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Impact of blood transfusion on gene expression

in human reticulocytes: Blood transfusion and
gene expression
Article in American Journal of Hematology July 2016
Impact Factor: 3.8 DOI: 10.1002/ajh.24470

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Jean-Daniel Tissot

University of Lausanne

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Impact of blood transfusion on gene expression in human reticulocytes

Olivier Salamin1, Laura Barras1, Neil Robinson1, Norbert Baume1, Jean-Daniel Tissot2, Yannis
Pitsiladis3, Martial Saugy1, Nicolas Leuenberger1*
1

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and
Geneva, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland
2

Transfusion interrgionale CRS, site dEpalinges, Switzerland

University of Brighton, United Kingdom

Running head: Blood transfusion and gene expression

*Corresponding author:
Nicolas Leuenberger, PhD
Swiss Laboratory for Doping Analyses
Ch. Des Croisettes 22
1066 Epalinges
Email: Nicolas.leuenberger@chuv.ch
Phone: +41 21 31470 95
Word count: 980
Number of figures and tables: 1
Clinicaltrials.gov identifier: NCT02423135

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
Accepted Article, doi: 10.1002/ajh.24470
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American Journal of Hematology

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To the Editor: Blood transfusion is a frequently performed therapeutic procedure that

requires regular evaluation, particularly for its indications, effectiveness and risks. There are
indeed an increasing percentage of blood transfusions considered to be inappropriate and their
efficiency has raised questions. The identification of new specific biomarkers of blood
transfusion would be of particular relevance for the monitoring of this method. These markers
would also be applicable for anti-doping purposes.
Infusion of blood results in a rapid increase of circulating red blood cells (RBCs), which
impairs endogenous production and release of immature RBCs. This impact of transfusion is
mediated by the suppression of erythropoietin (EPO) [1, 2]. We hypothesized that blood reinfusion may cause a decrease in the expression of genes related to structural and functional
components of reticulocytes and red blood cells (RBCs). The present study aimed to investigate
the transcriptional response of a subset of genes, whose functions are related to reticulocyte
metabolism, after autologous blood transfusion (ABT) using the digital multiplex mRNA
profiling.
Seven healthy male volunteers (age range, 20-35 years; body mass index, 18-30), that were
eligible for blood donation according to national regulations were included in the study. Details
regarding the clinical trial (NCT02423135) were previously described [2]. Briefly, during the
control phase, all volunteers were infused with saline solution. Fourteen days later, all volunteers
donated one full bag of blood (approximately 500 mL). The concentrated RBCs were stored at
~4C until re-infusion 36 days later. RNA expression was measured using Nanostring nCounter
Analysis System (Nanostring Technologies, Seattle, WA, USA). Gene expression analysis was
performed at baseline (D-4 and D-1) and after the infusion of 0.5 L of saline solution (0.9%
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American Journal of Hematology

NaCl, BBraun, Cressier, Switzerland) on day 3, 6, and 9 and after re-infusion of ones own blood
(0.28 L) on day 6, 9, and 15. Blood samples were drawn into Tempus Blood RNA tubes (Life
Technologies, Carlsbad, CA, USA) to stabilize genomic material and were stored at -20C until
further extraction. A one-way ANOVA followed by post-hoc pairwise comparisons (t-tests
adjusted by Bonferroni corrections and Tukeys Honestly Significant Difference) were used to
test differences between samples taken during saline or transfusion phase.
Based on the blood transcriptional signature of recombinant human erythropoietin [3], a
subset of 45 genes (following removal of the ten housekeeping genes) was selected for
evaluation of their expression following ABT. Agglomerative clustering (heat maps) resulted in
the identification 27 genes that were commonly down-regulated 6, 9, and 15 days after blood reinfusion in seven volunteers (S1 Table and S1 Fig). Delta-aminolevulinate synthase 2 (ALAS2),
carbonic anhydrase (CA1) and solute carrier family 4 member 1 (SLC4A1) were observed to have
the greatest fold-change following blood re-infusion than at baseline. A marked decrease in gene
expression was observed 6 days after ABT, although the number of transcripts for each gene was
significantly decreased 9 days, after re-infusion of blood (Fig 1A). At day 15, gene expression
remained low, and was not significant. During the control phase, the number of transcripts of the
candidate genes did not vary significantly 3, 6 and 9 days and stayed steady after saline infusion
(Fig 1B). Finally, the expression of the housekeeping genes (ACTB, ACTR10, MRFAP1, TBP,
TRAP1) remained constant throughout the phases, indicating that the changes observed in gene
expression are not cell count-based (S2 Fig). It suggests that the variations of gene expression
observed during the transfusion phase are specific to the re-infusion of blood.
Although they have shed their nucleus, circulating blood reticulocytes still retain
quantities of functional residual acid material which is essential for their maturation into

