Received: 29 November 2020 Revised: 8 March 2021 Accepted: 9 March 2021

DOI: 10.1111/trf.16382

ORIGINAL RESEARCH

Pathogen reduction of whole blood: Supplementing

fibrinogen partly corrects clot formation in a massive
transfusion model

Ahmad F. Arbaeen1,2,3 | Peter Schubert1,2,4 | William P. Sheffield5,6 |

1,2,4
Dana V. Devine

1
Department of Pathology & Laboratory
Medicine, University of British Columbia, Abstract
Vancouver, British Columbia, Canada Background: The use of whole blood (WB) to treat trauma patients is
2
Centre for Blood Research, University of becoming more common. Similar to the treatment of individual compo-
British Columbia, Vancouver, British
nents, pathogen inactivation (PI) technologies are available to treat
Columbia, Canada
3
Faculty of Applied Medical Sciences,
WB. The impact of PI on WB function is not well understood. This study
Department Laboratory Medicine, Umm investigated the impact of PI of WB with riboflavin/ultraviolet (UV) light
al-Qura University, Makkah, Saudi Arabia on its hemostatic function by modeling transfusion scenarios for trauma
4
Centre for Innovation, Canadian Blood
patients and assessing transfusion efficacy by rotational thromboelastometry
Services, Vancouver, British Columbia,
Canada (ROTEM). As fibrinogen is affected by PI of WB, the effect of fibrinogen supple-
5
Centre for Innovation, Canadian Blood mentation commonly used in trauma patients was also analyzed in this model.
Services, Hamilton, Ontario, Canada Study Design and Methods: Trauma transfusion scenarios were simulated by
6
Department of Pathology and Molecular mixing untreated WB or WB treated with the Mirasol PI technology (riboflavin/
Medicine, McMaster University,
Hamilton, Ontario, Canada UV) in different ratios with hemodiluted blood, and the thromboelasticity was
monitored by ROTEM. The impact of supplementation with the fibrinogen con-
Correspondence
centrate RiaSTAP was investigated in this model.
Dana V. Devine, CBS – Centre for Blood
Research, University of British Columbia, Results: Pathogen-inactivated WB (PI-WB) showed decreased activity in
2350 Health Sciences Mall, Vancouver, the hemostatic profile compared to the untreated control. Hemodiluted
British Columbia V6T 1Z3 Canada.
blood at a hematocrit (hct) of 20%, which was reconstituted with PI-WB or
Email: dana.devine@blood.ca
untreated WB, exhibited increased alpha values, maximum clot firmness,
Funding information and clot formation time. Simulating transfusion scenarios by blood replace-
Burroughs Wellcome Fund, Grant/Award
ment with PI-WB resulted in a significant difference in ROTEM parameters
Number: 1012479; Canadian Blood
Services between reconstituted PI-treated and -untreated WB (p ≥ .05). The effect of
PI treatment waned when PI-WB was enriched with fibrinogen.
Conclusion: ROTEM investigations suggest that PI treatment has a negative
impact on WB clot formation unless fibrinogen supplementation is used.

KEYWORDS
fibrinogen, massive transfusion, pathogen inactivation technology, riboflavin and ultraviolet
light, rotational thromboelastometry (ROTEM), whole blood (WB)

