August 200545813421348Original Article

Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322005 American Association of Blood Banks 4∞C STORAGE OF WHOLE BLOOD FOR FFP PRODUCTIONCARDIGAN ET AL.

BLOOD COMPONENTS

The quality of fresh-frozen plasma produced from whole blood

stored at 4∞C overnight

Rebecca Cardigan, Andrew S. Lawrie, Ian J. Mackie, and Lorna M. Williamson

I
n the United Kingdom, fresh-frozen plasma (FFP) is
BACKGROUND: The aim of this study was to assess clinically indicated for single coagulation factor defi-
whether the quality of FFP produced from whole blood ciencies where an appropriate concentrate is not
stored at 4∞C overnight is adequate for its intended available (factor [F] V and FXI), acute disseminated
purpose. intravascular coagulation, plasma exchange for throm-
STUDY DESIGN AND METHODS: Fresh-frozen plasma botic thrombocytopenic purpura (TTP), and in some
(FFP) separated from whole blood (n = 60) leukodepleted instances of warfarin reversal, liver disease, cardiopulmo-
(LD) after storage at 4∞C overnight (18-24 hr from nary bypass, and massive transfusion.1 Despite the fact
donation, Day 1 FFP) was compared with that LD within that most patients for whom FFP is indicated have normal
8 hours of donation (Day 0 FFP, the current standard or high FVIII levels, quality control of FFP is currently
method). based on FVIII measurement, with Council of Europe
RESULTS: In more than 95 percent of Day 1 FFP units, Guidelines requiring levels of greater than 0.70 IU per mL2
levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and and UK guidelines specifying that 75 percent of units pro-
FXII were greater than 0.50 U per mL except for von duced meet this FVIII level.3 To meet this requirement,
Willebrand factor (VWF) antigen and FVIII, where 92 and European guidelines mandate that plasma be separated
87 percent of units, respectively, contained greater than from whole blood and frozen preferably within 6 hours
0.50 IU per mL. Compared with historical data on FFP and certainly within 18 hours of collection. Rapid cooling
stored for 8 hours, fibrinogen, FV, FVIII, and FXI were of blood and holding at 20 to 24∞C for up to 24 hours
reduced by 12, 15, 23, and 7 percent, respectively, but before processing is also permitted by European guide-
other factors were not significantly reduced. Levels of lines, because such plasma also meets the FVIII require-
VWF-cleaving protease activity were not different ment, but is not yet permitted by the UK Medicines and
between FFP prepared from paired units of blood (n = 3) Healthcare Regulatory Agency.
held for 8 or 24 hours, but were below the reference range
in an additional 2 of 6 units held for 24 hours. The
ABBREVIATIONS: a2-AP = a2-antiplasmin; APTT = activated
activities of protein S, protein C, antithrombin III, and a2-
partial thromboplastin time; ATIII = antithrombin III; C1-INH =
antiplasmin were reduced by less than 10 percent in
C1-inhibitor amidolytic activity; FXIIa = activated FXII; FVIIa =
Day 1 FFP (n = 20), but with final levels above the lower
activated FVII; LD = leukodepleted; MB = methylene blue; PC =
limit of the normal range in greater than 95 percent of
protein C; Pro F1 + 2 = prothrombin fragment 1 + 2; PS =
units. Activated FXII antigen was not significantly raised
protein S; PT = prothrombin time; TTP = thrombotic
in plasma stored for 18 to 24 hours, but levels of
thrombocytopenic purpura; VWF:Ag = von Willebrand factor
prothrombin fragment 1 + 2 were slightly increased
antigen; VWF:CB = VWF collagen-binding (activity); VWF:CP =
(0.88 ng/mL, 18-24 hr; 0.65 ng/mL, < 8 hr).
von Willebrand factor–cleaving protease (activity).
CONCLUSION: These data suggest that there is good
retention of relevant coagulation factor activity in plasma From the National Blood Service, England & North Wales; the
produced from whole blood stored at 4∞C for 18 to Department of Haematology, University College London,
24 hours and that this would be an acceptable product for London; and the Department of Haematology, University of
most patients requiring FFP. Cambridge, Cambridge, United Kingdom.
Address reprint requests to: Rebecca Cardigan, PhD National
Blood Service, Long Road, Cambridge CB2 2PT, UK; e-mail:
rebecca.cardigan@nbs.nhs.uk.
Received for publication November 15, 2004; revision
received January 27, 2005, and accepted January 27, 2005.
doi: 10.1111/j.1537-2995.2005.00219.x
TRANSFUSION 2005;45:1342-1348.

1342 TRANSFUSION Volume 45, August 2005


Production of FFP after overnight hold of whole blood
greatly increases operational flexibility, which can indi-
rectly enhance component safety. For example, because
UK hemovigilance data demonstrate that HLA antibody–
positive female donors are the principle cause of transfu-
sion-related acute lung injury (TRALI) associated with
FFP,4 overnight hold could contribute to reduction of the
TRALI risk by increasing the number of “male-only” dona-
tions available for FFP production. An alternative to over-
night hold at 20 to 24∞C is to store whole blood overnight
at 4∞C for processing the following day (Day 1).
