Download Bacterial contamination in platelet concentrates

Vox Sanguinis (2014) 106, 256–283
© 2013 International Society of Blood Transfusion
DOI: 10.1111/vox.12098
INTERNATIONAL FORUM
Bacterial contamination in platelet concentrates
R. N. I. Pietersz, H. W. Reesink, S. Panzer, S. Oknaian, S. Kuperman, C. Gabriel, A. Rapaille, M. Lambermont, V. Deneys,
D. Sondag, S. Ramírez-Arcos, M. Goldman, G. Delage, F. Bernier, M. Germain, T. Vuk, J. Georgsen, P. Morel, C. Naegelen,
L. Bardiaux, J.-P. Cazenave, J. Dreier, T. Vollmer, C. Knabbe, E. Seifried, K. Hourfar, C. K. Lin, M. Spreafico, L. Raffaele,
A. Berzuini, D. Prati, M. Satake, D. de Korte, P. F. van der Meer, J. L. Kerkhoffs, L. Blanco, J. Kjeldsen-Kragh,
A.-M. Svard-Nilsson, C. P. McDonald, I. Symonds, R. Moule, S. Brailsford, R. Yomtovian & M. R. Jacobs
Septic reactions after transfusion, particularly of platelet
concentrates, still occur and belong to the most serious
transfusion reactions. From a previous International
Forum [1] on the subject, it could be concluded that in
part of the countries that participated in the forum, platelet concentrates (PCs) were tested for bacterial contamination and that culture-based methods, particularly the
BacT/Alert system, were used.
In recent years, several rapid bacterial detection methods, such as surrogate measurements of the pH or glucose, the detection of bacteria with a scan system or
PCR tests that detect bacterial RNA, have been developed. These tests can either be performed immediately
prior to transfusion of the PC or at a variety of test
moments at which culture and release tests are combined.
Pathogen inactivation (PI) methods also affect bacterial
contamination of PCs. In 2007 [1], in some countries, the
Intercept method of PI of PCs was implemented instead
of bacterial screening.
It seemed of interest to evaluate the present state of
the art of this subject. In order to obtain the desired
information, the following questions were sent to experts
in the field.
Question 1: How long do you store PC and is there a
difference between whole-blood-derived PC and apheresis
PC? Which method of preparation do you use for wholeblood-derived PC? Are PCs leuco-reduced?
Question 2: Do you use a culture method to detect bacterial contamination of PC? If so,
• Which method do you use?
• Do you apply aerobic as well as anaerobic culture
methods?
• Which volume of PC is cultured?
• When is the culture taken if day 0 is blood collection?
• How often are the results of the cultures checked
and how is this result linked with issuing the PC
(manually or automated)?
256
•
•
•
•
If the test results are negative, for how many days
are the cultures of the issued units continued?
What are your definitions of initially positives, true
positives and false positives and do you have
(annual) data of culture results, including time of
initially positives and type of bacteria?
Do you have data of cases that the culture became
positive after a PC had been issued as negative to
date (and eventually had been transfused) and the
follow-up of the patient, including type of bacteria
and level of proof?
Any other aspects?
Question 3: Have you experience with any of the rapid
detection methods of bacterial contamination? If so, With
which method(s)?
• Did you find this (these) method(s) satisfactory with
regard to the handling and time it takes to perform
it (them) and with the percentage of false positives
and false negatives, etc?
• What is the sensitivity of the method(s) as compared
to the culture methods? Have you evidence that bacterial contamination can be detected by the test(s)
before the culture becomes positive?
• Any other aspects?
Question 4: Instead of using one of the above methods,
have you chosen to use pathogen inactivation to prevent
transfusion of bacterially contaminated PC? If so,
• Did you culture or apply a rapid bacterial detection
method before and after pathogen inactivation for
validation of the method? Do you have data to show
in how many cases it has not prevented post-transfusion bacteriaemia, and is it known in such cases
which bacteria were involved and the impact on the
patient?
• Any other aspects?
We obtained 18 contributions to this forum from 16
countries. Data concerning the preparation and storage of
International Forum 257
PCs are summarized in Table 1, and information about
the screening method is given in Table 2. For detailed
information, please see the individual contributions. It
should be kept in mind that these do not always reflect
national policies.
In most cases (Table 1), both pooled PCs and apheresis
PCs are used except in Japan and one German centre
where only apheresis PCs are provided. For the preparation of pooled PCs, 13 of 16 participants used the BC
method and 3 the PRP method. Despite bacterial screening, the majority of both apheresis and (pooled) PCs have
a maximum shelf life of 5 days (range 35–7 days). Virtually all PCs are leucoreduced.
It is clear that 11 of 16 participants submit all PCs
to bacterial screening by a culture method (the BacT/
Alert system) as a release test. In most countries, only
aerobic culture bottles are inoculated 24–48 h after collection. The positively signalled PCs (and corresponding
components) are removed from inventory and further
investigated. The cultures are continued for up to
7 days if they remain negative even if the PCs have
been issued.
Both German centres combine late sampling, that is,
48–72 h after collection with a flow cytometric bacterial
screening assay: the BactiFlow method to release PCs,
in one German centre also a 16S PCR test is applied to
pools of 10 units of PCs. In Croatia, Germany (both centres), Italy and Spain, bacterial screening with the BacT/
Alert system is only applied for quality control. Japan
and France do not screen.
For the BacT/Alert screening, definition of ‘initial positive’ is clear: ‘positive signal of culture bottle’. However,
the definition of ‘true positive’ is not uniform. It varies
from simply ‘identification of micro-organisms in initially positive culture bottle’ to ‘same strain of bacteria
in original culture bottle and in repeat culture of PC’ or
‘same strain of bacteria in original culture bottle and in
repeat culture of PC and in retention sample of PC or in
Table 1 Methods of preparation and storage time of PCs
Platelet concentrates
Pooled PC
Apheresis PC
Country
Method
%
Storage
time (days)
Leucoreduced
%
Storage
time (days)
Leucoreduced
Argentinaa
Austriaa
Belgiumb
Canadaa
Canadaa
Hema Quebec
Croatia
Denmarka
France
Germanya,c
Bad Oeyenhausen
Germanya,c
Frankfurt
Hong Konga
Italy
Japan
The Netherlandsa
Spaina
Swedena
PRP
BC
BC (TACSI PL syst)
BC
BC
60
60
?
?
16
5
5
7
5
5
Yes
Yes
Yes
Yes
Yes
40
40
?
?
84
5
5
7
5
5
Yes
Yes
Yes
Yes
Yes
BC
BC
BC
–
82
99
50
–
5
7
5
–
84%
Yes
Yes
–
18
Rare
50
10–15
5
7
5
5
99%
–
Yes
Yes
BC
93
5
Yes
7
5
Yes
PRP
BC
–
BC
BC (TACSI)
BC
Orbisac system
BC
PRP
?
Nearly 100
–
90–95
90%
?
5
5
–
7
7
–
No
Yes
–
Yes
Yes
Yes
?
Rare
100
10–5
10
?
5
Part.
35
7
7
7
Yes
Yes
Yes
Yes
?
7
7
5
Yes
Yes
?
93
7
5
Yes
Yes
UKa
USAd
a
The 100% (pooled PCs and apheresis PCs) bacterial screening.
No detection method is implemented in France either on treated platelets (about 10% of the distributed platelets) or on untreated.
c
5 days only if bacterial screening has been performed, otherwise 4 days.
d
Data from Brecher et al. [2]. (From the reference list of the US contribution on page 280-283).
b
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
258 International Forum
Table 2 Method of 100% screening for bacterial contamination and data of execution method
Culture bottle
Inoc vol. (ml)
Result
Country
Method
Aer.
Anaer.
Pool
Aph.