Transfusion. 2021;1–10. wileyonlinelibrary.com/journal/trf © 2021 AABB 1

2 ARBAEEN ET AL.
1 | INTRODUCTION
Hemorrhage resulting from severe injury is among the
leading causes of death worldwide.1,2 Ideally, trauma
patients should receive early balanced transfusions of
blood components to restore hemostasis following mas-
sive bleeding.3–7 In a clinical setting, whole blood
(WB) might have superiority over the combination of
individual blood components8 as the recombination
approach results in a greater impact of anticoagulants
and additives, possibly leading to a higher chance for
coagulopathy. Component therapy also might be less
beneficial due to lower oxygen-carrying capacity com-
pared to fresh blood.9,10
However, hemostatic resuscitation with WB is being
used in military settings and in civilian medicine in some
Middle East countries and increasingly in hospitals in
developed countries, with the WB stored for up to 21 days
in the civilian setting for the treatment of massively
hemorrhaging patients and pediatric cases.11,12 This
potential new trend has been called out in a statement
from the US NIH Heart, Lung, and Blood Institute, mak-
ing this topic a research priority.13
The recent in vitro and in vivo studies on cold WB
have inspired physicians to reconsider WB transfusion for
patients with severe hemorrhage. It is more convenient in
a busy trauma setting to use one product to resuscitate a
bleeding patient rather than using multiple components.11
While it has been postulated that the negative effects of
cold temperature on the platelets in WB (4°C) could harm
the recipient, clinical studies indicate that there is no
increase in thrombosis level or adverse events when com-
pared to WB at 20°C.14,15 Moreover, resuscitation of
trauma patients with WB leads to exposure to one donor
per equivalent unit of blood instead of up to six donors in
the case of reconstituted WB from one red blood cell
(RBC) unit, a unit of WB-derived platelets pooled from
four donors, and a plasma unit. A recent study in pediatric
patients undergoing cardiac surgery concluded that the
fewer the number of donor exposures to the recipient, the
better the outcome for the trauma patient.16 In addition,
transfusion of WB stored at 4°C for 10–14 days to patients
with life-threatening bleeding would significantly lower
the logistic burden and both equipment-related and
staffing costs and extend the shelf-life compared to a
processed platelet concentrate for transfusion.11,17 How-
ever, some concern has been raised that a prolonged stor-
age time runs the risk of introducing infectious agents into
the patient, which would jeopardize blood safety.18
Several pathogen inactivation (PI) technologies have
been developed for application to plasma and platelet
concentrates (PCs),19 but the riboflavin and ultraviolet
(UV) light process (Mirasol PRT System Terumo BCT,
Lakewood, Colorado, USA) is the only PI system cur-
rently available for the treatment of WB units.20 This sys-
tem was reported as being an alternative to gamma
irradiation to prevent transfusion-associated graft-versus-
host disease,21 as well as effective in inactivating viruses,
bacteria, and parasites.20,22–25 Furthermore, during the
Coronavirus disease 2019 (COVID-19) pandemic, the PI
of COVID convalescent plasma has been advised by regu-
latory authorities.
Using a dose of 80 J/mlRBC of UV light, treatment of
WB with Mirasol results in an increase in RBC mean cor-
puscular volume (MCV), hemolysis levels, potassium
release, and microparticle production, in addition to
about 44% decreased activity of coagulation proteins; for
instance, fibrinogen levels decreased by as much as
30%.27,28 Studies also demonstrated that different RBC
additive solutions may overcome the negative impact of
the treatment. It was still suggested that WB cold storage
in conjunction with PI should only be performed for up
to 21 days to avoid compromising WB quality.24,25
Custer et al. concluded that pathogen-reduced WB
has a lower cost-effectiveness of quality-adjusted life-year
compared to current regulations of individual PI treat-
ment to plasma and PC in Canada.26 In vitro quality of
platelet concentrates from WB treated with PI seems to
be less negatively impacted than treatment of the PC
component.27 Previous studies assessing the in vitro qual-
ity of pathogen-inactivated WB (PI-treated WB) demon-
strated a reduction in fibrinogen activity.28,29 RiaSTAP
(CSL Behring) is a lyophilized purified fibrinogen con-
centrate made from human plasma administered to
patients with hypofibrinogenemia or afibrinogenemia.
For safety precautions, this product undergoes intensive
microbial inactivation and testing for hepatitis A, B, and
C; HIV-1/2; and parvovirus B19.30–32 In addition, it has
the potential for more rapid, safer, and predictable dosing
than cryoprecipitate and requires less time to prepare
than cryoprecipitate.30 It has been used to increase clot
firmness in patients with fibrinogen deficiencies.33 Cur-
rently, hypofibrinogenemic and afibrinogenemic patients
are given purified fibrinogen concentrates (RiaSTAP) at
doses of 70 mg/kg while monitoring clot firmness.34
In different clinical trials and studies, both ROTEM
and thromboelastography (TEG) technologies have been
able to identify ineffective blood components as a cause
for impaired clot strength.35,36 ROTEM was used success-
fully to assess the reconstituted hemodiluted blood.37
We aimed to determine whether ROTEM could be
used to test the effect of PI-treated WB in a trauma
model. We initially sought to establish whether ROTEM
could detect the effect on WB of riboflavin/UV light using
the hemostatic potential. To model actual transfusion
scenarios conducted in severe trauma cases, we used a
ARBAEEN ET AL. 3

slightly modified version of the transfusion model we hct of about 20%, a level chosen to reflect a realistic clini-
have previously reported.37 In this study, the hemostatic cal situation for severe hemorrhage.
function of PI-treated WB with hemodiluted fresh blood
to different hematocrit (hct) levels was assessed using
ROTEM. In addition, the impact of RiaSTAP supplemen- 2.2 | Pathogen reduction of WB and
tation of PI-treated WB was investigated in this model. plasma