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erythrocyte [4]. These remaining copies contained in circulating reticulocytes are hypothesized
to reflect gene expression activity of erythroblast into bone marrow [5]. Erythroid precursors
express at their surface a receptor specific to EPO. When secreted upon hypoxia, EPO targets
developing erythroblasts and controls their differentiation and proliferation. It also contributes to
the release of reticulocytes through a diminution of the normal marrow-peripheral blood barrier.
Previously, Durussel and colleagues demonstrated that EPO injection influenced the gene
expression in human reticuloctytes [3]. As blood transfusion suppresses erythropoiesis through
the decrease of EPO concentration [1, 2], similar changes in mRNA expression specific to
reticulocytes were expected.
Our study demonstrates that the transfusion of autologous blood triggers a downregulation of genes that are involved in biological processes related to reticulocytes and RBCs.
ALAS2 is involved in the heme synthesis, whereas CA1 and SLC4A1 are responsible for the
transport of oxygen and carbon dioxide. Thus, these candidate genes are specific to
erythropoiesis. The magnitude of the changes of the genes transcripts was more important
compared to the small physiological effect of ABT on peripheral blood markers [2].
In anti-doping field, autologous blood transfusion is assessed by measuring
hematological parameters via the Athlete Biological Passport (ABP) which involves brittle
biological materials. It requires costly investments in the pre-analytical steps to ensure the
validity of the analyses [6]. To overcome actual challenges, our study proposed the inclusion of
transcriptomic biomarkers, whose sensitivity is greater than that of classical variables, into the
adaptive model of the ABP, coupled with easy-to-use collection blood tubes that stabilize
genomic material for up to 5 days at room temperature and for years when kept frozen. However,
before a potential integration these three innovative biomarkers into the adaptive model of the
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American Journal of Hematology

ABP, intrinsic and extrinsic factors that may affect the expression of these genes must also be
fully characterized.
In summary, our results demonstrate that autologous blood transfusion triggered a downregulation of genes whose function is linked to the metabolism of immature RBCs. Following
autologous infusion of stored RBCs, expression of the genes ALAS2, CA1 and SLC4A1 was
markedly decreased. The profiling of reticulocytes transcriptome offers a new clinical way for
the study of erythroid biology in response to blood transfusion. Futhermore, these transcriptomic
biomarkers may also serve for the detection of autologous blood transfusion in an anti-doping
context as they appeared to be more sensitive than classic hematological biomarkers.

Acknowledgements
The authors would like to thank the Genomic Technologies Facility at the University of
Lausanne for Nanostring analyses and their assistance for the understanding of nSolver software.
This study was financially supported by the World Anti-Doping Agency (WADA) (Grant No.
12C14NL) and the Dpartement Universitaire de Mdecine et Sant Communautaire (DUMSC)
(Grant No. 06/2015).

Conflict of interests
The authors declare that they have no conflicts of interest relevant to the manuscript submitted to
American Journal of Hematology.

