Effect of storage time and additive solution on post-transfusion clearance and metabolic recovery

Red blood cell (RBCs) transfusions are frequently administered to patients that
require increase in circulating blood oxygen delivery capacity to improve
tissue oxygenation. In most countries shelf-life of RBCs has been maximized to
35 - 42 days as blood products undergo changes referred to as the *storage
lesion* during aging. The *storage lesion* is characterized by RBC
vesiculation, membrane loss and lysis, reduced glutathione, cellular levels of
2,3-diphosphoglycerate, adenosine triphosphate and nitric oxide, decreased
membrane expression of CD47, and increased oxidation of cellular lipids and
proteins. These processes induce storage dependent decrease in pH, increase of
potassium and loss of hemoglobin. The *storage lesion* effects post-transfusion
survival and function. Therefore, the United States Food and Drug
Administration (FDA) requires submission of satisfactory in vitro biochemical
and hematological characteristics and in vivo recovery data before approval of
RBC systems. This includes tests of in vitro hemolysis, pH, glucose, lactate,
white blood cell concentrations, RBC morphology and other parameters of RBC
transfusion product quality. Manufacturers of RBCs are required to quantify
24-hour survival in healthy volunteers of their transfusion products. In at
least 20 or more 24-hour recoveries on average at least 75% of RBCs have to
survive to receive approval.

However, even though these products meet quality criteria in healthy
volunteers, several studies have reported <75% survival of RBCs after 24 hours
in patients. It is unclear which factors are implicated in clearance of these
transfused RBCs. Age of RBCs may influence this process: as RBCs age they
express enhanced levels of *eat me* signals, as for example phosphatidylserine
(PS); in a murine model stored RBCs were found to adhere to the vasculature due
to conformational changes of the RBC; red cell deformability of stored RBCs
impedes microcirculation; red cell deformability diminishes in stored blood and
a study investigating the relation between survival and storage time found
reduced recovery with aged RBCs. This study reported a mean 24 hour post
transfusion recovery of 86.4% in RBCs stored < 10 days. For RBCs stored 25-35
days the average survival after 24 hours was 73.5%.

Over the last decade an enormous impulse was given for research into the
storage lesion of red cells. Observational studies showed an association
between storage time of RBCs and mortality and morbidity in the recipients of
these products. Recently, results of several large RCTs were published, which
showed that standard stored RBCs (median 21 days) is not associated with higher
mortality and morbidity compared to fresh stored RBCs (<8days). An important
limitation of these studies is that previous studies showed an association
between maximum stored RBCs and the onset of transfusion-related morbidity and
mortality, but in the recent RCT studies the RBCs were stored for the standard
storage time and not maximum storage time. Furthermore, it is known that the
quality of RBCs products deteriorates during storage which is undesirable from
both the blood bank and bedside perspective.

In the past decade, several alkaline, chloride-free storage solutions have been
developed, based on the original work of Meryman et al. but all with different
compositions. In vitro there are some differences among the three solutions
(AS-7, E-Sol and PAGGGM), with PAGGGM (Sanquin development) showing the lowest
hemolysis after 35 days and the highest 2,3-DPG and ATP levels (results to be
published). AS-7 (also known as SOL-X) is already approved for red cell storage
in the US and both E-Sol and PAGGGM are available for research purposes. For
both AS-7 and PAGGGM, metabolomics results are published, but so far, these
findings are not confirmed with enzyme activity tests. Moreover, very little is
known about the recovery of metabolism after transfusion of the stored red
cells. Insight in the mechanism of the RBC storage lesion may result in
development of improved storage conditions, which in turn results in improved
product quality with decreased side-effects of transfusion, and/or the
possibility to store products longer than the current standard.

RBC clearance and metabolic recovery after transfusion can be investigate with
biotin labelling of RBCs. This method has proven to be safe in healthy
volunteers study completed in the AMC. Biotin labelling facilitates infusion
of several populations of RBCs by labelling them with separate concentrations
of biotin.