Units of WB and plasma were illuminated according to

2 | MATERIALS AND METHODS
2.1 | WB unit collection and preparation
This study was approved by the research ethics board of
the Canadian Blood Services (CBS), and healthy volun-
teers gave informed consent. WB was collected at the
CBS netCAD facility (Vancouver, BC, Canada), and all
units were held overnight on butanediol cooling plates
(Fresenius-Kabi) for a minimum of 18 h. In addition,
plasma units were produced and stored at 4°C for up to
5 days. Small volumes of WB were collected from healthy
donors in citrated Vacutainer tubes (Becton Dickinson,
Franklin Lakes, NJ) immediately before conducting the
experiment. Hemodiluted blood was prepared by diluting
the citrated WB sample with 0.9% saline solution (pH 5.5;
Baxter Corp., Mississauga, Ontario, Canada) to obtain an
the manufacturer's instructions. PI was achieved with
riboflavin and UV light (Mirasol system, TerumoBCT,
Lakewood, CO) in which 35 ml of riboflavin solution
(500 μmol/L) was added to the WB or plasma prior to
illumination. Two ABO-matched units were pooled and
split, with one being PI-treated while the other one
received 35 ml of saline as a volume control (Figure 1).
Following the treatment, the contents of the illumination
bag were drained into the attached storage bag and tested
within 6 h or were retained at 4°C for 48 h.
2.3 | WB sampling and preparation for
hemostatic functionality
Units of WB and plasma were sampled aseptically in bio-
safety cabinets and the platelet, RBC count, and hct were

