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3.  Red blood cell subpopulations in freshly drawn blood: application of proteomics and metabolomics to a decades-long biological issue 
Blood Transfusion  2013;11(1):75-87.
Background
It has long been known that red blood cells comprise various subpopulations, which can be separated through Percoll density gradients.
Materials and methods
In this study, we performed integrated flow cytometry, proteomic and metabolomic analyses on five distinct red blood cell subpopulations obtained by Percoll density gradient separation of freshly drawn leucocyte-depleted erythrocyte concentrates. The relation of density gradient fractions to cell age was confirmed through band 4.1a/4.1b assays.
Results
We observed a decrease in size and increase in cell rugosity in older (denser) populations. Metabolomic analysis of fraction 5 (the oldest population) showed a decrease of glycolytic metabolism and of anti-oxidant defence-related mechanisms, resulting in decreased activation of the pentose phosphate pathway, less accumulation of NADPH and reduced glutathione and increased levels of oxidized glutathione. These observations strengthen conclusions about the role of oxidative stress in erythrocyte ageing in vivo, in analogy with results of recent in vitro studies. On the other hand, no substantial proteomic differences were observed among fractions. This result was partly explained by intrinsic technical limitations of the two-dimensional gel electrophoresis approach and the probable clearance from the bloodstream of erythrocytes with membrane protein alterations. Since this clearance effect is not present in vitro (in blood bank conditions), proteomic studies have shown substantial membrane lesions in ageing red blood cells in vitro.
Conclusion
This analysis shows that the three main red blood cell subpopulations, accounting for over 92% of the total RBC, are rather homogeneous soon after withdrawal. Major age-related alterations in vivo probably affect enzyme activities through post-translational mechanisms rather than through changes in the overall proteomic profile of RBC.
doi:10.2450/2012.0164-11
PMCID: PMC3557492  PMID: 22871816
red blood cell; population; density gradient; proteomics; metabolomics
5.  Red blood cell storage in SAGM and AS3: a comparison through the membrane two-dimensional electrophoresis proteome 
Blood Transfusion  2012;10(Suppl 2):s46-s54.
Background
SAGM is currently the standard additive solution used in Europe, while AS-3 is the third additive solution that has been licensed in the USA, and is also the one used in part of Canada. Although AS-3 is based on a saline-adenine-glucose solution, it also contains citrate and phosphate. Storage of red blood cell concentrates in CPD-SAGM is known to lead to the accumulation of a wide series of storage lesions, including membrane protein fragmentation and vesiculation, as we could previously determine through 2-dimensional gel electrophoresis.
Materials and methods.
Through 2D-SDS-IEF-polyacrilamide gel electrophoresis we performed a time course analysis (day 0, 21 and 42 of storage) of red blood cell membranes from leukocyte-filtered concentrates either stored in CPD-SAGM or CP2D-AS-3.
Results and discussion.
From the present study it emerges that the membrane protein profile of red blood cells stored in presence of AS-3 appears to be slightly different from (better than) previous reports on SAGM-stored counterparts. However, the increase of total membrane spot number due to the presence of fragments at day 21 and the significant decrease at day 42 are suggestive of a universal phenomenon which is not efficiently tackled by either of the two additive solutions investigated in the present study.
Conclusion
To further delve into the storage lesion issue for RBCs stored in AS-3, it would be interesting in the future to assay metabolic changes over storage progression as well.
doi:10.2450/2012.008S
PMCID: PMC3418620  PMID: 22890268
red blood cell; storage; SAGM; AS-3; proteomics
6.  Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping 
Blood Transfusion  2012;10(Suppl 2):s71-s77.
Background
Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions.
Materials and methods.
We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion.
Results
Membrane Prdx2 was visible in day 30 samples albeit not in day 0, while it was still visible in the 1:10 dilution of day 30 in day 0 RBCs. Cell gradients also highlighted changes in the profile of the RBC subpopulations upon dilution of stored RBCs in the fresh ones.
Discussion.
From this preliminary in vitro investigation it emerges that Prdx2 and RBC populations might be further tested as candidate biomarkers of blood doping through autologous transfusion, though it is yet to be assessed whether the kinetics in vivo of Prdx2 exposure in the membrane of transfused RBCs will endow a sufficient time-window to allow reliable anti-doping testing.
doi:10.2450/2012.011S
PMCID: PMC3418622  PMID: 22890272
blood doping; red blood cell; population; peroxiredoxin 2
7.  Clinical Metabolomics: the next stage of clinical biochemistry 
Blood Transfusion  2012;10(Suppl 2):s19-s24.
doi:10.2450/2012.005S
PMCID: PMC3418628  PMID: 22890264
Clinical biochemistry; metabolomics; mass spectrometry
8.  Troubleshooting in platelet storage temperature and new perspectives through proteomics 
Blood Transfusion  2010;8(Suppl 3):s73-s81.
doi:10.2450/2010.012S
PMCID: PMC2897204  PMID: 20606754
platelets; platelet concentrates; storage; cold activation
9.  Red blood cell storage: the story so far 
Blood Transfusion  2010;8(2):82-88.
doi:10.2450/2009.0122-09
PMCID: PMC2851210  PMID: 20383300
red blood cell; storage lesion; blood transfusion; adverse effect; oxidative stress
10.  The role of antenatal immunoprophylaxis in the prevention of maternal-foetal anti-Rh(D) alloimmunisation 
Blood Transfusion  2010;8(1):8-16.
doi:10.2450/2009.0108-09
PMCID: PMC2809506  PMID: 20104273
antenatal immunoprophylaxis; maternal-foetal alloimmunisation; haemolytic disease of the newborn; Rh(D)

Results 1-10 (10)