A number of studies have been performed to investi-
gate extended storage of blood for FFP production,5-11 but
these have generally focused on a limited range of coagu-
lation factors or on a small number of units or have sepa-
rated plasma before its storage at 4∞C. Given the clinical
indications for FFP, it is important that a wide range of
coagulation factors is preserved, including von Willebrand
factor–cleaving protease (VWF:CP, ADAMTS13) activity,
the presumed moiety of therapeutic value in FFP for
plasma exchange for TTP.12 There are currently no pub-
lished data available on levels of VWF:CP in plasma sepa-
rated from whole blood more than 8 hours from
collection.
The primary aim of this study was to measure a wide
range of coagulation factors and their inhibitors, as well
as markers of contact activation (activated FXII [FXIIa])
and thrombin generation (prothrombin fragment 1 + 2
[Pro F1 + 2]) in plasma produced from whole blood stored
overnight at 4∞C. Our hypothesis, based on previous liter-
ature, was that this plasma would be unlikely to meet
current UK or European standards for FVIII content, yet
retain substantial activity of this and other coagulation
factors. A secondary objective was to compare the effect
of three different whole-blood leukodepletion (LD) filters
that are currently used in England, on levels of coagula-
tion factors in FFP separated on Day 1.
MATERIALS AND METHODS
Blood collection and processing
Sixty units of whole blood (450 ± 45 mL) were collected
into citrate phosphate dextrose anticoagulant (63 mL)
according to Guidelines for UK Transfusion Services, 2002.
Because levels of VWF and FVIII are lower in group O
donors, an equal number of group A and group O dona-
tions were selected so that this did not bias results. Units
were stored overnight at 4∞C and then LD 18 to 24 hours
after donation with one of three filters (n = 20 for each of
RZ2000, Baxter Healthcare, Newbury, UK; LST2, Maco-
Pharma Ltd, Middlesex, UK; NPBI T2926, Fresenius
Hemocare, Abingdon, UK). Twenty units for each filter
were selected based on power calculations that this would
detect a 5 percent difference in mean results, with
90 percent power at the 5 percent significance level. After
4∞C STORAGE OF WHOLE BLOOD FOR FFP PRODUCTION
filtration, units were centrifuged (Beckman-Coulter High
Wycombe, UK, 2800 ¥ g, 10 min) and processed to red cells
and plasma with an automated blood component extrac-
tor (Optipress II system, Baxter Healthcare). Samples of
plasma were taken by sterile connection of a sample
pouch, frozen, and stored at -80∞C for later analysis. For
protein C (PC), antithrombin III (ATIII), protein S (PS),
and a2-antiplasmin (a2-AP), samples were collected with
the RZ2000 filter only after 8 and 18 to 24 hours of storage
(n = 20). Three of the latter units were also assessed for
VWF:CP activity.
We compared the levels of coagulation activity in
Day 1 FFP to a reference range based on 66 samples from
a previous study of LD plasma frozen less than 8 hours
from collection of whole blood with a variety of LD filters
currently approved for use in the NBS (Pall LPSI or Baxter
plasma filters Pall, Portsmouth UK, Baxter; RZ2000, Bax-
ter; NPBI T2926; WBSP, Terumo, Knowsley, UK; and
MacoPharma LST1, whole blood filters).13 Samples used to
establish the reference range were also from a 1:1 mix
of group O and group A donors. Differences in results
between test samples and historical controls attributed to
slight variations in assay methods were minimized by the
use of reference preparations calibrated against interna-
tional standards where they exist. Reference ranges were
calculated with the mean ± 2SD for normally distributed
data and the geometric mean with 95 percent confidence
interval (CI) for skewed data. Day 0 and Day 1 FFP were
compared with the U test. For the following assays paired
samples were collected after 8 and 18 to 24 hours of stor-
age and compared because no prior data was available on
Day 0 FFP: VWF:CP, PC, ATIII, PS, and a2-AP.
Laboratory analysis
Coagulation factors FII, FV, FVII, FVIII, F IX, FX, FXI, and
FXII were assayed by clotting assay at three dilutions (1/
10, 1/20, 1/40) with deficient plasma from Technoclone
Ltd (Dorking, UK) and Innovin as a prothrombin time (PT)
reagent or actin FS as an activated partial thromboplastin
time (APTT) reagent (both Dade Behring, Marburg, Ger-
many). Fibrinogen was measured by Clauss technique
with reagents from Dade Behring and VWF antigen
(VWF:Ag) by latex immunoturbidometric assay (STA Liat-
est, Diagnostica Stago Ltd, Asnieres, France). PC, ATIII,
and a2-AP were measured by chromogenic assay (all Ber-
ichrom, Dade Behring). Free PS antigen was measured
by latex immunoturbidometric assay (Liatest, Diagnostica
Stago), and PS coagulant activity was measured with
two methods (Staclot, Diagnostica Stago and Bioclot
protein S-300, Biopool AB, Trinity Biotech, Ireland). All
assays above were performed with an analyzer (CA-1500,
Sysmex UK Ltd, Milton Keynes, UK), standardized with
coagulation reference plasma and controlled with coagu-
lation control A (both Technoclone Ltd). APTT and PT
Volume 45, August 2005 TRANSFUSION 1343
CARDIGAN ET AL.