F I G U R E 1 Schematic overview of the study design. In study arm (A), two ABO-matched whole blood (WB) units were pooled and split
into two identical units (n = 24); all were kept on a cooling tray overnight (O/N). One unit was treated with riboflavin and ultraviolet (UV) light
(Mirasol), and one was kept untreated (Figure 2). In an arm (B), two ABO-matched plasma units were pooled and split into two identical units.
One unit was treated with riboflavin and UV light (Mirasol), and one was kept untreated (Figure 3). Units from study arm (A) were sampled as
pathogen inactivation (PI)-treated WB and untreated WB. Plasmas were obtained from these units and sampled as plasma from PI-treated WB
and plasma from untreated WB. Units from study arm B were sampled as PI-treated plasma and untreated plasma [Color figure can be viewed
at wileyonlinelibrary.com]
4 ARBAEEN ET AL.
obtained with a hematology analyzer (ADVIA 120, Sie-
mens, Mississauga, ON, Canada). Twenty-four units of
WB were used. Platelet-poor plasma (PPP) was prepared
from WB and plasma units to determine the coagulation
profile, as described earlier.37
2.4 | The preparation of the transfusion
model following illumination
The effect of PI treatment on WB was assessed by monitor-
ing the hemostatic profile in the trauma transfusion model.
One day after WB illumination, reconstitution was carried
out using ABO-matched hemodiluted blood samples in the
following ratios: (a) hemodiluted WB with untreated WB
unit and (b) hemodiluted WB with PI-treated WB units at
different ratios: 30% blood replacement achieved by com-
bining 70% hemodiluted WB and 30% WB resulting in
approximately 27.5% hct; 50% blood replacement achieved
by combining 50% hemodiluted WB and 50% WB resulting
in approximately 33.5% hct; and 70% blood replacement
achieved by combining 30% hemodiluted WB and 70% WB
resulting in approximately 37.5% hct. Eight independent
experiments were conducted on the entire series.
2.5 | The hemostatic profile generation
by ROTEM
ROTEM (Tem International GmbH, Munich, Germany)
was used to determine the hemostatic profile of the differ-
ent samples and the blood samples reconstituted with
hemodiluted blood and those enriched with RiaSTAP. Each
sample was incubated in a water bath at 37°C to mimic the
physiological temperature of human blood. A volume of
30 μl of 0.2 M CaCl2 was added to recalcify the samples
loaded into each ROTEM cup. Kaolin (Haemonetics Corp.,
Braintree, MA, USA) was used to initiate the contact activa-
tion pathway of coagulation as recommended by the manu-
facturer. Due to the interchangeable nature of TEG and
ROTEM results in the presence of kaolin, any differences
between ROTEM maximum clot firmness (MCF) and TEG
maximum amplitude MA disappeared.37
The mechanical and electronic calibration of each
ROTEM channel was checked before each study
according to the manufacturer's recommendations.
2.6 | Enriching the fibrinogen level
using the fibrinogen concentrate RiaSTAP
To assess whether additional fibrinogen has an impact on
clot firmness, RiaSTAP (CSL Behring GmbH, USA) was
added to both the control WB and the PI-treated WB, and
samples were reconstituted at a final concentration of
1 μg/μl. The MCF parameter was monitored to assess the
overall clot strength; the MCF is a reflection of fibrinogen
efficacy. The response of RiaSTAP to PI-treated WB with
or without reconstitution with hemodiluted blood was
determined and reported as the delta in the individual
MCFs. Eight independent experiments were performed.
2.7 | Statistical analysis
First, the normality of the distribution of the data was
tested using GraphPad 6 Prism software (GraphPad Soft-
ware, Inc., 2016, La Jolla, CA, USA). A statistical analysis
was performed using a one-way ANOVA to determine
the differences between PI-treated and control WB and
the delta of the MCF response to RiaSTAP, and the two-
way ANOVA was used to compare WB, hemodiluted
blood, and its replacement with different ratios of WB. In
cases where the Johnson transformation was not possi-
ble, nonparametric analyses were carried out using the
Kruskal-Wallis test on different WB samples before and
after their dilution with hemodiluted blood. Data were
reported as the mean and one standard deviation (±SD).
The Bonferroni correction was used to adjust the p-value
to account for multiple comparisons.
3 | RESULTS
3.1 | The hemostatic profile of PI-treated
WB versus control WB
The illumination of the WB samples showed different hemo-
static profiles, with clotting time (CT) and clot formation
time (CFT) increased significantly in the PI-treated WB com-
pared to the control WB, at 369.0 ± 49.6 versus 423.5 ± 35.8
(p < .01) and 112.5 ± 26.3 versus 175.3 ± 16.4 (p < .001),
respectively, using kaolin (n = 24; Figure 2(A),(B)). There
was a significant decrease in the rate of the fibrin–platelet
interaction observed expressed by the alpha value showing
58.3 ± 1.2 in the PI-treated WB versus 67.8 ± 2.3 in the con-
trol WB (Figure 2(C)). The MCF was reduced (p < .01) in
the PI-treated WB with 55.6 ± 3.1 mm compared to the con-
trol WB with 48.8 ± 2.9 mm (Figure 2(D)).
3.2 | The coagulation profile of
pathogen-inactivated plasma
It was crucial to determine the impact of the treatment
on the quality of PPP obtained from PI-treated WB and
ARBAEEN ET AL. 5

F I G U R E 2 Hemostatic profile
of pathogen inactivation (PI)-
treated WB versus control whole
blood (WB). Untreated and PI-
treated WB are displayed as white
and black bars, respectively.
Statistical analysis by one-way
analysis of variance, and (*)
represents a significant difference
between the two study arms
(p < .001). Results are displayed as
the mean ± SD of 24 replicates

F I G U R E 3 Hemostatic profile of pathogen-inactivated versus untreated plasma and whole blood (WB). Rotational thromboelastometry
profiles displaying plasma obtained from untreated and pathogen inactivation (PI)-treated WB (n = 24) in white and black bars and
untreated and PI-treated plasma (n = 12) in white and dark gray bars, respectively. Parameters monitored: CFT (Figure 2(A),(D)), fibrin–
platelet interaction (Figure 2(B),(E)), and MCF (Figure 2(C),(F)). Results are displayed as the mean ± 1 SD. Statistical analysis by one-way
analysis of variance, and (*) represents a significant difference (p < .001)

PI-treated plasma. The PPP obtained from PI-treated WB
showed a significant (p < .05) threefold prolongation in
the CFT compared to the PPP from the control WB
(Figure 3(A)). The rate of clot building decreased signifi-
cantly in the PPP obtained from PI-treated WB compared
to the PPP from the control WB (Figure 3(B)). The MCF
had a slight decrease, which did not achieve significance
(Figure 3(C)). Treating plasma units resulted in a nearly
fivefold increase (p < .001) in the CFT compared to that
of the control plasma unit (Figure 3(D)). The alpha and
MCF values decreased (p < .05) as seen in Figure 3
(E) and (F), respectively.
3.3 | Modeling the use of PI-treated WB
in the treatment of trauma
The control samples were collected from the WB of
healthy donors and hemodiluted with 0.9% normal saline
to an hct of 20%. The replacement of hemodiluted blood
6 ARBAEEN ET AL.