Time from
Collection
Argentina
Austria
Belgium
Canada
Canada HQ
Croatia
Denmark
France
Germany
Bad Oeyenhausen
Germany
Frankfurt
BacT/Alert
BacT/Alert
BacT/Alert
BacT/Alert
BacT/Alert
BacT/Alerta
BacT/Alert
No
BacT/Alerta
BactiFlowb
BacT/Alerta
BactiFlowb
16S PCRb
BacT/Alert
BacT/Alerta
No
BacT/Alert
BacT/Alerta
BacT/Alert
BacT/Alert
BacT/Alert
eBDS Pall
+
+
+
+
+
+
+
–
+
–
–
–
+
–
8
8
4–8
8–10
10
7–10
5–10
4
8
4–8
8–10
10
7–10
–
20–24 h
20–24 h
1 day
24–30 h
18–24 h
48 h
3–30 h
7
7
7
6
7
7
days
days
days
days
days
days
+
+
+
–
10
6 days
7 days
+
+
+
75–10
75–10
4 days
7 days
+
+
+
–
+
592
5
–
–
?
day 6
48 h
7 days
+
+
+
+
+
+
+
+
–
+
–
5–10
8–10
10
8
8–10
3–4
5–10
–
–
–
8–10
3–4
16–26
day 7
day 1
36–48
24–25
24–25
Hong Kong
Italy
Japan
The Netherlands
Spain
Sweden
UK
USAc
h
h
h
h
Max
Cult time
7 days
7 days
7 days
5 days
5 days
Man.
Auto.
+
+
+
+
+
+
–
+
+
+
+
–
+
+
+
a
These participants only use BacT/Alert in quality control.
BactiFlow = flow cytometric bacterial assay; 16S bacterial PCR applied as a release test of all PCs.
c
Most PCs are tested with either BacT/Alert or eBDS system.
b
blood culture from the patient’. Accordingly, ‘false positive’ knows many definitions as shown in the contributions.
From the results of bacterial screening reported by the
participants, it appears that already a very large number
(more than 1 200 000) of PCs have been tested and that
the frequency of confirmed positive cultures is very low
ranging from 001% to 008%. Many participants highlight the importance of skin disinfection and the introduction of the diversion pouch in the blood bag systems
as effective measures to reduce bacterial contamination
in PCs. Since the introduction of these preventive
actions together with bacterial screening, hardly any
cases of post-transfusion bacteriaemia have been diagnosed, even if PCs were transfused which later proved
to have been contaminated. Presumably, in these cases,
the type of bacteria was not virulent or the number of
bacteria in the PCs at the time of transfusion was too
small.
A total of 10 cases of post-transfusion bacteriaemia are
reported. In nine of these, the culture result was false
negative. For further details, the reader is referred to the
answers.
Several rapid detection methods have been tested
experimentally. Although some of these were found to be
indeed quick and easy to perform, none of them was
found to be more sensitive than the BacT/Alert system.
Combining late sampling with a rapid but less sensitive
test may be an option. In Germany, an interlaboratory
comparison of various rapid detection methods revealed
promising results of the BactiFlow assay and the 23S
rRNA RT-PCR screening. Both methods detected all samples correctly positive (12 of 12) and negative (8 of 8),
respectively [2]. The BactiFlow is now routinely applied
in the two German centres, and further studies will be
conducted.
In Austria, an in-house-developed bacterial PCR was
found to be more sensitive than other rapid methods.
Harm et al. [3] tested 70 561 PCs with the Pan Genera
Detection (PGD) test. There were only seven true positive
results, that is, an overall contamination rate of 99 per
106 PC. This rate is much lower than that obtained with
the BacT/Alert system (536–969 per 106 PC). It was concluded that the PGD test is clearly less sensitive than the
culture system. In France, the BacTx and the PGD tests
are also being evaluated.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 259
In a survey of methods used to detect bacterial contamination of PCs in the US [4], data were obtained from
40 blood centres and 184 hospital transfusion centres or
blood banks. Apheresis platelets were predominantly
screened with the BacT/Alert system (895%) and 952%
with at least 8 ml of the product. Rapid immunoassays
were used to screen a substantial proportion of wholeblood PCs.
Following extensive studies, France decided not to
implement bacterial screening. On the French Reunion
Island, pathogen inactivation (the Intercept method)
has been implemented in March 2006, mainly because
of the risk of chikungunya transmission. The method is
now implemented in all the French overseas departments (Reunion, Martinique, Guadeloupe and Guyana).
It is also used in Alsace since 2006. Various studies
have been performed in Alsace to see whether pathogen
inactivation can be applied instead of bacterial screening. After the transfusion of 129 183 PCs treated with
Intercept, no cases of transfusion-transmitted bacteriaemia were diagnosed, whereas (retrospectively) 41 such
cases, 7 of which were fatal, occurred after the transfusion of non-treated (and not bacterially screened) PCs
(from a total number of 1 759 780 PCs). In the Frenchspeaking part of Belgium, 90% of the PCs are treated
with Intercept since 2009, and the bacterial screening
was then reduced from 100% to only the non-treated
PCs.
Clinical trials in the Netherlands revealed that platelets
treated with amotosalen–HCL and ultraviolet-A (Intercept) had significantly decreased post-transfusion increments, and the results of the last trial also suggested
decreased haemostatic efficacy [5].
In conclusion, 11 of the 18 participants routinely use a
culture system (the BacT/Alert system) as a release test to
screen all PCs for bacterial contamination. Two participants apply other bacterial assays to screen all PCs, sometimes combined with late sampling. One participant
applied pathogen inactivation on 90% of the PCs and
only screened for bacteria in the remaining PCs, and four
participants only screen for quality control or not at all.
More than 1 200 000 PCs have now been screened
with BacT/Alert. The number of confirmed positive cultures was low. Since the introduction of bacterial screening, hardly any cases of post-transfusion bacteriaemia
have been diagnosed. Nine cases of this transfusion reaction due to a false-negative result of the culture have
been reported. In several countries, experimental testing
of rapid methods to detect bacterial contamination has
shown that none of these techniques are more sensitive
than the BacT/Alert system. Later sampling (day 2 or 3 of
storage of the PC) with a moderately sensitive but faster
assay might be also efficient.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
References
1 Pietersz RNI, Engelfriet CP, Reesink HW, et al.: Detection of
bacterial contamination of platelet concentrates. Vox Sang
2007; 93:260–277
2 Vollmer T, Hinse D, Schottstedt V, et al.: Inter-laboratory
comparison of different rapid methods for the detection of
bacterial contamination in platelet concentrates. Vox Sang
2012; 103:1–9
3 Harm SK, Delaney M, Charapata M, et al.: Routine use of a
rapid test to detect bacteria at the time of issue for nonleukoreduced, whole blood-derived platelets. Transfusion 2013;
53:843–850
4 Brecher ME, Jacobs MR, Katz LM, et al.: for the AABB Bacterial Contamination Task Force. Survey of methods used to
detect bacterial contamination of platelet products in the
United States in 2011. Transfusion 2013; 53:911–918
5 Kerkhoffs JLH, van Putten WLJ, Novotny VMJ, et al.: van
Rhenen DJ on behalf of the Dutch – Belgian HOVON cooperative group. Clinical effectiveness of leukoreduced, pooled
donor platelet concentrates, stored in plasma or additive
solution with and without pathogen reduction. Br J Haematol
2010; 150:209–217
Ruby N. I. Pietersz
Guest Editor
Emeritus Manager R&D
Sanquin Blood Bank NorthWest Region
Amsterdam
The Netherlands
E-mail: [email protected]
Henk W. Reesink
International Forum Editor
Assistant Professor
Emeritus Manager R&D
Sanquin Blood Bank NorthWest Region
Amsterdam
The Netherlands
E-mail: [email protected]
Simon Panzer
International Forum Editor
Professor
Department for Blood Group Serology and Transfusion Medicine
Medical University Vienna
Vienna
Austria
E-mail: [email protected]
S. Oknaian & S. Kuperman
Question 1
In the Blood Donor Center of Hospital de Pediatrıa
S.A.M.I.C. Prof. Dr. J. P. Garrahan, whole-blood-derived
PCs and apheresis PCs are stored up to 5 days.
260 International Forum
Whole-blood-derived PCs are prepared by plasma-rich
platelet method (PRP). Apheresis PCs and pool of three or
four whole-blood-derived PCs are leucoreduced between
20 and 24 h after collection.
using 10% iodopovidone plus 70% alcohol and a diversion
pouch integrated in the blood system to divert the first
30 ml of blood during blood collection in order to reduce
the risk of bacterial contamination.
Question 2
All PCs are tested in order to detect bacterial contamination using BacT/Alert 3D Microbial Detection System
[1]. We apply only aerobic culture (BacT/Alert BPA Culture Bottles bioMerieux, Inc, Durham, NC, USA). Eight, 4
and 25 ml of apheresis PCs, pooled PCs and individual
whole-blood-derived PCs are inoculated, respectively.