With this study we will use a healthy volunteer transfusion model of biotin
labelled RBCs to investigate the effect of 1) maximum storage time and 2)
different RBC storage solutions on the clearance of transfused RBCs and
metabolic recovery of transfused RBCs.


The advantages of such a model are the following;
1) an autologous transfusion model makes it possible to investigate the effect
of storage time on the clearance of RBCs;
2) transfusion of both 2 days autologous stored RBCs and 35 days autologous
stored RBCs in one volunteer enhances the power of the study as volunteers
serve as their own control and eliminated inter-volunteer confounding;
3) biotin labelling makes detailed clearance studies possible and makes it
possible to isolate transfused cells from the receiver*s circulation and
subject them to additional investigations.

1) Compare post-transfusion RBC clearance of fresh and stored RBCs and
investigate whether glycolytic enzyme function, glycolysis, pentose phosphate
pathway function and related metabolic pathways recover after transfusion of a
35 day stored RBC product in humans.
2) Investigate whether glycolysis (G6PD and other enzymes) and energy
metabolism are better preserved during storage in PAGGGM or a similar alkaline
additive solution compared to storage in SAGM.
3) Compare post-transfusion RBC clearance of RBCs stored in PAGGGM or a similar
alkaline additive solution to RBCs stored in SAGM and investigate whether
post-transfusion RBC clearance, glycolytic enzymes function and metabolites of
glycolysis, pentose phosphate pathway and related metabolic pathways recovery
after transfusion of a 35 day stored RBC product in humans is improved in RBCs
stored in PAGGGM or a similar alkaline additive solution compared to SAGM.

Study type: Open label randomized intervention trial.
Subjects: Healthy volunteers.

Methods:

Screening and RBC donation:
All subjects will be screened (medical history, physical examination, ECG,
blood and urine examination) by the research physician of our hospital and
must meet Sanquin Blood Bank Donor Criteria prior to involvement in the
experiment (Screening Phase). Twenty healthy volunteers, aged 18-35, will be
randomized to one of two groups and donate one unit of full blood 35 days
before the study day at Sanquin Blood Bank. A second donation of 200 ml
(miniature whole blood donation processed with a whole blood leukodepletion
filter) will be completed 2 days before the study day. Before the study day
volunteers screening and blood donation will result in collection of
approximately 735 ml of whole blood in three sessions (screening, donation 35
days prior to the study day, dondation 2 days prior to the study day). This
equals to 15% of circulating volume of 175 cm tall healthy males and is
according to Sanquin Blood Bank standards. However, volunteers will receive
this donated blood during the study day which results in a small netto loss of
blood. Processing and storage of donated blood will be according to Sanquin
Blood Bank (SBB) protocol in either SAGM or a new alkaline solution like
PAGGGM. During storage blood products will be sampled weekly with a sterile
coupler. These samples will be stored until further analysis.

Randomization groups:
Group 1a: Standard RBCs in SAGM - 2 days stored RBCs labelled with high density
biotin, 35 days stored RBCs labelled with low density biotin
Group 1b: Standard RBCs in SAGM - 2 days stored RBCs labelled with low density
biotin, 35 days stored RBCs labelled with high density biotin
Group 2a: RBCs stored in alkaline addative solution - 2 days stored RBCs
labelled with high density biotin, 35 days stored RBCs labelled with low
density biotin
Group 2b: RBCs stored in alkaline addative solution - 2 days stored RBCs
labelled with low density biotin, 35 days stored RBCs labelled with high
density biotin

