BRIEF REPORT

Hemostatic profile and safety of pooled cryoprecipitate

up to 120 hours after thawing

Parvez M. Lokhandwala,1 Adrian O’Neal,1 Eshan U. Patel,1 Patricia A. R. Brunker,1,2

Eric A. Gehrie,1 Gang Zheng,1 Thomas S. Kickler,1 Paul M. Ness,1 and Aaron A. R. Tobian1

BACKGROUND: AABB standards state that
cryoprecipitate should be transfused within 4 to 6 hours
after thawing. We evaluated coagulation factor levels and
sterility of thawed pooled cryoprecipitate to assess
whether shelf life can be safely extended.
STUDY DESIGN AND METHODS: Donor
cryoprecipitate pools (n 5 20, 10 group A, 10 group O)
were held at ambient temperature and sampled at 0, 4, 8,
24, 48, 72, 96, and 120 hours post-thawing for fibrinogen,
Factor (F)VIII, and von Willebrand factor (vWF) levels.
Samples were tested at 0 and 120 hours for sterility
(BacT/Alert system). Sixty additional cryoprecipitate
pools were evaluated after 72 hours. Longitudinal
differences in component levels were determined by
linear fixed-effects regression.
RESULTS: Group O cryoprecipitate had significantly
lower FVIII (p 5 0.002) and vWF activity (p 5 0.006)
compared to group A at 0 hours, but were not statistically
different in fibrinogen levels (p 5 0.33). Fibrinogen levels
were stable over 5 days: 501 6 81 mg/unit
(mean 6 standard deviation) at 0 hours to 506 6 102 mg/
unit at 120 hours (p 5 0.73). Similarly, there was no
decline in vWF activity: 200 6 53 IU/unit at 0 hours to
209 6 57 IU/unit at 120 hours (p 5 0.084). The FVIII
activity significantly declined on average by 9.6 IU (95%
confidence interval, 5.5-13.8) between 0 hours (111 6 33
IU/unit) and 120 hours post-thaw (101 6 33) (p < 0.001).
No organisms were detected when cryoprecipitate pools
were cultured at 0 hours, but at 120 hours
Staphylococcus epidermidis was identified from one
pool, potentially a contaminant introduced during
repeated sampling. No cultures were positive among the
60 additional cryoprecipitate pools assessed at 72 hours.
CONCLUSION: Extended cryoprecipitate storage at
ambient temperature did not affect fibrinogen levels over
120 hours. Sterility of products held at ambient
temperature for an extended period of time could be
assessed by secondary culture.
C
ryoprecipitated antihemophilic factor, or
cryoprecipitate, is the insoluble precipitate formed
when fresh frozen plasma (FFP) is thawed at 1
to 68C. This product is enriched in Factor
(F)VIII, fibrinogen, von Willebrand factor (vWF), FXIII,
and fibronectin.1-3 Cryoprecipitate is primarily used for
replacement of fibrinogen in acquired hypofibrinogenemia,4
during major bleeding due to either consumptive or
dilutional coagulopathy,5-7 and to control bleeding in
patients with uremia.8
AABB standards require that each unit of cryoprecipi-
tate contain a minimum of 150 mg of fibrinogen and 80
IU of FVIII; cryoprecipitate that is pooled prestorage is
required to contain at least 150 mg of fibrinogen and 80
IU of FVIII multiplied by the number of units in the pool.9
AABB standards also state that cryoprecipitate must be
used within 6 hours after thawing for single units or units
that were pooled using a sterile connector, and within 4
hours after thawing for units that were pooled in an open
system.9 Because of this short mandated shelf life, cryo-
precipitate is thawed at most institutions only after it has
been requested, that is, on an “as needed basis.” This
results in a critical delay in the administration of cryopre-
cipitate in acutely bleeding patients, which puts patient
safety at risk. Unused thawed cryoprecipitate is often dis-
carded, resulting in wastage of a donated blood product
and a significant monetary loss to the organization.
From the 1Department of Pathology, Johns Hopkins University
School of Medicine; and the 2Biomedical Services, Greater
Chesapeake and Potomac Region, The American Red Cross,
Baltimore, Maryland.
Address reprint requests to: Parvez M. Lokhandwala, MD,
PhD, Department of Pathology, Johns Hopkins University
School of Medicine, 550 North Broadway Building, Suite 810,
Baltimore, MD 21205; e-mail: plokhan1@jhmi.edu.
Received for publication November 10, 2017; revision
received January 9, 2018; and accepted January 9, 2018.
doi:10.1111/trf.14550
C 2018 AABB
V
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Volume 00, February 2018 TRANSFUSION 1