Culture is taken between 20 and 24 h after collection. No
quarantine period is established. Personnel is permanently
attending audible and visual alarms of the BacT/Alert
device, which is located in a laboratory in our Donor
Center. Positive results are manually linked to a PC stored
or already sent to a transfusion medicine location. BPA
bottles are incubated for 7 days. A positive signal of the
BacT/Alert device is considered as an initial positive
result. Positive bottles are sent to the microbiology laboratory to identify the micro-organism. On the other hand,
if the PC is available, another sample is taken for an
additional culture using an aerobic bottle for 7 days. If
the second culture detects the same micro-organism as
the first one, the result is assigned as true positive. If no
micro-organism is detected in the original positive bottle
or the second culture is negative, the result is considered
false positive. Any other situation different from the previous ones is assigned as indeterminate, including PCs
that have already been issued as ‘negative to date’. In our
Donor Center, samples were collected from 11 702 PC
units produced between July 2010 and June 2012. The
PCs involved are 4185 apheresis PCs, 1757 pools equivalent to 6089 individual PCs and 1428 individual wholeblood-derived PCs. Thirty-six (031%) samples showed
initially positive results. Twenty-four (021%) exhibited
false-positive results and 11 (011%) indeterminate results.
There was one true-positive result (TPR; 0008%). The following species were found in initial positive cultures:
Staphylococcus coag. neg. (11), Micrococcus spp (4), Propionibacterium acnes (2), Bacillus sp (2), Flarimones spp (1),
Enterobacter cloacae (1), Corynebacterium sp. (1), Proteus
mirabilis (1), Facklamia ssp (1) and Pseudomona stutzerii
(1). Median detection time was 257 h. Staphylococcus
coag. neg. was detected in the TPR in 195 h. About 50%
of PCs with an initial positive culture had already been
transfused. Eleven false-positive results were due to a positive signal without growth of micro-organisms. Adverse
reactions in patients were reviewed if they had been transfused with a unit negative ‘up to now’, which later became
positive. None of these patients had a transfusion reaction
reported. We performed a two-step skin disinfection protocol
Question 3
Surrogate measurements of pH and glucose using dipstick
(Multistix SG; Bayer Health Care, Elkhart, IN, USA) [2]
were assessed in a research study in 2004. We tested 569
PCs (109 whole-blood-derived PCs and 460 apheresis PCs)
before issuing. Ten (175%) PCs showed a positive result
(pH < 7, glucose < 250 mg/dl). Positive PCs were cultured
using BacT/Alert aerobic and anaerobic bottles. Although
two PCs had positive culture result (Propionibacterium
acnes and Corynebacterium sp.), both were negative in a
second confirmatory culture. Dipstick method is rapid,
but showed a high rate of false-positive result. The study
did not include a parallel comparison with a culture
method.
Question 4
Up to now, we did not choose any pathogen inactivation
method. They are not yet available in our country.
References
1 Munksgaard L, Albjerg L, Lillevang ST, et al.: Detection of
bacterial contamination of platelet components: six years’
experience with the BacT/ALERT system. Transfusion 2004;
44:1166–1173
2 Werch JB, Mhawech P, Stager CE, et al.: Detecting bacteria
in platelet concentrates by use of reagent strips. Transfusion
2002; 42:1027–1031
Sebastian Oknaian & Silvina Kuperman
Blood Donor Center
Hospital de Pediatria S.A.M.I.C. Prof. Dr. J. P. Garrahan
Combate de los Pozos 1881 Ciudad de Buenos Aires
Buenos Aires
Zip code 1245 Argentina
Emails: [email protected] and
[email protected]
C. Gabriel
Question 1
We store all products irrespective of the type of donation
up to 5 days. For whole-blood-derived PC pools, we use a
top–top configuration to produce buffy coats and spin a
second time to produce a single platelet unit. These single
units are kept up to 3 days on a shaking device. Six units
with the same blood group are pooled and filtered. Single
units exceeding the 3-day storage period are discarded,
because filtration is inefficient and we follow a policy of
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 261
distributing fresh platelets. About 40% of our PCs are
apheresis PCs and all PCs are leucoreduced.
Question 2
According to a recent publication, we combine two methods: automated blood culture and bacterial PCR [1]. All
PCs are tested with aerobic and anaerobic bottles in the
BacT/Alert system (bioMerieux). Samples of apheresis PCs
are drawn on day 1, that is, 16–24 h postdonation, and
the sample volume is 8 ml for each bottle [2, 3]. Samples
of pooled platelets are drawn with the same procedure.
All bottles are cultivated for 7 days, with the exception
of cord blood, which is cultivated 14 days. An automated
data transfer system sends results every 20 min to the
dispatch centre, which issues all blood products after
checking the system if the required PC is negative to date.
Remaining PCs on day 3 or, with some exceptions on day
4 (on holidays), are resampled and tested in our bacterial
PCR test (Patent: WO2007AT00420 20070904). Initial
positives are all samples, which were positive either by
PCR or by automated blood culture. Initially positive
samples of the BacT/Alert system are cultivated on conventional agar plates (COS/Sch€adler/MacConkey/PVX). If
these cultures are negative, we retest the sample in the
bacterial PCR. If positive, we start microbial identification
by Gram staining, catalase/oxidase/coagulase tests and
biochemical identification (API, bioMerieux).
Initially PCR-positive samples are resampled, retested and
confirmed in the same way. If retesting shows that conventional methods are negative, but PCR positive, we add bacterial sequencing. All donations, in which bacterial strains
were identified (by conventional methods or sequencing),
are considered as true positive. All samples without positive
bacterial identification are considered as false positives.
All inflicted PCs, the corresponding red cell concentrates and plasma units are restrained in the blood centre
or retrieved from hospitals. We discard all PCs and red
cell concentrates, which are initially positive, and plasma
is released for industrial use only if the donation was
classified as false positive. So far we had no case of sepsis
or related serious transfusion event in the last 2 years.
Our general policy is focused on issuing PCs, as fresh as
possible. Therefore, apheresis PCs are restricted to specific
indications and have to be ordered by hospital physicians,
normally 1 day before the planned transfusion. Most
apheresis PCs are issued within 9–32 h after donation.
This policy reduces the risk of serious events from bacterial contamination and ensures better transfusion efficiencies as well as less frequent PC transfusions. In 2012, we
registered 12 PCs as initially positive, 3 had a negative
and 9 a positive bacterial confirmatory results. Interestingly, there were 23 corresponding products already
issued to hospitals, of which 15 were not retrievable.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
None of these products (most of them were erythrocyte
concentrates) were either corresponding to the inflicted
PCs or caused a clinical case of bacterial contamination.
Question 3
The sensitivity of bacterial cultures is still unbeatable.
Rapid assays are usually compared with this deathless biological amplification assay, although their purpose is quite
different. It seems that a photoflash is compared with a
Hollywood film. Either these assays are point-of-care tests
prior to transfusion or performed in line with the production process. As there is also the possibility that bacterial
cultivation methods might not detect bacteria with long lag
phases or slow growth in the medium, it is advisable to retest PCs after day 3. The major goal – in our opinion – is to
use rapid assays as a supplement, when products are on
shelf after day 3. A rapid assay reduces the residual risk,
which remains if a bacterial cultivation system is still negative. We have tested several rapid assays, especially flow
cytometric assays, which have not been sensitive enough.
We have developed a bacterial PCR assay with an additional specific extraction method for Gram-positive
strains, which are usually less efficiently extracted by
common methods (Patent: WO2007AT00420 20070904).
Our multiplex real-time PCR method enables the detection
of more than 420 bacterial species with a sensitivity that
varies between 5 and 30 CFU/ml. Amplification efficiency
is highly dependent on the multiplicity of bacterial genomes, Gram positivity and the homogeneity of primer
binding sites. Therefore, differences in sensitivities may
occur. False positives by PCR are quite frequent if caused
by the Escherichia coli DNA contamination of recombinant reagents. Meticulous purification of enzymes is a prerequisite. In a validation study of the PCR assay, we have
not found a positive PCR result prior to the BacT/Alert
assay. But this comparison is not permissible, as positive
BacT/Alert results may show up long after PCR has been
performed.