ratios were calculated in the usual way as the ratio of test
result to the geometric mean result of at least 20 normal
plasma samples anticoagulated with trisodium citrate.
Commercially available kits were used to determine VWF
collagen-binding (VWF:CB, Technoclone Ltd) activity, C1-
inhibitor amidolytic activity (C1-INH, Technoclone Ltd),
Pro F1 + 2 (Dade Behring), and FXIIa antigen (Axis-Shield
Ltd, Dundee, UK). VWF multimers and cleaving protease
activity were assayed as previously described.14,15 VWF:CP
results were expressed as a ratio to that of a pooled normal
plasma.
Statistical analysis
Paired data were compared with the Wilcoxon-rank test.
Results are presented as median with range for data sets
including variables with non-Gaussian distribution and
mean with SD for normally distributed data. A p value of
less than 0.05 was considered significant.
RESULTS
A summary of levels of coagulation factors in plasma pro-
cessed from whole blood after overnight hold at 4∞C
followed by LD is shown in Table 1. Compared to FFP
processed on Day 0, plasma processed on Day 1 showed
losses of fibrinogen (12%), FV (15%), FVIII (23%), and FXI
(7%) but not FII, FVII, F IX, FX, or FXII. This was associated
with an increase in the APTT ratio from a mean of 1.08
(Day 0) to 1.25 (Day 1) but there was no change in the PT
ratio. More than 95 percent of the 60 FFP units processed
on Day 1 had levels of FII, FV, FVII, FVIII, F IX, FX, FXI, and
FXII above the lower limit of the reference range calcu-
lated from Day 0 LD FFP (Table 1). Furthermore, for these
factors, more than 98 percent of the 60 FFP units pro-
cessed on Day 1 had levels above 0.50 IU per mL, except
FVIII for which 92 percent of units were greater than
0.50 IU per mL (Table 1). Only 33 of 60 (55%) of units con-
tained greater than 0.70 IU per mL FVIII activity, thus fail-
ing to meet the UK specification of more than 75 percent
of units greater than 0.70 IU per mL. Mean levels of FXII
(1.29 U/mL) and FX (1.17 U/mL) were surprisingly high in
plasma processed on Day 1.
The only unit containing less than 0.50 IU per mL FVII
(0.42 IU/mL) had no other factors below the reference
range. In 4 of 5 units where levels of FVIII were less than
0.50 IU per mL, levels of VWF:Ag and VWF:CB were also
below the reference range. In 8 of 9 units with levels of
VWF:Ag below the reference range, levels of VWF:CB were
also below range. In 2 of 3 units with levels of FXI of less
than 0.60 U per mL, there were no other factors below
range, but the third unit also had lower levels of CI-INH
(0.60 U/mL).
Mean levels of VWF:Ag were 1.02 IU per mL in plasma
processed on Day 1 (Table 1), and 95 percent of units con-
tained greater than 0.50 IU per mL VWF:Ag. There was no
significant difference in levels of VWF:CB between plasma
processed on Day 0 and plasma processed on Day 1
(Table 1). Six units of FFP processed on Day 1 were
assayed for VWF:CP, with a mean value of 0.74 (range, 0.42-

TABLE 1. Coagulation variables in plasma frozen within 8 hours from donation or after storage of whole blood at 4∞C
overnight (18-24 hr from donation) in comparison to reference ranges*
Reference range based on Standard hematology
FFP separated < 8 hr reference ranges
Percent of units Percent of units
Whole blood storage time stored 18-24 hr stored 18-24 hr
Factor <8 hr (n = 66) 18-24 hr (n = 60) Range (n = 66) in range Range in range
PT ratio (n = 110) 1.04 (0.94-1.18) 0.99 (0.89-1.17) 0.95-1.16 98 Varies
APTT ratio (n = 110) 1.08 (0.83-1.97) 1.25 (1.03-1.46)b 0.86-1.36 83 Varies
Fibrinogen (g/L) 2.69 (1.54-5.00) 2.37 (1.54-3.90)a 1.10-4.30 100 1.5-4.0 100
Prothrombin (IU/mL) 0.96 (0.72-1.26) 1.05 (0.82-1.40)b 0.70-1.20 100 0.50-2.00 100
FV (U/mL) 0.94 (0.35-1.48) 0.80 (0.53-1.12)b 0.50-1.40 100 0.50-2.00 100
FVII (IU/mL) 1.01 (0.58-1.56) 1.07 (0.42-2.65) 0.60-1.40 98 0.50-2.00 98
FVIII (IU/mL) 1.00 (0.48-1.85) 0.77 (0.38-1.40)b 0.40-1.60 98 0.50-2.00 92
F IX (IU/mL) 0.98 (0.60-1.45) 1.05 (0.60-1.58) 0.60-1.40 100 0.50-2.00 100