LOKHANDWALA ET AL.
Two prior studies have evaluated the hemostatic
properties of thawed cryoprecipitate held at ambient tem-
perature for an extended period of time. A study per-
formed in Canada tested cryoprecipitate units up to 24
hours after thawing and showed adequate coagulation
factors in pooled and single units.10 Another study, per-
formed in the United Kingdom, showed that a pool of cry-
oprecipitate retained its hemostatic ability, based on
UK standards, for as long as 72 hours after thawing.11
Due to methodological differences in cryoprecipitate
manufacturing, pooling, and storage in different countries,
it is important to validate these data at a national level.
If one could demonstrate that thawed cryoprecipitate
retains its coagulation factor activity and is not at
increased risk of bacterial contamination with prolonged
storage, it could greatly benefit patient care. Extending the
outdate would allow blood banks to keep thawed cryopre-
cipitate ready to be supplied on demand, reducing turn-
around times and wastage.
This study investigated the hemostatic profile, as well
as bacterial growth, in pooled cryoprecipitate manufac-
tured in the United States after storage for up to 5 days at
ambient temperature.
MATERIALS AND METHODS
Study design
Twenty cryoprecipitate pools, 10 group O and 10 group A,
were obtained from the regional blood collection center.
The regional blood center manufactured cryoprecipitate
from platelet-rich plasma that was separated from whole
blood collected in citrate-phosphate-dextrose. The
platelet-rich plasma was frozen in a blast freezer at
2508C. The frozen platelet-rich plasma was thawed over-
night at 48C in a temperature-monitored cold room. The
cryoprecipitate was manufactured by centrifuging thawed
plasma at 48C and resuspending the precipitate in approx-
imately 25 mL of residual plasma. Five ABO-identical indi-
vidual cryoprecipitate units were pooled together in a
closed system using a sterile connector, which was refro-
zen and stored at less than 2188C.
Each pool contained a volume of 129 6 6 mL (mean 6
standard deviation). The pooled cryoprecipitate was
thawed in a (plasma thawer, Helmer Scientific) at 378C 6
28C for 25 minutes and held at ambient temperature for 5
days. Ambient temperature of 20 to 248C was continu-
ously monitored using an (Isensix Advanced Remote
Monitoring System, ISENSIX).
Samples (approx. 2 mL) for coagulation testing were
collected at 0, 4, 8, 24, 48, 72, 96, and 120 hours in a non-
coated, no additive tube (Becton-Dickinson) and refrozen
at 2808C until testing was performed in batches. Addition-
ally, 8-mL samples for bacterial cultures were collected at
0 hours and again at 120 hours to detect any bacterial
2 TRANSFUSION Volume 00, February 2018
growth. All samples were collected using a (SampLok
Sampling Kit, ITL Biomedical).
Testing of coagulation factors
All coagulation testing was performed on the (BCS XP
platform, Siemens Medical Solutions USA, Inc.) in a Clini-
cal Laboratory Improvement Amendments-certified labo-
ratory using an assay that has been validated for reporting
of clinical test results. Because of the anticipated high
concentrations of the coagulation factors in cryoprecipi-
tate samples, each sample was diluted 1:5 prior to testing.
The raw results were multiplied by 5 (the dilution factor)
and converted to either activity or concentration/individ-
ual cryoprecipitate unit. Fibrinogen was measured using a
modified Clauss method. The samples were diluted 1:5
with saline prior to testing. The diluted sample was then
mixed with (Multifibren U reagent), and time to clot for-
mation was detected. Factor VIII was measured using a
one-stage activated partial thromboplastin time–based
assay. The cryoprecipitate sample was diluted 1:5 with
FVIII deficient plasma. An activated partial thromboplas-
tin time test was performed on the diluted cryoprecipitate
samples using (Actin FSL reagent) and calcium chloride
reagent. The percentage of activity was read from a stan-
dardized curve generated by the BCS XP instrument using
standard human plasma. vWF activity was measured
using the ristocetin cofactor activity assay by mixing the
diluted (with saline) cryoprecipitate samples with BC von
Willebrand reagent and measuring the ability to aggluti-
nate platelets in the presence of ristocetin.
Bacterial culture
An 8-mL aliquot was collected into an aerobic (BacT/Alert
BPA blood culture bottle) at 0 and 120 hours, which was
incubated in the (BacT/Alert system, BioMerieux, Inc.) for
5 days. An additional 60 cryoprecipitate pools were sam-
pled at 72 hours.
Statistical analysis
A two-tailed unpaired t test with unequal variance was
used to determine if the fibrinogen, FVIII, and vWF mean
levels were significantly different in group O versus group
A cryoprecipitate pools at 0 hours. A linear fixed-effects
(within) regression model was used to determine if the
fibrinogen, FVIII, and vWF levels significantly decreased
over time.12,13 This type of longitudinal model accounts
for the autocorrelation caused by repeated measures. The
Hausman test for endogeneity further indicated random
effects were not needed. A two-tailed p value less than
0.05 was considered significant. Statistical testing was
done using Stata SE 14.2. (StataCorp). Graphpad Prism
version 7 (GraphPad Software, Inc.) was used to generate
graphs.