In any case, a rapid assay might not compete with a bacterial cultivation assay if used as a screening assay on day
1. However, bacterial PCR is highly sensitive in comparison
with other rapid detection methods and a very valuable
method to test PCs on day 3. At this time, automated blood
culture might not catch positive samples in time, and there
is a great chance that these products might be issued to hospitals. This strategy avoids the release of PCs, which might
not have been found positive by BacT/Alert at on day 1,
and overcomes the time-consuming testing time of
automated blood culture.
Question 4
We have not chosen to use pathogen reduction methods,
as our testing scheme is sufficient, and rapid use of PCs
262 International Forum
(TerumoBCT). The 100% whole-blood-derived PCs and
100% apheresis PCs are leucoreduced.
is in our opinion the most efficient method to avoid serious events with the benefit to serve the patient with
the best possible PC. Improving PC logistics is cheaper
and more efficient and a good alternative to pathogen
reduction methods.
Question 2
YES
A known volume of 4–8 ml of PCs is cultured in aerobic
bottle by the BacT/Alert system on day 1 after collection.
Results are checked automatically every 10 min on BacT/
Alert. When a positive culture is detected, an audible
alarm rings and the PC is taken from the stock. The culture is continued during 7 days for all negative bottles.
All the initially positive (IP) bottles are sent for germ
identification. At the same time, the PCs are seeded again
in an aerobic bottle as duplicate test, and the germ in the
positive bottle is identified. A positive result with the
same germ is considered as true positive (TP). If a discrepancy exists between the tests, the result is considered
as false positive (FP).
Seven-year results are presented in Table 3. Up to May
2009, 100% of PCs were cultured. After this date, more
than 90% were PI treated.
A total of 63 594 PCs have been tested during this period; 008% of PCs have been issued with a negative-todate result and had a positive result after that, and 32%
of these have been transfused. No evidence of transfusion
reactions has been reported in these cases.
Time of initially positive and type of bacteria are
recorded.
References
1 Su LL, Kamel H, Custer B, et al.: Bacterial detection in
apheresis platelets: blood systems experience with a twobottle and one-bottle culture system. Transfusion 2008;
48:1842–1852
2 Brecher ME, Hay SN: Investigation of an isolate of Staphylococcus lugdunensis implicated in a platelet fatality: a possible
advantage of the use of an anaerobic bottle. Transfusion
2007; 47:1390–1394
3 Sireis W, R€
uster B, Daiss C, et al.: Extension of platelet shelf
life from 4 to 5 days by implementation of a new screening
strategy in Germany. Vox Sang 2011; 101:191–199
Christian Gabriel
Red Cross Transfusion Service of Upper Austria
Krankenhausst 7
4017 Linz
Austria
E-mail: [email protected]
A. Rapaille, M. Lambermont, V. Deneys & D. Sondag
Question 3
YES
Question 1
Non-PI-treated and PI-treated PCs are stored for 7 days.
There is no difference between whole-blood-derived PCs
and apheresis PCs. Whole-blood-derived buffy coats are
pooled and treated on the TACSI platelet system
We evaluated the scan system in parallel with routine
BacT/Alert culture during 6 months. The scan system
needs more time to perform the test than the BacT/Alert.
Table 3 Culture results
IP
FP
TP
First positive
Identification
negative and/or
second culture
negative
Identification
positive and
second culture
positive but not
the same ID
Identification
positive and
same ID second
culture
Year
No. of cultures
n
%
n
%
n
%
n
%
2006
2007
2008
2009
2010
2011
2012
Total
17 059
16 214
16 753
5675
1564
2928
3401
63 594
30
30
16
18
2
14
9
119
018
019
010
032
013
048
023
019
4
12
8
2
0
6
7
39
002
007
005
004
000
020
018
006
22
16
6
16
2
6
2
70
013
010
004
028
013
020
005
011
4
3
2
0
0
2
0
11
002
002
001
000
000
007
000
002
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 263
With the scan system, two different problems were
encountered. Test results delay of the BC-PC, in routine
was 2–3 days after collection. For BC-PC, contrary to PC
from apheresis, a background higher than the positive
controls prevented any automated outcomes. The sensitivity was 100 CFU/ml on the scan system compared to 1–
10 CFU/ml on BacT/Alert. We did not have any evidence
of quicker detection with the scan system compared to
BacT/Alert culture.
Question 4
YES
A total of 615 outdated PI-treated PCs were cultured in
aerobic and anaerobic bottles at 7 days from May 2009
to December 2012. No positive culture has been observed.
Andre Rapaille, Micheline Lambermont, Veronique Deneys &
Daniele Sondag
Service du Sang
Belgian Red Cross
Rue du Fond du Marechal 8/1
5020 Suarlee
Belgium
Email: [email protected]
S. Ramirez-Arcos & M. Goldman
Question 1
At Canadian Blood Services, both whole-blood-derived
and apheresis PCs are leucoreduced and stored under constant agitation at 20–24°C for a maximum of 5 days.
Whole-blood-derived PCs are prepared by the buffy coat
(BC) method by pooling BC fractions from four donors,
which are suspended in a plasma unit derived from one
of the four donations [1].
Question 2
Canadian Blood Services uses the automated culture
BacT/Alert 3D system for routine bacterial testing of
PCs. Approximately 15 ml of PCs is transferred into an
integral secondary bag between 24 and 30 h postcollection during week days in most of the Canadian Blood
Services sites. Sampling may occur later for Saturday collections. During sampling, between 8 and 10 ml of PCs is
inoculated into aerobic BacT/Alert culture (BPA) bottle;
anaerobic cultures are not used at Canadian Blood
Services. BPA bottles are then placed in the BacT/Alert
3D incubator for a maximum of 6 days until they are
flagged as either positive or negative. Once sampling is
performed, the PCs are quarantined pending completion
of other tests. Once all required tests are completed, the
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
PCs are labelled and put into inventory. Therefore, PCs at
Canadian Blood Services are released as ‘negative to date’
for bacterial culture. If a BPA culture is flagged as positive, the bottle is sent to a microbiology laboratory for
bacterial identification. If the PCs have been issued at the
time they are flagged as positive, the hospital blood bank
is notified to ensure appropriate patient monitoring and
testing. When positive PCs have not been transfused, they
are removed from the hospital or blood centre inventory
and, if possible, resampled for confirmatory cultures.
Co-components of BC-PC are also removed from the
inventory and cultured if possible [2, 3].
The follow-up of initial BPA-positive cultures is performed according to the criteria outlined in the AABB
Bulletin #04-07 (http://www.aabb.org/resources/publica
tions/bulletins/Documents/ab04-7.pdf): (1) initial positive,
when a positive result is obtained during initial testing;
(2) confirmed positive, when an initial test is confirmed
using Gram staining and a repeat culture from the blood
product, a retention sample and/or samples from the recipient. The same micro-organism must be isolated from all
samples; (3) discordant positive, if a different microorganism is identified during repeat testing; (4) false positive due to contamination during sampling (i.e. user false
positives), if there is microbial growth in the BacT/Alert
3D bottle, but no growth in remaining product or retention sample; (5) false positive due to instrument error,
if the culture is flagged positive by the system, but does
not demonstrate microbial growth; (6) indeterminate,
when confirmatory tests cannot be performed or interpreted; and (7) false negative, if the initial test is negative, but a remaining sample is found positive following
testing of a product that has caused a post-transfusion
reaction. The same micro-organism should be isolated
from the component and the recipient.
Between March 2004 and March 2013, 251 928
apheresis PCs have been tested at Canadian Blood Services with 373 (02%) initial positive results. Out of
these, 35 (94%) were true-positive cultures, while 221
(592%) and 55 (147%) were instrument errors and user
false positives, respectively. A total of 350 729 buffy
coat PCs have been screened between October 2005 and
March 2013 with 201 (01%) initial positive results.
From these, 40 (199%) were true positives, while 78
(388%) and 53 (264%) were false positives due to
instrument errors and user contamination, respectively.
True-positive cultures have been detected within the first
2 days of incubation of BacT/Alert cultures, while false
positives have taken up to 4 days of incubation to be
detected. Interestingly, two apheresis PCs, contaminated
with Escherichia coli, collected from the same donor at
two different donations, were captured during routine
PC screening at Canadian Blood Services. Medical
264 International Forum
follow-up of the donor revealed asymptomatic multiple
colonic diverticula, and therefore, the donor was permanently deferred.
Out of the 602 657 PCs tested at Canadian Blood Services between March 2004 and March 2013, including
apheresis units and BC pools, a total of seven adverse
transfusion reactions (all false-negative BacT/Alert
cultures) have been reported for a rate of 001/1000.