FX (IU/mL) 0.99 (0.71-1.29) 1.17 (0.80-1.77)b 0.70-1.30 100 0.50-2.00 100
FXI (U/mL) 0.95 (0.66-1.44) 0.88 (0.51-1.36)a 0.60-1.30 95 0.50-2.00 100
FXII (U/mL) 0.98 (0.20-1.47) 1.29 (0.51-2.39)b 0.40-1.50 100 0.50-2.00 100
C1-INH (U/mL) 0.87 (0.60-1.64) NA† NA 0.70-1.30 92
FXIIa antigen (ng/mL) 1.89 (0.40-4.12) 1.83 (0.67-3.51) 0.50-5.00 100 <2.9 92
Pro F1 + 2 (nmol/L) 0.65 (0.35-1.30) 0.88 (0.37-1.82)b 0.20-1.10 72 0.40-1.10 72
VWF:Ag (U/mL) 1.13 (0.66-1.75) 1.02 (0.39-2.21) 0.60-1.65 85 0.50-2.00 95
VWF activity (U/mL) 1.01 (0.57-1.63) 0.97 (0.30-1.85) 0.50-1.50 87 0.50-2.00 87
* Data are given as median (range). ap < 0.05 and bp < 0.0001 compared with FFP separated less than 8 hours. Data from FFP separated less
than 8 and 18 to 24 hours are not paired (see Materials and methods). Reference ranges have been calculated with mean ± 2SD for
normally distributed data and the geometric mean with 95 percent CI for skewed data. In range is defined as above the lower limit for
coagulation factors and below the upper limit for PT and/or APTT and activation markers.
† NA = not available.

1344 TRANSFUSION Volume 45, August 2005


0.96). The two units with the lowest VWF:CP results (0.42
and 0.62) were from group A donors with high levels of
VWF (1.76 and 1.66 IU/mL, respectively). Because 2 of 6
results were below the reference range of the assay (0.80-
1.20) determined with citrated plasma, three additional
units of blood were assessed where paired plasma samples
from the same whole-blood unit were taken less than 8
and 24 hours after donation. In these 3 units, levels of
VWF:CP were 1.03, 1.03, and 0.95 after 8 hours and 1.06,
1.06, and 0.94 after 24 hours, respectively, and the VWF
multimeric distribution was normal at both time points.
Levels of FXIIa antigen were not significantly higher
in FFP processed on Day 1 compared to Day 0 (Table 1)
and levels of C1-INH, the main inhibitor of FXIIa in
plasma, were greater than 0.70 IU per mL in 92 percent of
Day 1 FFP units. Levels of FXIIa antigen were not above
the reference range in units where C1-INH levels were less
than 0.70 IU per mL. Levels of Pro F1 + 2, a marker of
thrombin generation, were on average 35 percent higher
in plasma processed on Day 1 compared to Day 0
(Table 1). Twenty-eight percent of FFP units processed on
Day 1 had a Pro F1 + 2 level higher than the upper limit of
the reference range based on either Day 0 LD FFP or cit-
rated plasma (Table 1). Of the 14 units with a Pro F1 + 2
level of greater than 1.10 nmol per L, 9 were processed
with the MacoPharma LST2 filter.
Storage at 4∞C for 18 to 24 hours did not result in a
significant reduction in ATIII, but did result in a significant
(<10%), but clinically insignificant, reduction in PC, PS,
and a2-AP (Table 2). The loss of PS coagulant activity was
greater when assayed with the Bioclot assay (8%) than the
Staclot assay (3%). Levels of ATIII, PS, and a2-AP were
above the lower limit of the reference range in more than
95 percent of units processed on Day 1 (Table 2).
The only major difference observed among the three
different whole-blood filters was that levels of FXI were
10 percent lower, and levels of Pro F1 + 2 were 20 percent
higher with the MacoPharma filter.
TABLE 2. Coagulation inhibitors in plasma frozen less than 8 hours from donation or after storage of whole blood at
4∞C overnight (18-24 hr from donation) compared with reference ranges*
FFP storage time
Factor <8 hr (n = 20) 18-24 hr (n = 20)
ATIII (IU/mL) 0.96 ± 0.10 0.95 ± 0.10
PC (IU/mL) 0.98 ± 0.23 0.96 ± 0.22b
a2-AP (U/mL) 1.02 ± 0.08 0.97 ± 0.11b
PS Bioclot (IU/mL) 0.99 ± 0.13 0.91 ± 0.09b
PS Staclot (IU/mL) 1.12 ± 0.16 1.09 ± 0.16a
PS free antigen (IU/mL) 0.80 ± 0.15 0.77 ± 0.15a
* Data are given as mean (SD). ap < 0.05 and bp < 0.01 compared with FFP separated less than 8 hours. Data from FFP separated less than
8 and 18 to 24 hours are paired. FFP was LD with a RZ2000 whole-blood filter.