Study day:
Prior to the study day autologous 2 days (*fresh*) and 35 days (*stored) RBCs,
will be labelled with two different densities of biotin (Vitamin B8).25,26
Preparation will be done under sterile conditions. To exclude any effect of
biotin label concentration half of each group will receive fresh RBCs labelled
with a low biotin concentration and stored RBCs labelled with a high
concentration. The other half will receive fresh RBCs labelled with a high
concentration biotin and stored RBCs labelled with a low concentration of
biotin. Cultures will be taken of labelled products to confirm sterile
conditions. Detection of the biotinylated RBCs can then be performed in blood
samples taken after transfusion, after staining with streptavidin-FITC by flow
cytometry. Subsequently on the study day at the AMC healthy volunteers will
first receive a indocyanine green infusion. This will be used to calculate the
volunteers circulating volume according to previously published protocols and
is required to detect clearance of transfused RBCs directly after infusion.29
After collection of samples for calculation of circulating volume, the
volunteers will receive autologous transfusion of the full unit of fresh and
stored biotin labelled RBCs (BioRBCs). Blood samples will be drawn from an
indwelling venous canula prior to indocyanine green infusion, 5, 10 and 20
minutes after indocyanine green infusion, directly before transfusion and 10
minutes, 0.5, 1, 2, 4, 6, 8 and 24 hours and 2, 7, 30 and 90 days after
transfusion. In the course of the study day, in total 135 ml blood will be
drawn to obtain the required volume to calculate circulating volume and to
detect and sort the two populations of transfused RBCs with flow cytometry.
Metabolomics will be performed on the sorted populations and on samples of
stored blood products, in combination with measurement of the activity of
selected glycolytic enzymes, to investigate the effect of storage on
enzyme-activity in the stored RBC and the effect of transfusion on the recovery
of the stored cell.

Post study day follow-up
Twenty-four and forty-eight hours after transfusion volunteers will return to
the AMC for an additional blood sample (12 ml). Seven days, 30 and 90 days
after transfusion a venous blood sample (12 ml) will be collected to monitor
RBC clearance and to detect development of biotin antibodies (AMC Visit 4 and
5). This data will be used to monitor antibody prevalence after exposure to
biotin. Antibodies will be tested with a IgG gel card test (Ortho Clinical
Diagnostics, MTS* Anti-IgG Card).

Screening
All subjects, healthy volunteers aged 18-35 will be screened at the AMC and at
Sanquin.
Investigation of the medical history, a physical examination, ECG and blood and
urine examination will be performed to determine volunteer eligibility to
participate in the study. Volunteers will be excluded if they have any
abnormalities at the screening, if they use any medication on doctor*s
prescription, if they have lost >500 ml blood in the past 3 months, regardless
of the cause and if they participate in another randomized trial during the
course of our study. If the volunteers are found to have no exclusion criteria
to participate in the study, they will be enrolled into the study.

Study
All included healthy volunteers (n=20) will donate 1 unit of whole blood at
Sanquin Blood Bank which will be processed into 1 unit of RBCs (approximately
300ml) 35 days before the experiment. Processing and storage will be according
to Sanquin Blood Bank protocol and products will be stored in either SAGM or an
alkaline storage solution like PAGGGM, AS-7or E-Sol (to be selected). Every
week, a sample will be collected from these products with a sterile coupler.
Two days before the experiment healthy volunteers will donate a second smaller
blood product (approximately 200 ml; miniature whole blood donation processed
with a whole blood leukodepletion filter; validated Sanquin product) which will
be processed into a small RBC concentrate. Both donations will be labelled with
biotin, using two densities of biotin according to previously published
protocols. To exclude any effect of biotin label concentration half of each
group will receive fresh RBCs labelled with a low biotin concentration and
stored RBCs labelled with a high concentration. The other half will receive
fresh RBCs labelled with a high concentration biotin and stored RBCs labelled
with a low concentration of biotin.

Before the study day volunteers screening and blood donation will result in
collection of approximately 735 ml of whole blood in three sessions (screening,
donation 35 days prior to the study day, dondation 2 days prior to the study
day). This equals to 15% of circulating volume of 175 cm tall healthy males and
is according to Sanquin Blood Bank standards. The collected RBCs will be used
to produce an autologous transfusion product and volunteers will receive their
own RBCs back at the study day. This results in a small netto loss of blood.