Fig. 1. Coagulation factor levels in group O and group A cryo-
precipitate at 0 hours. (A) Fibrinogen levels (mg/unit), dotted
line (150 mg/unit) designates the AABB standard requirement.
(B) FVIII activity (IU/unit), dotted line (80 IU/unit) designates
the AABB standard requirement. (C) vWF activity (% activity/
unit). Symbols and error bars represent data mean and stan-
dard deviation respectively. *Significant difference between
group O and group A CRYO is designated as p < 0.05.
RESULTS
Comparison between group O and group
A cryoprecipitate immediately after thawing
There was no significant difference in fibrinogen levels in
the group O cryoprecipitate (519 6 100 mg/unit) (mean 6
THAWED POOLED CRYOPRECIPITATE SHELF LIFE
standard deviation) versus group A cryoprecipitate (481 6
77 mg/unit) (p 5 0.33), at 0 hours (i.e., immediately after
thawing) (Fig. 1). The FVIII activity in group O cryopreci-
pitate (90 6 22.5 IU/unit) was 42.6 IU/unit (95% confi-
dence interval [CI], 18.4-66.9 IU/unit) lower than the
group A cryoprecipitate (132 6 28.5 IU/unit) (p 5 0.002),
at 0 hours. The vWF activity in group O cryoprecipitate
(168 6 23% activity/unit) was lower than the group A cryo-
precipitate (232 6 56% activity/unit) by 63.8% activity/
unit (95% CI, 22.2-105.5) (p 5 0.006).
Coagulation factor levels in pooled cryoprecipitate
up to 120 hours after thawing
There was no decline in the fibrinogen levels after 120
hours (506 6 102 mg/unit) at ambient temperature com-
pared to 0 hours (501 6 89 mg/unit) (p 5 0.723) (Fig. 2).
FVIII levels decreased by approximately 9.6 IU (95% CI, 5.5-
13.8) over the 5-day period, from a mean of 111 6 33 IU/
unit at 0 hours to 101 6 33 IU/unit at 120 hours (p < 0.001).
Of the pools tested, 75% of products (15 of 20) met the
AABB required standard for FVIII of 80 IU/unit at the time
of thawing, which decreased to 60% at 120 hours. All group
A cryoprecipitate pools met the AABB required standard for
FVIII of 80 IU/unit at 0 hours, declining to 80% of cryopre-
cipitate pools meeting the standard at 120 hours. In con-
trast, 50% of the group O cryoprecipitate pools met the
standard requirement of 80 IU/mL at 0 hours, which
decreased to 40% of cryoprecipitate pools meeting the
standards at 120 hours.
There was no significant change in the vWF activity
levels after 120 hours (209 6 57% activity/unit) compared
to 0 hours (200 6 53% activity/unit) (p 5 0.084).
Sterility of thawed cryoprecipitate held at ambient
temperature
All 20 aerobic cultures that were sampled at 0 hours had no
organisms detected. After 120 hours of storage at ambient
temperature, one out of the 20 cryoprecipitate pools (5%)
cultured grew bacteria after approximately 10 hours of
incubation in the BacT/Alert system instrument. The
organism was identified as Staphylococcus epidermidis and
confirmed with repeat culture.
Sixty additional pools of cryoprecipitate cultured after
being held for 72 hours at ambient temperature did not
detect any organisms.
DISCUSSION
In severe trauma, early administration of fibrinogen may
help control bleeding, improve coagulopathy, and reduce
transfusion requirements.14-16 The current practice in the
United States, Canada, and the United Kingdom is to
transfuse cryoprecipitate within 4 to 6 hours of thaw-
ing,3,9,17,18 which makes it difficult to maintain an
Volume 00, February 2018 TRANSFUSION 3
LOKHANDWALA ET AL.
Fig. 2. Box and whisker plot of coagulation factor levels in
thawed pooled cryoprecipitate over time. (A) Fibrinogen
(mg/unit); dotted line (150 mg/unit) designates the AABB
standard requirement. (B) FVIII (IU/unit) over time post-
thawing; dotted line (80 IU/unit) designates the AABB stan-
dard requirement. (C) vWF (% activity/unit). Horizontal bar,
box, and whisker represent data mean, standard deviation,
and range, respectively. Significant difference compared to 0
hours is designated as p < 0.05.
inventory of prethawed cryoprecipitate in the blood bank.
This leads to a critical delay in the administration of cryo-
precipitate in severe trauma settings and increases wast-
age. Consequently, lyophilized fibrinogen concentrate,
which has been approved for the indication of congenital
4 TRANSFUSION Volume 00, February 2018
fibrinogen deficiency, is gaining popularity in trauma set-
tings primarily because of the ability to resuspend and
administer quickly. Compared to cryoprecipitate, however,
lyophilized fibrinogen concentrate is very expensive and
lacks other coagulation factors (FVIII, vWF, and FXIII). A
clinical trial comparing the feasibility and efficacy of
fibrinogen concentrate and cryoprecipitate to replace
fibrinogen early in severe trauma is ongoing.19
Our study demonstrated the stability of cryoprecipi-
tate for up to 5 days post-thawing and is the first study to
test for the sterility of cryoprecipitate held for an extended
period of time. Our study shows that even after 5 days of
storage, fibrinogen levels remain stable, and all cryopreci-
pitate pools meet the AABB requirements of 150 mg/unit.
In contrast, FVIII levels showed a small (approx. 10%) but
appreciable decline in both Group A and Group O cryo-
precipitate, similar to a prior study.11 FVIII activity shows
a greater decline in thawed plasma upon storage at 1 to
68C for 5 days (nearly 50% decline).10 A greater decline in
FVIII levels at 1 to 68C compared to ambient temperature
has been postulated to be due to additional precipitation
of coagulation factors.11,20 The requirement to meet the
AABB threshold of 80 IU/unit is primarily a concern with
Group O cryoprecipitate because of lower concentration
of FVIII in Group O compared to Group A. AABB stand-
ards do not specify the blood group of the cryoprecipitate
to be tested. Therefore, while not all cryoprecipitate units
meet AABB standards for FVIII after 120 hours of storage,
this is less of a concern because cryoprecipitate is no lon-
ger routinely transfused for FVIII replacement in the
United States. There was no significant decline in the vWF
activity levels after 5 days.
There are limitations to this study. Our study did not
investigate single cryoprecipitate units; however, a prior
study has shown an equivalency between single and
pooled cryoprecipitate for up to 24 hours.10 Consequently,
we would predict that single cryoprecipitate units like
pooled cryoprecipitate units will have stable coagulation
factors for up to 5 days.
A major patient safety concern with extending shelf
life of cryoprecipitate, which is held at ambient tempera-
ture, is the potential growth of bacteria and the risk of sep-
tic transfusion reactions. The risk of septic transfusion
reactions to products that have been frozen is extremely
rare,21 since a small amount of bacteria in a product that
has previously cleared infectious disease testing at the
time of collection are unlikely to survive the freeze-thaw
process. However, bacteria introduced into thawed cryo-
precipitate can continue to proliferate when stored for
longer than 4 hours.22 In our study, one of the 20 pools of
cryoprecipitate grew a positive culture, which was speci-
ated as S. epidermidis. S. epidermidis is primarily a skin
contaminant. Although a sterile connecting device was
utilized for sampling, we speculate that bacteria may have
been introduced into the cryoprecipitate pool during