4∞C STORAGE OF WHOLE BLOOD FOR FFP PRODUCTION
DISCUSSION
In our study, holding blood at 4∞C overnight resulted in a
more than 20 percent loss of FVIII and modest reductions
in levels of fibrinogen, FV, and FXI, but no significant
change in FII, FVII, F IX, FX, or FXII compared to plasma
frozen within 8 hours of donation. This was associated
with an increase in the APTT ratio, but not the PT ratio.
The lack of effect on the PT ratio is not unsurprising, given
that there was only slight loss of factors in the tissue factor
pathway. One previous study has reported a 12 percent
loss of FV and 8 percent loss of fibrinogen between plasma
separated immediately after donation or after whole
blood is stored for 8 hours at 22∞C.6 Others, however, have
shown that FV and fibrinogen are stable in whole blood
stored at 4∞C for at least 24 hours, whereas there is a loss
of 20 to 50 percent FVIII activity.5,8,9 In addition, O’Neill
and colleagues10 reported no significant difference
between the activity of FV, FVII, or FX in plasma separated
from whole blood stored at 4∞C for 24 hours compared
with 8 hours. One limitation to our study was that data on
plasma processed on Day 1 were compared with historical
data. Slight differences in testing methods between stud-
ies might have accounted for why we have observed a loss
of FV, fibrinogen, and FXI while most others have not.
As well as quantifying losses, it is also important to
consider the residual coagulation factor activity in the
plasma that patients receive. Levels of all coagulation fac-
tors studied in plasma separated on Day 1 were above the
lower limit of the reference range based on Day 0 plasma
in at least 95 percent of units and above 0.50 IU per mL in
92 percent of units, suggesting good retention of activity.
The quality of plasma produced was comparable among
the three types of whole blood LD filter studied, with only
small differences observed among them. Despite FVIII
activity being the worst affected factor, in 100 units of FFP
produced in a routine environment from blood stored at
4∞C overnight, levels of FVIII were on average 0.87 IU per
Based on standard hematology reference ranges
Range Percentage of Day 1 units in range
0.80-1.20 95
0.70-1.30 95
0.80-1.20 95
0.55-1.60 100
0.77-1.43, men 100
0.55-1.23, women
0.70-1.48, men 100
0.50-1.34, women
Volume 45, August 2005 TRANSFUSION 1345
CARDIGAN ET AL.
mL with 96 percent of units greater than 0.50 IU per mL.
These levels are similar to, if not higher than, those found
in FFP pathogen inactivated by methylene blue (MB), sol-
vent/detergent (S/D), or amotosalen methods, all prod-
ucts that have been considered clinically acceptable.16-18
One theoretical concern with whole blood filtered
after storage at 4∞C is the potential for increased contact
activation, because the activity of C1-inhibitor (the main
inhibitor of FXIIa in plasma) is known to be reduced at
temperatures lower than 37∞C.19 The retention of C1-
inhibitor in FFP processed on Day 1 was good, however,
with 92 percent of units containing greater than 0.70 IU
per mL; in addition, levels of FXIIa antigen (measured with
an assay that detects free FXIIa in plasma) were not signif-
icantly raised. A higher degree of contact activation can-
not be absolutely excluded, however, because any FXIIa
generated would be expected to complex to plasma inhib-
itors, which the FXIIa antigen assay does not detect.20
The mean levels of FXII and FX in FFP processed on
Day 1 were surprisingly high, which could be attributable
to coagulation factor activation, although this was not
reflected by increased levels of FXIIa. Our findings are
consistent with those of Nilsson and coworkers,7 who
observed a marked increase in FXII coagulant activity
after whole blood had been stored for 2 weeks at 4∞C. They
observed no such increase in plasma-depleted of platelets
(PLTs) or separated before storage, indicating that the
presence of cells (most probably PLTs) is responsible for
this phenomenon.
A high mean FVII level (1.25 IU/mL) was also
observed in FFP filtered on Day 1 with the LST2 filter. This
could be due to an increase in levels of the activated form
of FVII (FVIIa), which is known to influence FVII clotting
assays.21 FVIIa is unlikely to be generated in plasma in the
absence of tissue factor, but theoretically F IXa and FXIIa
generated by contact activation could cleave FVII directly
to form FVIIa. Levels of Pro F1 + 2, a marker of thrombin
generation, appeared to be increased in plasma separated
on Day 1 compared with Day 0. This was mainly attribut-
able to plasma from one whole-blood filter type (LST2),
which we have previously shown results in greater gener-
ation of Pro F1 + 2 after filtration compared to the other
filters studied.13 The clinical significance of this is not
clear, but final levels of Pro F1 + 2 are similar or lower to
those observed in S/D-treated plasma.22
Levels of VWF:Ag and VWF:CB activity appeared to
be well preserved 24 hours after donation. Our data are
consistent with those of Hughes and associates,9 who
observed approximately 90 percent recovery of VWF:Ag
and ristocetin cofactor activity up to 24 hours from dona-
tion, irrespective of whether blood was stored at 22 or
4∞C.9 Our study is the first to assay VWF:CP in Day 1 FFP.
