF, Menz V, Burtscher M, Gnaiger E (2017) The effect of lifestyle on respiration of blood cells. Abstract MiP2017.


== References ==
::::::We thank [[Droescher S|Stephanie Droescher]] and [[Dikova V|Valentina Dikova]] for technical assistance and [[Laner V|Verena Laner]] for project administration. Supported by K-Regio project [[MitoFit]] (GSLF, ZS, VM) and Action Austria-Slovakia (BV). Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MITOEAGLE.

Revision as of 17:35, 19 October 2017

Zuzana Sumbalova
Purity of blood cells fractions in respirometric studies: peripheral blood mononuclear cells and platelets.

Link: MiP2017

Sumbalova Z, Garcia-Souza LF, Velika B, Menz V, Gatterer H, Burtscher M, Gnaiger E (2017)

Event: MiP2017

COST Action MITOEAGLE

Respiration of peripheral blood mononuclear cells (PBMC) and platelets (PLT) can be a valuable tool for functional diagnostics of mitochondrial disorders. The isolation of blood cells from whole blood is relatively easy, but a common problem is the impurity of the target cell fraction. PLT and neutrophils are the main contaminants in the PBMC fraction; isolated PLT fraction can contain traces of lymphocytes and monocytes. Metabolically active contaminating cells may significantly contribute to the respiration typically expressed per cell count of the target cells.

In our study, we aimed to 1) increase purity of the PBMC fraction by improving the isolation method; 2) establish a contamination threshold for the necessitiy of O2 fluxes correction; 3) develop a method for the correction of O2 fluxes to contribution by contaminating cells.

The PBMC and PLT were isolated from the same blood samples [1]. The purity of each preparation was determined by counting on hematology analyzer Sysmex XN-350 (Sysmex Corporation). The same respirometric protocols were applied for both cell types in O2k-FluoRespirometer (Oroboros Instruments, Austria) at 37°C. The contribution of PLT respiration to respiration of PBMC fraction was calculated for each individual from the ratio of PLT to PBMC in PBMC fraction (NPLT/NPBMC)PBMC and O2 fluxes in PLT fraction measured in parallel. The contribution of PBMC to respiration of PLT fraction was calculated from the ratio of PBMC to PLT in PLT fraction (NPBMC/NPLT)PLT and corrected O2 fluxes of PBMC.

The ratio of PLT to PBMC in whole blood, (NPLT/NPBMC)blood, varied significantly between individuals in the range of 60 – 212 (average 120.6 ± 35 sd, n=77). The PBMC fraction isolated with our standard method [1] contained about 5.8% of the whole blood ratio, being (NPLT/NPBMC)PBMC in the range of 1.8 – 18.3, with the average 7.1 ± 3.5 sd. Relative contribution of PLT respiration to respiration of PBMC fraction was in each respiratory state slightly different and accounted in average to ~ 2% per unit of (NPLT/NPBMC)PBMC. As the average (NPLT/NPBMC)PBMC was around 7, the average contribution of PLT to total O2 flux in PBMC fraction was around 14%, but varied between individuals from ~4 to 50% [2].

The ratio of PBMC to PLT in PLT fraction (NPBMC/NPLT)PLT varied between 0.00021 – 0.0021 with an average 0.0008 ± 0.0005 sd. (n=23). Relative contribution of PBMC to respiration of PLT fraction differed in each respiratory state and accounted in average to ~ 4.3% per 0.001 unit of (NPBMC/NPLT)PLT.

Minor alterations on the isolation method, such as higher volume of washing medium (with a concomitantly higher g) or one extra washing step did not significantly improve the purity of PBMC fraction. If a threshold for the correction of O2 fluxes from contaminating cells is set to 5% of total O2 flux, the correction of PBMC respiration is necessary if (NPLT/NPBMC)PBMC > 2.5 in PBMC fraction. The correction of PLT respiration is necessary if (NPBMC/NPLT)PLT >0.0012 in PLT fraction. Normalization of O2 fluxes per cell count of target cells gives the most consistent results, as contaminating cells contribute also to citrate synthase activity or protein concentration.

The purity of target cell preparation is an important parameter in respirometric studies, that should be determined in every preparation for quality control and data comparison between laboratories. A device counting all cell types should be used for that and O2 fluxes should be corrected if contamination exceeds certain threshold. The purity of PBMC fraction could be improved by addition of more washing steps, the effect of which on PBMC respiration should be tested.


Bioblast editor: Kandolf G O2k-Network Lab: AT Innsbruck Oroboros, SK Bratislava Sumbalova Z, AT Innsbruck Burtscher M, AT Innsbruck Gnaiger E


Labels: MiParea: Respiration 


Tissue;cell: Blood cells, Platelet 



HRR: O2k-FluoRespirometer"O2k-FluoRespirometer" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 


Affiliations

Sumbalova Z(1,2), Garcia-Souza LF(1,3), Velika B(1,4), Menz V(3), Gatterer H(3), Burtscher M(3), Gnaiger E(1,5)
  1. Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
  2. Pharmacobiochemical Lab, 3rd Dept Internal Medicine, Fac Medicine, Comenius Univ, Bratislava, Slovakia
  3. Inst Sport Science, Univ Innsbruck, Austria
  4. Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik Univ Košice, Slovakia
  5. Oroboros Instruments, Innsbruck, Austria. – [email protected]

References and acknowledgement

  1. Sumbalova Z, Hiller E, Chang S, Garcia L, Droescher S, Calabria E, Volani C, Krumschnabel G, Gnaiger E (2016) Isolation of blood cells for HRR. Mitochondr Physiol Network 21.17:1-15. »»Bioblast Link
  2. Sumbalova Z, Garcia-Souza LF, Menz V, Burtscher M, Gnaiger E (2017) The effect of lifestyle on respiration of blood cells. Abstract MiP2017.
We thank Stephanie Droescher and Valentina Dikova for technical assistance and Verena Laner for project administration. Supported by K-Regio project MitoFit (GSLF, ZS, VM) and Action Austria-Slovakia (BV). Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MITOEAGLE.