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References
1.
Leuenberger N, Schumacher YO, Pradervand S, et al. Circulating microRNAs as
biomarkers for detection of autologous blood transfusion. PloS one 2013;8:e66309.
2.
Leuenberger N, Barras L, Nicoli R, et al. Hepcidin as a new biomarker for detecting
autologous blood transfusion. American journal of hematology 2016;91:467-472.
3.
Durussel J, Haile DW, Mooses K, et al. The blood transcriptional signature of
recombinant human erythropoietin administration and implications for anti-doping strategies.
Physiological genomics 2016:physiolgenomics 00108 02015.
4.
Lee E, Choi HS, Hwang JH, et al. The RNA in reticulocytes is not just debris: it is
necessary for the final stages of erythrocyte formation. Blood cells, molecules & diseases
2014;53:1-10.
5.
Goh SH, Josleyn M, Lee YT, et al. The human reticulocyte transcriptome. Physiological
genomics 2007;30:172-178.
6.
Robinson N, Giraud S, Schumacher YO, et al. Influence of transport and time on blood
variables commonly measured for the athlete biological passport. Drug testing and analysis
2016;8:199-207.

Figures legends
Fig 1. (A) Transfusion phase. Expression of ALAS2, CA1 and SLC4A1 before (D-4 and D-1)
and 6, 9, and 15 days (n=7) after autologous blood transfusion. The dashed line indicates
transfusion. Gene expression fold-changes were calculated on original/normalized mean data and
are annotated below the time points 6, 9, and 15 days post-transfusion relative to baseline (mean
of the data from 1 and 4 days pre-transfusion). Raw counts were normalized to internal levels of
five reference genes, ACTR10, ACTB, MRFAP1, TBP, and TRAP1. The Y axis represents log2transformed normalized counts of the genes.

P 0.05, statistically significant difference

compared with baseline values. (B) Saline phase. Expression of ALAS2, CA1 and SLC4A1
before (D-4 and D-1) and 3, 6, and 9 days (n=7) after saline infusion. The dashed line indicates
saline infusion. Raw counts were normalized to internal levels of five reference genes, ACTR10,
ACTB, MRFAP1, TBP, and TRAP1. The Y axis represents log2-transformed normalized counts
of the genes. No statistically significant difference was observed between time points.
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American Journal of Hematology

Supplementary data
S1 Fig. Difference of expression in the 45 genes tested before (baseline) and after
autologous blood transfusion. Heat map of changes in expression in the 45 genes.

S2 Fig. Expression of the housekeeping genes used for normalization during both phases.
(A) Transfusion phase (B) Saline phase

S1 Table. List of the 27 genes commonly down-regulated after autologous blood

transfusion.

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American Journal of Hematology

Fig 1. (A) Transfusion phase. Expression of ALAS2, CA1 and SLC4A1 before (D-4 and D-1) and 6, 9, and 15
days (n=7) after autologous blood transfusion. The dashed line indicates transfusion. Gene expression foldchanges were calculated on original/normalized mean data and are annotated below the time points 6, 9,
and 15 days post-transfusion relative to baseline (mean of the data from 1 and 4 days pre-transfusion). Raw
counts were normalized to internal levels of five reference genes, ACTR10, ACTB, MRFAP1, TBP, and TRAP1.
The Y axis represents log2-transformed normalized counts of the genes. # P 0.05, statistically significant
difference compared with baseline values. (B) Saline phase. Expression of ALAS2, CA1 and SLC4A1 before
(D-4 and D-1) and 3, 6, and 9 days (n=7) after saline infusion. The dashed line indicates saline infusion.
Raw counts were normalized to internal levels of five reference genes, ACTR10, ACTB, MRFAP1, TBP, and
TRAP1. The Y axis represents log2-transformed normalized counts of the genes. No statistically significant
difference was observed between time points.
Main document
254x190mm (300 x 300 DPI)

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