Several of the units processed on Day 1 appeared to con-
tain low levels of VWF:CP. When this was repeated in a
small number of paired samples, however, there appeared
1346 TRANSFUSION Volume 45, August 2005
to be no difference in VWF:CP activity between whole
blood stored for 8 or 24 hours. The reasons for the initial
low results are unclear, because in 16 samples of Day 0 LD
and MB-treated FFP that we have previously assayed for
VWF:CP by the same technique, results outside the refer-
ence range (0.80-1.20) have not been observed (unpub-
lished data). It is interesting, however, that the two lowest
VWF:CP results were both from group A donors who had
high levels of VWF. It is well established that levels of VWF
are lower in group O donors, but a recent study has shown
that levels of VWF:CP are higher in group O donors and
there is a negative association of levels of VWF:CP with
VWF.23
In this study, the activity of the naturally occurring
anticoagulants ATIII, PC, or PS appeared to be only mini-
mally affected by extending the period before plasma was
separated from whole blood from 8 to 18 to 24 hours. This
is consistent with previous studies10,24 and is a clinically
important observation, because it has been suggested that
a propensity to deep vein thrombosis in TTP patients
undergoing plasma exchange with S/D FFP might have
been contributed to by low levels of PS in the product.25
Interestingly, the reduction in PS activity appeared to dif-
fer dependent on the coagulation assay used to measure
it. This might reflect the fact that the two assays use dif-
ferent activators (FVa for Staclot and FXa for Bioclot). We
did not observe a clinically significant decrease in the
main inhibitor of fibrinolysis in plasma (a2-AP) after
24 hours of storage, in agreement with others.7 The latter
study also showed that fibrinolytic activity is not
enhanced (as evidenced by the euglobulin clot lysis
method), when blood is stored at 4∞C for several weeks.
Despite good coagulation factor retention, Day 1 FFP
would not meet current UK specifications (>75% of units
must contain >0.70 IU/mL FVIII) or Council of Europe
guidelines (>0.70 IU/mL FVIII, percentage of units not
specified), because only 55 percent of units contained
greater than 0.70 IU per mL FVIII activity. The routine pro-
duction of this plasma would therefore require a change
to UK and European quality monitoring requirements for
FFP or for this plasma to have a separate specification. In
the United States, FFP produced less than 8 hours or more
than more 24 hours from donation are issued as different
products,26 although they may be used for the same indi-
cations (with the exception of FVIII replacement).
Although FFP is now never given for replacement of FVIII
alone, it is important that FFP contains a reasonable level
of FVIII for treatment of disseminated intravascular coag-
ulation and massive transfusion. Reducing UK and Euro-
pean specifications to greater than 0.50 IU per mL FVIII
activity, but ensuring a higher percentage of units (say
90%) meet this criteria would seem to satisfy this clinical
requirement. Alternatively, the requirement for any spe-
cific coagulation factor activity could be dropped, as is the
case in the United States, providing that time and/or tem-

perature of blood storage and/or freezing are well con-
trolled. An additional possibility is to use a different
marker of plasma quality altogether, one that is clinically
more relevant than FVIII.
Plasma produced from whole blood after an over-
night hold at 4∞C appears to contain levels of coagulation
factors that are equivalent to or higher than those seen in
most studies evaluating plasma treated with S/D, MB, or
amotosalen pathogen reduction systems.16-18 Both MB and
S/D-treated plasma are used in several European coun-
tries for all of the same clinical indications as FFP, without
apparent clinical detriment. S/D-treated plasma contains
greater than 0.50 U per mL of each coagulation factor,
which can be guaranteed because it is produced from a
large pool of donations and each batch can be tested.
Because of natural variability, plasma from individual
donations cannot be guaranteed to contain greater than
0.50 U per mL of each coagulation factor, but our results
suggest this will be achieved in a very high percentage of
donations processed 18 to 24 hours after collection. More-
over, we did not observe any individual units in which
multiple coagulation factors were severely reduced
together. Studies of S/D or amotosalen FFP do not suggest
either failure to correct coagulopathy or a requirement to
increase the dose above that recommended for FFP.27-32
Clinical data on MB FFP are more limited, but one study
has suggested increased overall usage following universal
implementation of MB FFP.33 Data on correction of coag-
ulation were not presented in the latter study. The loss
of fibrinogen, FVIII, and FXI (all 25%-30%), however, is
greater with MB treatment than after overnight 4∞C stor-
age and may have accounted for the increased usage.