F I G U R E 4 In vitro simulation of hemostatic functionality in vivo following replacement of hemodiluted blood with pathogen
inactivation (PI)-treated whole blood (WB). Clot forming time (A), rate of fibrin–platelet interaction (B), and clot maximum amplitude (C) in
hemodiluted blood reconstituted with treated or control WB. The symbol (■) refers to normal WB before or after the hemodilution with an
approximate hematocrit level of 40% or 20%, respectively. The symbols () and (●) refer to the in vitro replacement of the hemodiluted blood
with the control WB or the PI-treated WB, respectively. The replacement was at three different concentrations: 30% blood replacement (70%
hemodiluted whole blood + 30% WB) “HCT ≈ 27.5%”, 50% blood replacement (50% hemodiluted whole blood + 50% WB) “HCT ≈ 33.5%”,
70% blood replacement (30% hemodiluted whole blood + 70% WB) “HCT ≈ 37.5%”. The hemostatic functionality of the WB unit alone is
indicated by (◊) for the control WB and (♦), for the PI-treated WB. *Significant difference between the two study arms (p < .01). Results are
displayed as means ± SD of eight replicates

in the model with PI-treated or nontreated WB was con-
ducted at different ratios to simulate a scenario of 30%,
50%, and 70% blood replacement. The PI-treated or non-
treated WB were tested separately but without the use of
kaolin (Figure 3). ROTEM traces of the PI-treated WB
were diminished compared to the control WB as demon-
strated by the reduction in the fibrin–platelet interaction
rate and the MCF and delays in the CFT, p < .01. The
CFT was 115.3 ± 17.7 s and 163 ± 17.3 s (Figure 4(A)),
and the rate of the fibrin–platelet interaction was 68.3
± 4.6 versus 59.1 ± 2.0 (Figure 4(B)), while the MCF was
61.6 ± 3.0 versus 55.5 ± 2.4 mm (Figure 4(C)).
To model the worst potential trauma scenario, PI-
treated or nontreated WB was used in subsequent experi-
ments. Replacing the hemodiluted blood with the PI-
treated WB at 50% or 70% resulted in a shortened CFT
(Figure 4(A)) and an increasing alpha (Figure 4(B)). The
ROTEM profile of 30% blood replacement with control
WB but not PI-treated WB showed superior procoagulant
activity when compared to the hemodiluted blood. The
overall effect of PI treatment on WB does not disappear
with 30%–70% blood replacement or when comparing
with nontreated WB (p < .05; Figure 4).
3.4 | Supplementation of PI-treated WB
with RiaSTAP following illumination
Based on the dose response curve of RiaSTAP on the
MCF of PI-treated WB (Figure 5), a final concentration of
1 μg/μl was chosen for the experiments to evaluate the
addition of fibrinogen on MCF moiety.
The addition of RiaSTAP generally resulted in signifi-
cant (p < .01) improvements in the clot strength, reported
as delta MCF between the supplemented and control
groups (Figure 6). The delta of the clot strength was 6.8
± 0.5 mm between the PI-treated WB and the control
(Figure 6, black bar) and decreased (p < .01) to 1.4
± 0.5 mm after introducing the RiaSTAP (Figure 6, light
gray bar). The overall response to RiaSTAP supplementa-
tion resulted in a decrease in the delta of the MCF
between the PI-treated and nontreated WB. There was no
significant (NS) difference between the ratios of blood
replacement with PI-treated WB and enriched with
RiaSTAP (Figure 6, dark gray bars) as the delta between
the two groups of WB with or without reconstitution with
hemodiluted blood.
4 | DISCUSSION
This study investigated whether PI impacts the hemo-
static profile of WB in an in vitro model of transfusion in
trauma patients using ROTEM. These data support the
previous observation that PI has a negative impact on the
hemostatic characteristics of WB, but strategies can be
developed to minimize this effect, such as altering the
storage of the WB following treatment, the amount of
blood volume replaced, or the compensation of appropri-
ate blood clotting factors.28 Our investigations have
ARBAEEN ET AL.
F I G U R E 5 The dose response curve to RiaSTAP. The dose
response curve of RiaSTAP on PI-treated WB showed slightly
increased maximum clot formation, n = 1
F I G U R E 6 Supplementation pathogen inactivation (PI)-
treated whole blood (WB) with RiaSTAP following treatment. WB
responsiveness to RiaSTAP, (*) indicates a significant difference in