Three transfusion reactions were associated with apheresis
PCs and four with BC pools. Micro-organisms isolated
included virulent species such as Staphylococcus aureus
[3] and Serratia marcescens (1); the later resulted in a
fatality [4]. The common skin/mucosa contaminants coagulase-negative Staphylococcus (2) and group A Streptococcus (1) were also identified.
Question 3
A research study examining the incidence of missed bacterial detection during initial screening of BC pools using the
BacT/Alert 3D and the Verax PGD immunoassay was conducted at Canadian Blood Services [5]. Out of 4002 outdated (7–10 days old) BC pools tested, one (0025%) pool
was found to be contaminated with Staphylococcus epidermidis and was captured by both the BacT/Alert 3D system and the Verax PGD immunoassay during repeat
testing. The PGD test was found to be an easy-to-use
method able to detect contamination in this outdated BC
pool in <20 min, a considerably shorter actionable time
than the 4 h taken by the BacT/Alert 3D system to yield a
positive result. Eleven (027%) false-positive PGD tests
were observed in the Gram-positive window of the strip. It
was concluded that a combination of both systems, automated culture by the blood supplier and PGD or any other
rapid method at the hospital end, would ensure that the
occurrence of false-negative cases and the potential consequence of adverse transfusion reactions are minimized.
Question 4
Canadian Blood Services has not yet adopted pathogen
reduction methods to prevent adverse reactions due to
transfusion of bacterially contaminated PCs. However, a
clinical trial of pathogen inactivation of buffy coat platelets is under way.
References
1 Levin E, Culibrk B, Gy€
ongy€
ossy-Issa MI, et al.: Implementation of buffy coat platelet component production: comparison
to platelet-rich plasma platelet production. Transfusion 2008;
48:2331–2337
2 Ramirez-Arcos S, Jenkins C, Dion J, et al.: Canadian
experience with detection of bacterial contamination in
apheresis platelets. Transfusion 2007; 47:421–429
3 Jenkins C, Ramırez-Arcos S, Goldman M, et al.: Bacterial
contamination in platelets: incremental improvements drive
down but do not eliminate risk. Transfusion 2011; 51:2555–
2565
4 Ramirez-Arcos S, Chin-Yee I, Hume H, et al.: A fatal septic
shock case associated with transfusion-transmitted Serratia
marcescens. Transfusion 2006; 46:679–681
5 Ramırez-Arcos S, Kou Y, Mastronardi C, et al.: Bacterial
screening of outdated buffy coat platelet pools using a culture system and a rapid immunoassay. Transfusion 2011;
51:2566–2572
Sandra Ramırez-Arcos & Mindy Goldman
Canadian Blood Services
1800 Alta Vista Drive
Ottawa
Ontario
Canada K1G 4J5
Emails: [email protected] and [email protected]
G. Delage, F. Bernier & M. Germain
Question 1
The shelf life of all our platelet products is 5 days. Since
October 2010, we prepare our platelet pools using the
buffy coat method. All our products are leucoreduced. A
total of 84% of our platelet products are apheresis platelets.
Question 2
All our platelet products undergo bacterial cultures. We
use the BacT/Alert 3D (bioMerieux) blood culture system.
A known volume of 10 ml of product is inoculated into
an aerobic culture bottle; we do not use anaerobic bottles.
We culture platelets 18–24 h after collection. Occasionally, the delay can extend to 40 h, for platelets collected
on weekends.
Platelet products are labelled as cultured and put in
inventory immediately after initiating the incubation.
Culture monitoring is continuous. Immediately upon
detection of a positive signal by the instrument, the product is removed from inventory or recalled if it has already
been distributed to a hospital. Negative cultures are incubated for 7 days before a negative result is entered in our
information system.
All positive signals from the BacT/Alert system are
flagged as initially positive. An initially positive culture
is deemed to be a true positive if a micro-organism is
isolated following subculture of the bottle to growth
media and if the same micro-organism is found upon
reculture of the component or culture of one of the
co-components. It is deemed to be a false positive due
to false-positive alarm if no bacteria are found upon
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 265
subculture to growth media and if reculture of the component or culture of the co-components is negative. It is
deemed to be a laboratory contamination if bacteria are
found on subculture to growth media, but a negative
result is obtained on reculture of the product or culture
of co-components.
The following table summarizes the results of our bacterial cultures of platelets during the period of November
2010 (following introduction of buffy coat platelet pools)
to December 2012.
Gilles Delage, France Bernier & Marc Germain
Hema-Quebec
4045 boul. C^
ote-Vertu
Ville Saint-Laurent
Quebec, Canada
Email: [email protected]
T. Vuk
Question 1
No
True positives
False positives false-positive alarm
False positives laboratory contamination
Apheresis
platelets
Pooled
platelets
51 560
4
22
2
19 252
2
3
1
Time to positive culture varied from 52 h (E. coli) to
17 h (coagulase-negative Staphylococcus) in our true positives.
Of the 56 true-positive cultures we found since bacterial testing of platelets began in February 2005, 2 were
transfused before the positive culture was detected (both
were due to coagulase-negative Staphylococci), and none
of the recipients had any clinical manifestations. The
mean time to detection in these true-positive cultures was
193 h: in only five cases was the time to detection
>24 h.
Since February 2005, we have had two septic reactions following the transfusion of platelet products that
were false negative on culture, one due to Staphylococcus aureus (with patient survival) and one due to group
A Streptococcus that was fatal. Before February 2005,
when only apheresis platelets were cultured, we had a
septic reaction due to Salmonella spp following transfusion of a false-negative apheresis platelet: the patient
survived. In all three cases, the same micro-organism
was detected in both patient blood cultures and culture
of the product.
Question 3
We do not use rapid detection methods.
Question 4
We have not yet introduced pathogen reduction (PR)
methods because these methods have not yet received
regulatory approval in our country. However, we will
reconsider the option of introducing PR once a product is
licensed in Canada.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
In 2012 in Croatia, 18% of the total number of platelet
concentrates are prepared by apheresis. The buffy coat
(BC) method has been predominantly (86%) used in the
preparation of whole-blood-derived platelet concentrates.
All blood establishments employing this method prepare
four unit pools. Prestorage leucoreduction was performed
on 84% of whole-blood-derived PCs and 99% of apheresis PCs. The shelf life of these products is 5 days
irrespective of the method of preparation.
Question 2
In Croatia, universal screening of platelet concentrates for
bacterial contamination is not obligatory by legal provisions. At the national level, bacterial testing of platelet
concentrates is performed in the scope of statistical quality control, whereby German recommendations known as
Minimal Requirements for Sterility Testing of Blood Components [1] are applied on about two-thirds of the overall
production.
The method of aerobic and anaerobic cultivation is
employed for the detection of bacterial contamination, for
which the BacT/Alert (bioMerieux) automated system is
used in 90% of all tests. In these cases, 7–10 ml of the
sample is inoculated per bottle, and the incubation takes
7 days (or to positive result signal) at 36 – 1°C. The products intended for bacteriological control are aged – 3 days
of expiry date. This means that at least 48 h have to elapse
from blood collection (day 0) to cultivation.
At the Croatian Institute of Transfusion Medicine (CITM)
in Zagreb, where more than 60% of all platelet concentrates
are prepared at the national level, the results obtained by
the BacT/Alert system are checked at least twice daily. At
present, the system is not connected to the blood bank software, and thus, there is no automatic blockage or release of
blood products sampled for bacterial testing. In case of an
initially positive result, all blood products in storage are
blocked, while issued blood products are recalled. If the
blood product has already been transfused, intensive
patient monitoring is requested and clinicians are regularly
informed on testing status and results.
266 International Forum
On bacterial testing, a positive signal on the BacT/
Alert system is considered an initially reactive (IR) result.
Every IR sample is submitted to confirmation testing at
the laboratory of microbiology. A test result is considered
confirmed positive (CP) when the same agent is isolated
from the retention sample and/or another blood product
from the same donation. When a bacterial agent is found
in the IR sample and the retention sample cannot be
tested and products from the same donation are unavailable for testing or negative, then the finding is designated as ‘bacterial contamination possible’ (BCP). When
no bacterial agent is detected in the IR sample (instrument error), or when the presence of a bacterial agent in
the IR sample is not confirmed by testing the retention
sample and a product from the same donation (laboratory contamination), the result is designated as false
positive (FP).