It would appear therefore that Day 1 FFP is suitable
for use in all the situations of multiple acquired coagulop-
athy for which FFP is currently prescribed. There is no
evidence to suggest that Day 1 FFP would be unsuitable
for plasma exchange for TTP because levels of VWF:CP
were normal in three paired samples, as was VWF multi-
meric distribution. This is consistent with other reports
that VWF:CP is preserved in FFP after storage for 24 hours
at room temperature.34 This is also true of MB plasma,
however, a product with which reduced efficacy in TTP
has been reported.35,36 Further studies are therefore
required to determine the product-related variables that
determine outcome in plasma exchange for TTP.
A reduction of coagulation factor content in FFP by
pathogen reduction techniques has already been deemed
acceptable to achieve improvements in the safety of the
component. TRALI is an important cause of transfusion-
related morbidity and mortality. A key cause is HLA and/
or HNA antibodies present in 10 to 15 percent of female
donor plasma, usually as a result of pregnancy.37 In
England, considerable progress has been made toward
male-only FFP within the context of an 8-hour product,
with greater than 90 percent of FFP now produced from
4∞C STORAGE OF WHOLE BLOOD FOR FFP PRODUCTION
male donors. To extend this and to select male plasma for
resuspension of PLT pools will require greater operational
flexibility. This could be achieved by universal holding of
whole blood at 20∞C under controlled conditions, which
paradoxically conserves FVIII. Discussions on this practice
are being held with UK regulators, but in the event that
this is not permissible, FFP produced from whole blood
stored at 4∞C would offer a clinically viable alternative.
We have not assessed the suitability of 24-hour
plasma for the production of cryoprecipitate or cryosu-
pernatant because these can be adequately sourced from
FFP stored less than 8 hours. Finally, it may be that Day 1
FFP would not be an optimal product to use as a starting
material for pathogen reduction systems in view of the
additional loss of coagulation factors that these systems
cause.
ACKNOWLEDGMENTS
We thank Kim Smith, Claire Pergande, Graham Walters, and Rada
Kelly (all National Blood Service, England & North Wales) for help
in the collection of samples and Gordon Purdy (UCL) for labora-
tory analysis.
REFERENCES
1. O’Shaughnessy DF, Atterbury C, Bolton Maggs P, et al.
Guidelines for the use of fresh-frozen plasma,
cryoprecipitate and cryosupernatant. British Committee for
Standards in Haematology, Blood Transfusion Task Force. Br
J Haematol 2004;126:11-28.
2. Guide to the preparation, use, quality assurance of blood
components 9th ed. Strasbourg: Council of Europe
Publishing; 2003.
3. Guidelines for the blood transfusion services in the United
Kingdom. 6th ed. London: The Stationary Office; 2002.
4. Serious Hazards of Transfusion (SHOT) Annual Report 2003.
Manchester: Serious Hazards of Transfusion Steering Group;
2003.
5. Weisert O, Jeremic M. Preservation of coagulation factors V
and VIII during collection and subsequent storage of bank
blood in ACD-A and CPD solutions. Vox Sang 1973;24:126-
33.
6. Sohmer PR, Bolin RB, Scott RL, Smith DJ. Effect of delayed
refrigeration on plasma factors in whole blood collected in
CPDA-2. Transfusion 1982;22:488-90.
7. Nilsson L, Hedner U, Nilsson IM, Robertson B. Shelf-life of
bank blood and stored plasma with special reference to
coagulation factors. Transfusion 1983;23:377-81.
8. Kakaiya RM, Morse EE, Panek S. Labile coagulation factors
in thawed fresh frozen plasma prepared by two methods.
Vox Sang 1984;46:44-6.
9. Hughes C, Thomas KB, Schiff P, et al. Effect of delayed blood
processing on the yield of factor VIII in cryoprecipitate and
factor VIII concentrate. Transfusion 1988;28:566-70.
Volume 45, August 2005 TRANSFUSION 1347
CARDIGAN ET AL.
10. O’Neill EM, Rowley J, Hansson-Wicher M, et al. Effect of 24-
hour whole-blood storage on plasma clotting factors.
Transfusion 1999;39:488-91.
11. Smith JF, Ness PM, Moroff G, Luban NL. Retention of
coagulation factors in plasma frozen after extended holding
at 1-6 degrees C. Vox Sang 2000;78:28-30.
12. Rock GA. Management of thrombotic thrombocytopenic
purpura. Br J Haematol 2000;109:496-507.
13. Cardigan R, Sutherland J, Garwood M, et al. The effect of
leucocyte depletion on the quality of fresh frozen plasma
(FFP). Br J Haematol 2001;114:233-40.
14. Lawrie AS, Hoser MJ, Savidge GF. Phast assessment of vWF:
Ag multimeric distribution. Throm Res 1990;59:369-73.
15. Allford SL, Harrison P, Lawrie AS, et al. von Willebrand
factor-cleaving protease activity in congenital thrombotic
thrombocytopenic purpura. Br J Haematol 2000;111:1215-
22.
16. Hellstern P, Haubelt H. Manufacture and composition of
fresh frozen plasma and virus inactivated therapeutic
plasma: correlation between composition and therapeutic
efficacy. Thrombosis Res 2002;107:S3-8.