Two days after the second smaller donation subjects will be admitted to the
medium care of the AMC where they first will receive indocyanine green to
calculate circulating volume, followed by the autologous biotinylated RBCs
stored for 2D and 35D. At the study day, blood samples will be drawn from an
indwelling venous canula prior to indocyanine green infusion, 5, 10 and 20 min
after infusion, prior to the transfusion and 10 minutes, 0.5, 1, 2, 4, 6 and
8 hours after transfusions. Volunteers will return to the AMC 24 hours after
transfusion and 2, 7, 30 and 90 days later for follow-up samples. These samples
will be used to detect and sort the two populations of transfused RBCs with
flow cytometry.

Indocyanine green will be used to calculate circulating volume. This volume is
required to be able to detect any immediate clearance of transfused BioRBCs
direct after transfusion. This clearance cannot be detected without an
estimation of circulating volume on forehand.29 The counts of BioRBCs in the
circulation will be used to measure post-transfusion clearance. In cooperation
with a laboratory in Denver Colorado, USA, with staff trained in metabolomics,
the energy metabolism of all sorted BioRBC populations and samples of stored
blood products will be investigated. These analyses will be combined with
direct enzymatic measurements (spectrophotometry and cytofluorometry) of
selected enzymes in these samples, for example glucose-6-phosphate
dehydrogenase (G6PD). Thus we will be able to investigate the effect of storage
on enzyme-activity in the stored RBC and the effect of transfusion on the
metabolic recovery of the stored cell.

During the experiment subjects will be monitored for heart rate and blood
pressure by non-invasive continuous monitoring at the ICU.

One, two, seven, 30 and 90 days after transfusion a venous blood sample (12 ml)
will be collected to monitor RBC clearance and to detect development of biotin
antibodies. This data will be used to monitor antibody prevalence after
exposure to biotin. Antibodies will be tested with a IgG gel card test (Ortho
Clinical Diagnostics, MTS* Anti-IgG Card).

Benefits: none.
We recently showed that transfusion of one unit autologous aged biotin labelled
Red Blood Cells (RBCs) (METC protocol 2012-299) is safe in healthy volunteers
suffering endotoxemia. Transfusions will be prepared and transfused using the
standard clinical protocols by Sanquin and our hospital. Our study requires
donation of a standard full blood donation. We therefore feel that this study
has a lower volunteer burden and volunteer risk than our previous study.

Prior to transfusion stored RBCs will be biotinylated to allow their
identification with flow cytometry. In vivo and in vitro testing of
biotinylated RBCs and PLTs showed that survival and recovery of RBCs was not
affected by biotinylation. Although in a healthy volunteer study on
biotinylated RBCs 1 out of 8 subjects developed a transient positive test for
antibody to biotin, at 11 months post transfusion antibodies to biotin had
disappeared. The survival of RBCs was not altered noticeably in this subject by
the presence of this antibody; this is in line with previous reports. Three
months after the study day a last blood sample will be drawn to detect
development of biotin antibodies. Although the presence of absence of
antibodies had no clinical relevance and repeated intravenous exposure to
biotin did not produce adverse effects in previous studies, this data can be
used to monitor antibody prevalence after exposure to biotin. In our recently
completed study in healthy volunteers who received biotin-labelled RBCs, none
of the volunteers developed biotin antibodies (unpublished data).

Volunteers will donate approximately 735 ml of whole blood in three sessions
(screening, donation 35 days prior to the study day, dondation 2 days prior to
the study day) before the study day. This equals to 15% of circulating volume
of 175 cm tall healthy males which should not result in any adverse effects and
is according to Sanquin Blood Bank standards. However, as these donations are
not standard care at Sanquin, the Sanquin Medical Ethical Board is also
required to approve the study protocol. During the study day volunteers receive
1.5 autologous transfusion which negates any effect of whole blood collection
before the study day.