At CITM, results of bacterial testing of blood products
are part of the regular quality assurance reports, and
cumulative results for the 11-year period (2000–2010)
have recently been published in Transfusion Medicine [2].
Out of 8459 platelet products, there were 072% of IR,
026% of CP, 021% of FP (laboratory contamination) and
022% of FP (related to testing system) results. In CP cultures, Propionibacterium acnes and Staphylococcus epidermidis were the most commonly isolated agents (364%
each). The mean time to a signal of positive result was
184 (33–840) h in aerobic bottles and 522 (37–1320)
h in anaerobic bottles. This is no surprise considering the
relatively high proportion of bacteria of the genus Propionibacterium, the slow-growing anaerobes, in our platelet
concentrates (455%).
During 8 years (2005–2012), six cases were recorded
of issuing and transfusing a platelet concentrate with
bacterial contamination subsequently demonstrated on
routine quality control. These included P. acnes in four
cases and S. epidermidis in two cases, none of which
induced reaction in the recipients. Apart from the
usually low pathogenicity of P. acnes, other causes of
the favourable outcomes of these transfusions could
only be speculated (e.g. amount of bacteria in the
product, possible antibiotic therapy, patient immune
status, etc.).
Question 3
The rapid detection methods of bacterial contamination
are not employed. As part of routine statistical quality
control, pH is determined on the last storage day; at
CITM, microbiological testing is obligatory for all units
with pH < 64. During the 7-year period (2006–2012),
only 3 of 5004 (006%) platelet concentrates tested had
pH < 64. One of these three products was contaminated
(Staphylococcus aureus). In the same period, 3829 platelet
concentrates were tested for bacterial contamination, of
which 8 (021%) were confirmed positive. These data
indicate that pH as a surrogate marker of bacterial contamination is of limited value because the real risk of
bacterial contamination was 35-fold higher than the one
indicated by low pH. However, these data are not sufficient enough to make relevant conclusion.
Question 4
Decision on the implementation of pathogen inactivation
in platelet concentrates has not yet been made in
Croatia.
References
1 Arbeitskreis Blut: Mindestanforderungen zur Sterilit€atstestung von Blutkomponenten. 23. Sitzung des Arbeitskreises
Blut am 05.06.1997. Bundesgesundheitsblatt 1997; 8:307–
309
2 Vuk T, Barisic M, Hecimovic A, et al.: Bacterial contamination of blood products at the Croatian Institute of Transfusion
Medicine: results of eleven-year monitoring. Transfus Med
2012; 22:432–439
Tomislav Vuk
Croatian Institute of Transfusion Medicine
Petrova 3
HR-10000 Zagreb
Croatia
E-mail: [email protected]
J. Georgsen
Question 1
In Denmark, it is allowed to store platelets, whether
derived from whole blood or apheresis, for 7 days provided that a screening for bacterial contamination is
implemented. All whole-blood-derived platelets are prepared by the buffy coat method, and all are leukoreduced. Apheresis has until recently only been used for
HLA-compatible platelets and in connection with public
holidays (Christmas and Easter) due to lack of buffy coats.
However, due to the decreasing demand for RBC, apheresis platelets will soon be produced routinely as a supplement to buffy coat platelets in the capital region.
Question 2
BacT/Alert has been used in our institution since 1997.
Corresponding culture methods are used in other centres.
Only aerobic bottles are used, and 5–10 ml is the sample
volume.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 267
Sampling is performed >3 and <30 h after collection
of the whole blood (0–24 h after pooling of buffy coats
or collecting of apheresis units). Provided that results of
virus marker screen and blood group control are available, PCs are transferred to inventory of released products without a hold period. Since all blood banks are
hospital blood banks, the status of the culture is checked
manually immediately before PCs are issued to wards
(online connection to the BacT/Alert system in all hospitals issuing platelets). After issuing the PCs, culturing is
continued until the end of the product shelf life, and the
responsible clinician is notified if the culture becomes
positive.
If bacteria are found by confirmative culture, our conclusions (most plausible cause) are as shown in Table 4 as
is time of initially positives and types of bacteria for the
last 6 years.
For all PCs and red cells transfused after a positive
result (Table 4), the recipients’ records were looked
through. No transfusion complications or signs of sepsis
were recorded.
Question 3
No.
Question 4
No.
In 2010, the Danish Health and Medicines agency
appointed a working group regarding possible use of
pathogen inactivation. It was concluded that the reduced
activity of platelets induced by pathogen inactivation
techniques constitutes a greater risk for patients than the
remaining risk of transmissible infections.
Jørgen Georgsen
Medical Director
Department of Clinical Immunology
South Danish Transfusion Service and Tissue Center
Odense University Hospital
Sdr. Boulevard 29
DK-5000 Odense C
Denmark
E-mail: [email protected]
Table 4 Annual data, South Danish Transfusion Service
Year
No. of PCs
Species
Time to
detection
Bottle
PC
SAGM 1
SAGM 2
SAGM 3
SAGM 4
2007
6011
Staph.
Staph.
Negative
Negative
Staph. Epi.
Staph. Epi.
Staph.
Staph.
Negative
Staph. Epi.
Negative
Staph. Epi.
Penumo.
Negative
Staph. Epi.
Negative
Staph. Epi.
Staph. Epi.
Staph. Epi.
Staph.
Bac. spec.
Staph.
Staph.
Bac. cer.
Staph. Epi.
Staph.
Unknown
23 h
4h
10 days
28 h
22 h
Unknown
24 h
44 h
23 h
Unknown
26 h
15 h
Unknown
24 h
>6 days
Unknown
24 h
26 h
19 h
12 h
21 h
24 h
12 h
Unknown
20 h
+
+
–
–
+
+
+
+
–
+
–
+
+
–
+
–
+
+
+
+
+
+
+
+
+
+
–
+
–
Iss.
+
+
–
Iss.
–
–
Iss.
–
–
Iss.
+
Iss.
+
–
–
–
+
Iss.
–
+
+
–
+
Apheresis
–
Iss.
–
+
–
+
–
–
–
–
–
–
–
–
–
Apheresis
–
Apheresis
–
–
–
–
–
–
–
–
–
–
Iss.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Iss.
–
–
–
Iss.
–
–
–
–
–
–
–
Iss.
–
Disc.
Iss.
–
–
Disc.
Iss.
–
–
–
–
–
–
–
Iss.
Iss.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2008
2009
6017
5920
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
268 International Forum
Table 4 (Continued)
Year
No. of PCs
2010
Staph.
5766
2011
5961
2012
6605
Species
Staph.
Staph.
–
?
Staph. Epi.
Staph.
24 h
Staph.
Staph.
Staph.
Corynebac.
Staph. Epi.
Staph.
Staph.
Staph.
Strep.
Strep.
Staph.
Staph.
Bac. spec.
Staph.
Staph.
Staph.
Staph. Epi.
Bac. cer.
Staph. Epi.
Staph.
Time to
detection
Bottle
PC
SAGM 1
SAGM 2
SAGM 3
SAGM 4
24 h
+
–
Apheresis
24 h
+
+
–
–
48 h
Unknown
>7 days
+
>3 days
>5 days
21 h
Unknown
20 h
unknown
<24 h
<48 h
17 h
48 h
27 h
24 h
30 h
30 h
27 h
22 h
24 h
Unknown
24 h
29 h
–
?
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
?
+
+
+
+
+
+
–
–
–
–
–
Iss.
–
–
–
–
–
–
–
–
–
–
–
–
–
Iss.
–
–
–
–
–
–
–
–
–
–
–
–
Iss.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Bottle
overloaded
, Bottle overloaded;
Contamination by
sampling
, Contamination by sampling;
Contamination by TC
production
, Contamination by TC production;
Contamination by
collection/bacteriaemia
–
+
Iss.
–
–
Iss.
–
–
–
–
–
–
?
?
–
–
–
+
–
–
–
+
Iss.
?
–
–
–
–
–
Iss.
–
–
–
–
+
–
–
–
Apheresis
–
–
–
–
–
–
–
–
–
, Contamination by collection/bacteraemia.