17. Horowitz B. Pathogen inactivated transfusion plasma:
existing and emerging methods. Vox Sang 2002;83(Suppl
1):429-36.
18. Williamson LM, Cardigan R, Prowse CV. Methylene blue-
treated fresh-frozen plasma: what is its contribution to
blood safety? Transfusion 2003;43:1322-9.
19. Weiss R, Silverberg M, Kaplan AP. The effect of C1-inhibitor
upon Hageman factor autoactivation. Blood 1986;68:239-43.
20. Esnouf MP, Burgess AI, Dodds AW, Sarphie AF, Miller GJ. A
monoclonal antibody raised against human beta-factor XIIa
which also recognizes alpha-factor XIIa but not factor XII or
complexes of factor XIIa with C1 esterase inhibitor. Thromb
Haemost 2000;83:874-81.
21. Miller GJ, Stirling Y, Esnouf MP, et al. Factor VII-deficient
substrate plasmas depleted of protein C raise the sensitivity
of the factor VII bio-assay to activated factor VII: an
international study. Thromb Haemost 1994;71:38-48.
22. Hellstern P, Sachse H, Schwinn H, Oberfrank K.
Manufacture and in vitro characterization of a solvent/
detergent-treated human plasma. Vox Sang 1992;63:
178-85.
23. Mannucci PM, Capoferri C, Canciani MT. Plasma levels of
von Willebrand factor regulate ADAMTS-13, its major
cleaving protease. Br J Haematol 2004;126:213-8.
24. Inkster M, Sherman LA, Ahmed P, Benton MB, Gaston LW.
Preservation of antithrombin III activity in stored whole
blood. Transfusion 1984;24:57-9.
25. Yarranton H, Cohen H, Pavord SR, et al. Venous
thromboembolism associated with the management of
1348 TRANSFUSION Volume 45, August 2005
acute thrombotic thrombocytopenic purpura. Br J Haematol
2003;121:778-85.
26. Standards for Blood Banks and Transfusion Services. 21st ed.
Bethesda: American Association of Blood Banks; 2002.
27. Solheim BG, Svennevig JL, Mohr B, et al. The use of Octaplas
in patients undergoing open heart surgery. In: Muller-
Berghaus G, ed. DIC: pathogenesis, diagnosis and therapy of
disseminated intravascular fibrin formation. Amsterdam:
Elsevier; 1993. p. 253-62.
28. Horowitz MS, Pehta JC. SD plasma in TTP and coagulation
factor deficiencies for which no concentrates are available.
Vox Sang 1998;74(Suppl 1):231-5.
29. Williamson LM, Llewelyn CA, Fisher NC, et al. A randomised
trial of solvent/detergent and standard fresh frozen plasma
in the coagulopathy of liver disease and liver
transplantation. Transfusion 1999;39:1227-34.
30. Haubelt H, Blome M, Kiessling AH, et al. Effects of solvent/
detergent-treated plasma and fresh-frozen plasma on
haemostasis and fibrinolysis in complex coagulopathy
following open-heart surgery. Vox Sang 2002;82:9-14.
31. Mintz P, Steadman R, Blackall D, et al. Pathogen inactivation
of plasma using S-59 and UVA light is efficacious and well
tolerated in the treatment of end-stage liver disease
patients- the STEP AC trial. Transfusion 2002;42(Suppl):15S.
32. de Alarcon P, Benjam RJ, Shopnick R, et al. Patients with
congenital coagulation factor deficiencies demonstrate
consistent therapeutic responses to repeated transfusions of
plasma prepared with pathogen inactivation treatment
(INTERCEPT plasma). Blood 2003;102:815a.
33. Atance R, Pereira A, Ramirez B. Transfusing methylene blue-
photoinactivated plasma instead of FFP is associated with
an increased demand for plasma and cryoprecipitate.
Transfusion 2001;41:1548-52.
34. Yarranton H, Lawrie AS, Purdy G, Mackie IJ, Machin SJ.
Comparison of von Willebrand factor antigen, von
Willebrand factor-cleaving protease and protein S in blood
components used for treatment of thrombotic
thrombocytopenic purpura. Transfus Med 2004;14:39-44.
35. De la Rubia J, Arriaga F, Linares D, et al. Role of methylene
blue treated or fresh frozen plasma in the response to
plasma exchange in patients with thrombotic
thrombocytopic purpura. Br J Haematol 2001;114:721-3.
36. Alvarez-Larran A, Del Rio J, Ramirez C, et al. Methylene blue-
photoinactivated plasma vs. fresh-frozen plasma as
replacement fluid for plasma exchange in thrombotic
thrombocytopenic purpura. Vox Sang 2004;86:246-51.
37. MacLennan S, Lucas G, Brown C, et al. Prevalence of HLA
and HNA antibodies in donors: correlation with pregnancy
and transfusion history. Vox Sang 2004;87(Suppl 3):S2-
S16.