the response to the addition of RiaSTAP of the treated WB when
compared to the respective control (p < .01). Results are displayed
as the mean ± SD of eight replicates. The addition of RiaSTAP to a
final concentration of 1 μg/μl resulted in a significant improvement
in clot strength, reported as MCF (p < .01). The delta of clot
strength was steady between different ratio of blood replacement
when the WB is PI-treated and enriched with RiaSTAP (NS)
focused on the Mirasol PI treatment as applied to
WB. Mirasol treatment lowered the hemostatic profile of
the WB samples, but the PI-treated WB sample demon-
strated potential efficacy for transfusions as suggested by
in vitro and in vivo assessments.29,38
The decreases observed in the CT, CFT, and alpha
value of the PI-treated WB and plasma units might have
resulted from a reduction in the residual coagulation fac-
tor activity, particularly in fibrinogen and factor FVIII,
which are major contributors to the coagulation pathway
and are reported to be affected by Mirasol treat-
ment.27,28,39 The Mirasol treatment also seems to impact
the αIIbβ3 integrin functionality, leading to an overall
reduction in thrombus formation. Furthermore, this
treatment increases the anaerobic metabolism rate,
α-degranulation, and phosphatidylserine exposure.40
Those changes can affect the ability of platelets to inter-
act with fibrinogen41,42 and cause changes in their
7
biochemistry that lower the thrombus formation rate as
reflected in the alpha value and MCF.
However, the results reported here demonstrated that
treatment of WB could be superior to the reconstitution
of WB from PI-treated plasma and PC. Using PI-treated
and leukoreduced WB to treat trauma patients could
enhance the procoagulability and accelerate hemostasis
for several reasons. It is obvious that coagulation factors
can better maintain their functionality when comparing
PPP obtained from PI-treated WB and plasma (Figure 3).
Therefore, coagulation factors in PI-treated WB but not
in PI-treated plasma have a higher potential to oppose
coagulopathy and reduce bleeding needs. Moreover, the
moderate microvesiculation of platelets and RBCs, in
addition to an increased resistance to shrinkage caused
by energy depletion, emphasizes that the comprehensive
treatment could be better than individual component
treatment.43
Nonetheless, no study has been conducted to date
involving PI-treated WB samples in patients with severe
hemorrhage; it is thus important to determine the impact
of PI on the quality of the WB used for massive hemor-
rhage. This study attempted to mimic as closely as possi-
ble the in vivo state by replacing hemodiluted blood, hct
of 20%, with PI-treated versus control WB prepared for
massive transfusion scenario. This model has diluted
coagulation factors, and that results in a decrease in clot
firmness and platelet–fibrin interaction.44,45
The evaluation of these simulated transfusion config-
urations by ROTEM demonstrated that the parameters
measured in the clot profile were influenced in this
model by the use of PI-treated WB. The ratio of blood
replacement affected the CFT, alpha value, and MCF of
the hemostatic test. In addition, Mirasol treatment
reduced the efficacy in the groups with 30% blood
replacement and higher. This large delta value in MCF of
7.8 mm (p < .01) present between the hemodiluted blood
reconstituted with PI-treated and nontreated WB could
be related to the decrease in activity of the major coagula-
tion factors in the treated WB.
Simulating trauma transfusion scenarios, these study
results suggest that hemostasis would be more com-
promised by PI-treated WB than it was in the previous
study when a reconstituted WB containing PI-treated
plasma and buffy coat-derived PC but not RBC was
used.37
These studies suggest that RiaSTAP or similar fibrino-
gen concentrates could be a useful supplement for WB
after PI with Mirasol as it is currently available to treat
patients with dysfunctional fibrinogen or reduced fibrino-
gen levels.46–48 The normal plasma fibrinogen level is in
the range of 2. 0–4.5 μg/μl,49 while the critical plasma
fibrinogen level below which hemorrhages can occur is
8 ARBAEEN ET AL.
approximately 1.0 μg/μl.50 Our lab has observed a 29%
reduction in the plasma fibrinogen (2.62 ± 0.20 to 1.85