P. Morel, C. Naegelen & L. Bardiaux
Question 1
In France, platelet concentrates (PCs), apheresis PCs
(APCs) and whole-blood-derived PCs (WBPCs) are stored
for 5 days. The time at the end of donation is the time of
reference for the calculation of the exact expiry date of
the PCs. The WBPCs are obtained by pooling five buffy
coats. A 280-ml volume of PAS III is added, and the
TACSI method (Terumo, TF-CSPB3F02) provides a leucoreduced WBPC. Leucoreduction of PC is systematic, and
the guidelines require <106 residual leucocytes in a PC
(APCs and WBPCs).
Question 2
No detection by bacterial culture of PCs is now used in
France.
The automated culture of bacteria (i.e. BacT/Alert;
bioMerieux, Marcy L’Etoile, France) has been considered
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 269
as a potential method that could be implemented in
France. BacT/Alert was in use in several blood centres at
the end of 1990s and was evaluated in the middle of
2000s. The national feasibility study had shown the
necessity of significant changes in terms of production
and logistics to implement BacT/Alert, and in 2007, the
choice was not to implement this method in France. Such
a decision was also supported by the belief that pathogen
reduction for PCs would shortly be implemented. Again
in 2012, the different parameters for the use of BacT/Alert
(or another bacterial culture method) were redefined for a
possible implementation in France. If BacT/Alert was to
be implemented, the method would have to take into
account the difficulties (the main difficulty being the
false-negative results in relation to sampling) and to propose the best compromise that guarantees the detection
of a bacterial risk while maintaining a good availability
of PCs for patients. The time of sampling from PCs (APCs
and WBPCs) will have to be performed within 24 h postdonation. If the sampling occurs 24 h after donation,
quarantine will not be considered as necessary. Approximately 20 ml should be taken from PCs: 16 ml is used
for the culture in two aerobic bottles (8 ml each) and
4 ml for quality control (2 ml is enough for CQ checks).
Culture would be extended for 5 days, results are considered as ‘negative in course’ at the time of delivery, and
the final result is obtained by 5 days.
Question 3
Up to now, bacterial detection was not implemented in
France for the detection of contaminated blood products
by bacteria. The implementation of pathogen inactivation
(PI) is still under discussion. Consequently, the possibility
to implement a method for bacterial detection is reconsidered and is evaluated once again. Two rapid detection
methods are being currently evaluated in France, BacTx
(Immunetics, MA, USA) and PGD prime (Verax, MA,
USA). A national study is undertaken in order to confirm
the performance claimed by the manufacturers and to
establish the best conditions for using these rapid tests
based on their design and their constraints. The goal is to
offer rapid tests at the time of PC delivery with a minimum change in PC availability. The results of this study
will be available by the end of 2013.
Back in 2007, the different methods available at that
time for bacterial detection were evaluated and three
were serious candidates (BacT/Alert-BioMerieux; eBDSPall Corp; ScanSystem-Hemosystem). A feasibility study
of these three methods was carried out in seven regional
blood centres. It was shown that considerable adaptations would be needed with all three methods, to implement systematic bacterial detection in PCs at a national
level. In addition, despite these adaptations, the mean
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
time of PC availability would be systematically
increased. As mentioned previously, the choice at that
time was in favour of the implementation of PI for
PCs [1].
At the same time, it must be emphasized that several
additional measures contributing to the prevention of
transfusion-transmitted bacterial infection (TTBI) had
been implemented (skin disinfection, diversion of the first
30 ml, systematic leucoreduction of blood products and
postdonation alert system within the national haemovigilance network). Since then, technological awareness
remained active to identify new methods for detecting
bacteria in PCs and resulted in the rapid test assessments
mentioned above.
Question 4
As mentioned earlier, PI for PCs was experienced in 2007
as being on the verge of national implementation. Such
an implementation being justified not only to manage
residual infectious risk, notably the bacterial residual risk,
but also to prevent emerging risks and the supplementary
risks, especially in French overseas departments (Reunion
Island in the Indian Ocean, the Caribbean islands Martinique and Guadeloupe and Guyana in South America).
The Intercept method has been licensed in France since
2005. The regional blood transfusion centre of the
Reunion Island, due to the risk of chikungunya transmission, implemented the Intercept method as of March
2006, in order to enable the continuation of the local collection and delivery of PCs [2]. PI is now implemented in
all the French overseas departments and allows the use of
the local PC production even when local pathogen outbreaks occur (i.e. dengue fever, chikungunya, Chagas’ disease). The Intercept technology is also used in the
regional blood centre of Alsace since August 2006. As
such, EFS-Alsace is a pilot centre to universally introduce
PI-PCs in real-life conditions to all patients needing a
transfusion of PCs [3] in a region of 2 million inhabitants. The feasibility study of preparing and delivering PCs
(35% APCs and 65% buffy coat PCs) is accompanied by a
mandatory haemovigilance study to detect and report
acute transfusion reactions to the national regulatory
agency (ANSM). More than 129 183 PCs treated by Intercept were transfused to patients. No case of TTBI due to
bacterial contamination of PI-PCs occurred between 2006
and 2012. While during the same period, 1 759 780 of
non-inactivated PCs were delivered in France, and 41
TTBIs (including 7 deaths) were registered by the French
haemovigilance network [4].
When Intercept is used, no additional measure is necessary to prevent TTBI, neither bacterial culture nor rapid
bacterial detection test. Intercept method is deemed
effective to prevent the proliferation of most bacteria
270 International Forum
involved in blood contamination, with the exception of
spores, and to avoid transfusion-related sepsis.
References
1 Pietersz RN, Engelfriet CP, Reesink HW, et al.: Detection of
bacterial contamination of platelet concentrates. Vox Sang
2007; 93:260–277
2 Rasongles P, Angelini-Tibert MF, Simon P, et al.: Transfusion of platelet components prepared with photochemical
pathogen inactivation treatment during a Chikungunya virus
epidemic in Ile de La Reunion. Transfusion 2009; 49:1083–
1091
3 Cazenave JP, Isola H, Waller C, et al.: Use of additive solutions and pathogen inactivation treatment of platelet components in a regional blood center: impact on patient outcomes
and component utilization during a 3-year period. Transfusion 2011; 51:622–629
4 Cazenave JP, Isola H, Kientz D: Pathogen inactivation of
platelets; in: Sweeney J, Lozano M (eds): Platelet Transfusion
Therapy. Bethesda, MD, AABB Press, 2013:119–176
Pascal Morel, Christian Naegelen & Laurent Bardiaux
EFS-Bourgogne Franche-Comte
1, Boulevard Fleming
BP 1937, 25020 Besancon Cedex
France
Email: [email protected]
Jean-Pierre Cazenave
Etablissement Francßais du Sang (EFS)-Alsace
10, rue Spielmann
BP36, 67450 Strasbourg Cedex
France
Email: [email protected]
J. Dreier, T. Vollmer & C. Knabbe
Question 1
In Germany, the National Advisory Committee Blood
(NACB; Arbeitskreis Blut) of the German Federal Ministry of Health and Social Security is an advisory board
as stated in §24 of the Transfusion Law to advise the
federal government in matters of safety in the yield and
application of blood and blood products. In 2009, the
shelf life of both whole-blood-derived PCs and apheresis
PCs was reduced in Germany to 4 days (4 9 24 h) after
the day of production according to Vote 38. PCs treated
with pathogen reduction techniques (PRTs) were
excluded from this regulation. The application of bacterial screening methods is an alternative way of lowering
the infection risk of PCs. In recent studies, we demonstrated the implementation of the BactiFlow test as a
routine method for bacterial screening of PCs and introduced a new screening strategy using this assay, which
validated extension of platelet storage back to the
former German regulation of 5 days [1]. We were the
first transfusion facility in Germany to gain acceptance
to extend PC shelf life to 5 days. The Uni.Blutspendedienst OWL (ILTM, Bad Oeynhausen, Germany)
exclusively prepares apheresis-derived PCs (double PCs)
with standard processing using the Haemonetics MCS+
(Haemonetics
GmbH,
M€
unchen,
Germany).
This
plateletpheresis system uses an inline negatively charged
leucodepleting filter.
Question 2
Detection of bacterial contamination of PCs by culture
methods is utilized only for quality control screening at
the end of the PC shelf life using the BacT/Alert system
(bioMerieux, N€
urtingen, Germany) with aerobic and
anaerobic bottles. The current announcements of the
NACB describe the minimum requirements for the microbiological control of blood components for transfusion
(Vote 43, 2012).