repeated sampling for coagulation testing. Another poten-
tial source of contamination is the water bath used for the
thawing process.
Platelets are the only other blood product held at
ambient temperature prior to transfusion. Per the Food
and Drug Administration’s draft guidance for the industry,
bacterial contamination in platelets is primarily a concern
with transfusion of Day 4 and Day 5 stored platelets.23,24
Similarly, cryoprecipitate stored at ambient temperature
for an extended period of time might have a potential for
bacterial growth after Day 3. Sixty additional cryoprecipi-
tate pools held for 72 hours at ambient temperature with-
out interim sampling did not culture any organisms,
suggesting that cryoprecipitate is safe for the first 72 hours
of storage.
There are two possible interventions to ensure steril-
ity of the extended-shelf-life cryoprecipitate product. The
first is the use of pathogen reduction technology. It has
been shown that cryoprecipitate prepared from plasma
treated with riboflavin or amotosalen has comparatively
lower but acceptable levels of coagulation factors.25,26 The
pathogen reduction technology will not protect against
bacterial contamination that might occur after the
manufacturing process, for example, during the pooling
or thawing process. Another option is to perform a culture
for bacterial detection prior to releasing the extended-
shelf-life product, similar to methods proposed for aphe-
resis platelets.27 Either of these approaches may allow
rapid dispensing of cryoprecipitate, similar to other blood
products in a trauma setting.
In conclusion, our study supports the possibility of
extending the shelf life of thawed pooled cryoprecipitate.
It is anticipated that extending the shelf life of cryopreci-
pitate will significantly reduce wastage and improve turn-
around time while issuing cryoprecipitate in emergent
situations.
CONFLICT OF INTEREST
The authors have disclosed no conflicts of interest.
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