± 0.14 μg/μl) following illumination.27 However, RiaSTAP
was added at a final concentration of 1 μg/μl to the Mirasol-
treated WB and compared with the control WB without the
addition of RiaSTAP. The delta value decreased significantly
between the WB enriched with RiaSTAP pre- and post-
treatment with Mirasol, which supported the hypothesis that
RiaSTAP could be used to correct fibrinogenemia post-
treatment with Mirasol. Surprisingly, the delta of MCF,
reflecting the supplementation with RiaSTAP in the PI-
treated WB and WB without treatment or RiaSTAP, was
steady at all dilution ratios with hemodiluted blood and
despite increased blood replacement ratio.
Several different PI techniques are currently on the
market for the treatment of PC or plasma. To date, only
the Mirasol technology has been applied to WB to miti-
gate pathogen contamination and avoid the challenge of
using treated blood components in every massively bleed-
ing patient, although other PI technologies are reportedly
in development. Although in vivo radiolabel and recovery
studies on PI-treated WB in animals have demonstrated
variable changes in in vitro quality, none of these
changes have translated into significant alterations in
posttransfusion WB variables.27,38,51 A clinical trial test-
ing human RBCs following WB treatment with Mirasol
concluded that the treatment maintained acceptable cell
quality at least through 21 days of storage (IMPROVE II;
Identifier NCT01907906).29
As with all models, this study has its limitations.
ROTEM does not entirely reflect vascular or extravascu-
lar contributions to clotting. It may not identify all
aspects associated with primary hemostasis or platelet
dysfunction. The dynamic effects of blood loss in wounds,
which increases with increasing clotting times, were not
measured in this model.
In addition, this study did not assess the impact of
riboflavin/UV light on the component function in treated
WB as this is already published. However, non-
leukoreduced WB was used in this study, and although
the PI technologies inactivate white blood cells (WBCs),
any impact of the remaining WBCs was not accounted
for. Subsequently, a leukoreduction step would remove
some platelets even if using a platelet-sparing filter, possi-
bly impacting the hemostatic potential.
Fibrinogen levels for hemodiluted blood or PI-treated
WB were not measured. Whether these ROTEM signa-
tures are predictive of the clinical use of PI-treated WB
and WB spiked with RiaSTAP remains to be determined
with clinical trials. The study reported here suggests that
if WB is superior to a balanced transfusion strategy (rec-
onstituted WB: RBCs, plasma, and PCs in a 1:1:1 ratio)
for trauma patients,9,52 the effects of Mirasol treatment
on clot firmness might be modified by coagulation factor
supplementation posttreatment.
Importantly, this study was performed without
leukoreduction of the WB, and while Mirasol will inacti-
vate leukocytes and prevent their proliferation, it is
unknown whether the inactivated WBC could affect the
quality of the ROTEM hemostatic test. Even with these
limitations, this study suggests a potential solution to the
apparent reduction in hemostatic capability of WB cau-
sed by treatment with Mirasol; the use of fibrinogen sup-
plementation appears to largely correct the Mirasol
impact on clot formation.
ACKNOWLEDGMENTS
This work was funded by a Burroughs Wellcome Fund
Innovation in Regulatory Science Award to DVD and
Canadian Blood Services through a grant from the Gov-
ernment of Canada. AFA was funded by the Faculty of
Applied Medical Sciences, Umm al-Qura University,
Makkah Al Mukarramah, Saudi Arabia
CONFLICT OF INTEREST
DVD has received grant support from TerumoBCT for
studies of Mirasol-treated blood products; however, this
project was not supported by those funds. No other
author reports any conflict of interest.
ORCID
Peter Schubert https://orcid.org/0000-0002-3310-6819
Dana V. Devine https://orcid.org/0000-0002-9059-0344
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How to cite this article: Arbaeen AF, Schubert P,
Sheffield WP, Devine DV. Pathogen reduction of
whole blood: Supplementing fibrinogen partly
corrects clot formation in a massive transfusion
model. Transfusion. 2021;1–10. https://doi.org/10.
1111/trf.16382