Difference between revisions of "Sumbalova 2017 MiP2017 WG4"

From Bioblast
Jump to navigation Jump to search
m (Kandolf Georg moved page Sumbalova 2017 MiP2017 to Sumbalova 2017 MiP2017 WG4 without leaving a redirect)
Line 1: Line 1:
{{Abstract
{{Abstract
|title=[[Image:SumbalovaZ.JPG|left|90px|Zuzana Sumbalova]] Purity of blood cells fractions in respirometric studies: peripheral blood mononuclear cells and platelets.
|title=[[Image:SumbalovaZ.JPG|left|90px|Zuzana Sumbalova]] Purity of blood cell 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
|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) yields valuable information 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; the isolated PLT fraction can contain some lymphocytes and monocytes. Metabolically active contaminating cells may significantly contribute to the respiration typically expressed per cell count of the target cells.
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.  
In our study, we aimed at (1) increasing the purity of the PBMC fraction; (2) developing a method for the correction of O<sub>2</sub> fluxes by contaminating cells; and (3) establishing a contamination threshold for the necessity to correct O<sub>2</sub> fluxes.


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].  
PBMC and PLT were isolated from the same blood samples [1]. The purity of each preparation was determined by counting with a hematology analyzer Sysmex XN-350 (Sysmex Corporation). The same respirometric protocols were applied for both cell types using O2k-FluoRespirometers (Oroboros Instruments, Austria) at 37 °C. The contribution of PLT respiration to respiration of PBMC fraction was calculated for each preparation from the PLT/PBMC ratio in the PBMC fraction (''N''<sub>PLT</sub>/''N''<sub>PBMC</sub>)<sub>PBMC</sub> and O<sub>2</sub> fluxes in the PLT fraction measured in parallel. The contribution of PBMC to respiration of PLT fraction was calculated from the PBMC/PLT ratio in the 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 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>.  
(''N''<sub>PLT</sub>/''N''<sub>PBMC</sub>)<sub>PBMC</sub> was correlated with the PLT/PBMC ratio in whole blood (''N''<sub>PLT</sub>/''N''<sub>PBMC</sub>)<sub>blood</sub> which showed a large variation between individuals in the range of 60 – 212 (average 120.6 ± 35 SD, N=77). (''N''<sub>PLT</sub>/''N''<sub>PBMC</sub>)<sub>PBMC</sub> ~ 0.058 (''N''<sub>PLT</sub>/''N''<sub>PBMC</sub>)<sub>blood</sub> with a range of 1.8 18.3, and an average of 7.1 ± 3.5 SD. The relative contribution of PLT respiration to respiration of PBMC fraction was slightly different in each respiratory state, at ~2% per unit of (''N''<sub>PLT</sub>/''N''<sub>PBMC</sub>)<sub>PBMC</sub>. The average contribution of PLT to total O<sub>2</sub> flux in the PBMC fraction was around 14%, with a large variation between individuals from 4 to 50% [2].  


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.
(''N''<sub>PBMC</sub>/''N''<sub>PLT</sub>)<sub>PLT</sub> varied between 0.00021 – 0.0021 with an average of 0.0008 ± 0.0005 SD (N=23). The relative contribution of PBMC to respiration of PLT fraction differed in each respiratory state and accounted on average for ~4.3% per 0.001 unit of (''N''<sub>PBMC</sub>/''N''<sub>PLT</sub>)<sub>PLT</sub>


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.
Minor alterations of the isolation method, such as higher volumes of washing medium with concomitantly higher g, or an extra washing step, did not significantly improve the purity of the PBMC fraction. If a threshold for the correction of O<sub>2</sub> fluxes from contaminating cells is set at 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. The correction of PLT respiration is necessary if (''N''<sub>PBMC</sub>/''N''<sub>PLT</sub>)<sub>PLT</sub> >0.0012. Normalization of O<sub>2</sub> fluxes per cell count of target cells yields the most consistent results, since contaminating cells contribute also to citrate synthase activity or protein concentration.
 
The purity of target cells in blood preparations is an important parameter in respirometric studies, which should be determined in every preparation for quality control. All cell types have to be counted and O<sub>2</sub> fluxes should be corrected if contamination exceeds a certain threshold. The purity of the PBMC fraction can be improved by additional washing steps, which is effectively achieved in the course of cryopreservation of PBMC.
|editor=[[Kandolf G]]
|editor=[[Kandolf G]]
|mipnetlab=AT Innsbruck Oroboros, SK Bratislava Sumbalova Z, AT Innsbruck Burtscher M, AT Innsbruck Gnaiger E
|mipnetlab=AT Innsbruck Oroboros, SK Bratislava Sumbalova Z, AT Innsbruck Burtscher M, AT Innsbruck Gnaiger E

Revision as of 11:27, 20 October 2017

Zuzana Sumbalova
Purity of blood cell 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) yields valuable information 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; the isolated PLT fraction can contain some 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 at (1) increasing the purity of the PBMC fraction; (2) developing a method for the correction of O2 fluxes by contaminating cells; and (3) establishing a contamination threshold for the necessity to correct O2 fluxes.

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

(NPLT/NPBMC)PBMC was correlated with the PLT/PBMC ratio in whole blood (NPLT/NPBMC)blood which showed a large variation between individuals in the range of 60 – 212 (average 120.6 ± 35 SD, N=77). (NPLT/NPBMC)PBMC ~ 0.058 (NPLT/NPBMC)blood with a range of 1.8 – 18.3, and an average of 7.1 ± 3.5 SD. The relative contribution of PLT respiration to respiration of PBMC fraction was slightly different in each respiratory state, at ~2% per unit of (NPLT/NPBMC)PBMC. The average contribution of PLT to total O2 flux in the PBMC fraction was around 14%, with a large variation between individuals from 4 to 50% [2].

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

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

The purity of target cells in blood preparations is an important parameter in respirometric studies, which should be determined in every preparation for quality control. All cell types have to be counted and O2 fluxes should be corrected if contamination exceeds a certain threshold. The purity of the PBMC fraction can be improved by additional washing steps, which is effectively achieved in the course of cryopreservation of PBMC.


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.