Question 3
In the last 10 years, we have developed and/or evaluated several rapid screening methods for screening of
bacterial contamination of PCs, including a bacteriaspecific NAT assay, the BactiFlow flow cytometric assay
and the PGD immunoassay. The sensitivity of our initially developed 23S rRNA RT-PCR assay for the detection of bacterial contamination in PCs was further
enhanced by modifying sample volume, nucleic acid
input, probe technology and the nucleic acid extraction
methods [2]. High-volume nucleic acid extraction
improved the lower detection limit (LOD, 95%) of the
assay to 29 and 22 colony-forming units (CFU)/ml for
Staphylococcus epidermidis and Escherichia coli, respectively. Due to limitations of NAT screening in workflow,
flexibility and test performance (mainly time to result),
we developed a flow cytometric analysis method with a
diagnostic sensitivity of 300 CFU/ml, called BactiFlow
assay [3, 4]. In parallel, we evaluated a comparatively
new rapid screening method, the FDA-licensed Pan
Genera Detection assay. Our study demonstrates that the
PGD immunoassay is an easy-to-perform bedside test
for the detection of bacterial contamination in PCs, but
this assay has presented some shortcomings regarding
the interpretation of results and assay sensitivity, especially in the detection limits for some Gram-negative
strains [5]. Due to these shortcomings, we implemented
the BactiFlow flow cytometric assay as a routine
in-process quality control in our transfusion facility. We
have observed a percentage rate of false positives
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 271
ranging from 005% to 17% since the introduction of
the BactiFlow assay in 2009 [1, 3, 4]. Bacteria were
only detected by cultural methods in ten PCs, but retrospective analysis of bacterial growth kinetics indicates
that (1) bacteria were not able to proliferate in PCs
under PC storage conditions or (2) corresponding bacterial titres were most likely below the BF analytical
detection limit and probably had no transfusion relevance. The sensitivity of cultural method with approximately 1 CFU/ml is superior compared to the BactiFlow
assay (300 CFU/ml); however, the flow cytometric assay
is not considered as a sterility test, but rather as a
point-of-issue test to identify transfusion-relevant bacterial titres. Inoculation experiments revealed that results
obtained by the BactiFlow assay were available approximately 15 h postsampling, independent of the PC-contaminating bacterial titre. Culture methods require a
minimum of 3 h of incubation in the case of PC-contaminating bacterial titres >10E+06 CFU/ml, considerably
increasing with decreasing contaminating bacterial titres
[2]. BactiFlow proved sufficient as a rapid screening
method possibly promoting pretransfusion testing of
PCs, with an acceptable time to result of 15 h, a high
specificity and sensitivity.
In September 2010, we performed a national interlaboratory comparison of different rapid methods for the
detection of bacterial contamination in PCs [6]. Samples
were blinded with a random order for each screening
method, shipped to partners and analysed with different
rapid screening methods immediately after receipt. The
interlaboratory comparison revealed that the BactiFlow
assay and 23S rRNA RT-PCR screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan
Genera Detection (PGD) assay detected only four of the
positive samples. Four of the non-detected positive samples were below the assay’s detection limit. Another four
inoculated samples with comparatively high bacteria
counts were detected false negative, including strains of
E. coli, Klebsiella pneumoniae, and Staphylococcus
aureus. All rapid screening methods revealed no falsepositive results. In conclusion, both BactiFlow and 23S
rRNA RT-PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs.
We are currently planning a pilot collaborative trial for
the detection of bacteria in PCs with an external quality
control panel. Regular successful participation in proficiency testing demonstrates the efficiency of the analytical sensitivity and all processes of rapid bacterial
screening methods under routine conditions and is the
basis to extend platelet shelf life back to 5 days.
Question 4
Not applicable.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
References
1 Vollmer T, Engemann J, Kleesiek K, et al.: Bacterial screening
by flow cytometry offers potential for extension of platelet
storage: results of 14 months of active surveillance. Transfus
Med 2011; 21:175–182
2 Dreier J, Stormer M, Kleesiek K: Real-time polymerase chain
reaction in transfusion medicine: applications for detection
of bacterial contamination in blood products. Transfus Med
Rev 2007; 21:237–254
3 Dreier J, Vollmer T, Kleesiek K: Novel flow cytometry-based
screening for bacterial contamination of donor platelet preparations compared with other rapid screening methods. Clin
Chem 2009; 55:1492–1502
4 Vollmer T, Dreier J, Schottstedt V, et al.: Detection of bacterial contamination in platelet concentrates by a sensitive flow
cytometric assay (BactiFlow): a multicentre validation study.
Transfus Med 2012; 22:262–271
5 Vollmer T, Hinse D, Kleesiek K, et al.: The Pan Genera Detection immunoassay: a novel point-of-issue method for detection of bacterial contamination in platelet concentrates.
J Clin Microbiol 2010; 48:3475–3481
6 Vollmer T, Hinse D, Schottstedt V, et al.: Inter-laboratory
comparison of different rapid methods for the detection of
bacterial contamination in platelet concentrates. Vox Sang
2012; 103:1–9
J. Dreier & T. Vollmer
Institut f€
ur Laboratoriums-und Transfusionsmedizin
Herz- und Diabeteszentrum Nordrhein-Westfalen
Universit€atsklinik der Ruhr-Universit€at Bochum
Georgstrasse 11
32545 Bad Oeynhausen
Germany
Emails: [email protected] and [email protected]
C. Knabbe
Director
Institut f€
ur Laboratoriums-und Transfusionsmedizin
Herz- und Diabeteszentrum Nordrhein-Westfalen
Universit€atsklinik der Ruhr-Universit€at Bochum
Georgstrasse 11
32545 Bad Oeynhausen
Germany
Emails: [email protected]
E. Seifried & K. Hourfar
Question 1
In Germany, since the year 2009, the maximum shelf life
of platelet concentrates is reduced to 4 days. The German
competent authority for blood and blood products, however, allowed to extend the maximum shelf life of platelet
concentrates to 5 days if adequate screening methods
or pathogen reduction technologies are established.
272 International Forum
Regarding shelf life, there are no differences between
whole-blood-derived and apheresis PCs.
For preparation of whole-blood-derived platelets, four
buffy coats were pooled by sterile docking and mixed
with 200 ml of additive solution (T-Sol; Baxter, Frankfurt, Germany). Following soft-spin centrifugation at
300 g for 11 min, the platelet-rich plasma is transferred
to a storage container using an automated device (Compomat; Fresenius Hemocare, Bad Homburg, Germany). All
platelet concentrates are filtered for leucocyte reduction.
Question 2
At our blood donor service, culture methods are only used
for quality control. Approximately 1% of samples are tested
using the BacT/Alert system. The aerobic and anaerobic
bottles of the BacT/Alert are inoculated with 75–10 ml of
the PCs. Culture is taken at the end of platelet shelf life that
is at the moment day 4 after blood donation or later.
Samples were cultured for 7 days within the BacT/Alert.
An automated culture check is implemented, but this is
not relevant for PCs, because platelets used for quality
control are not issued for transfusion. However, RBC and
FFP produced from the same donations are held if the
result for the PC is positive.
PC units with a positive signal in at least one of the
two culture bottles of BacT/Alert, but without identification of bacteria in a reference laboratory, are considered
as being non-specifically reactive. PC units with a positive signal in at least one of the culture bottles and bacterial identification from this culture, but no confirmation
by a second culture from the sample bag or the original
PC unit, were considered as potentially positive. PC units
with positive signal in the culture, identification of bacteria and a positive signal in a second culture from the
sample bag and/or the original PC unit were considered
confirmed positive [1].
Based on our quality control measurements, 007% of
PCs are true positive (in 80% P. acnes).
Question 3
Our blood donor service has evaluated several rapid
detection methods regarding their eligibility for use in
routine screening of platelet concentrates. Besides
in-house-developed FACS-based and PCR methods [2],
several commercial methods such as Scansystem [3] and
BactiFlow [4, 5] have been studied.
From these methods, the in-house developed and now
CE-certified 16S PCR method and the FACS-based BactiFlow method have been shown to be suitable for use in
routine testing. We have implemented both methods into
routine screening at our blood centres. For both methods
we use the strategy of a late sampling (>48 h after donation for 16S-PCR; >72 h after donation for BactiFlow).
The PCR method for us has the benefit that it is also
validated for minipool screening in pools of 10. Additionally, the nucleic acid extraction of bacterial DNA can be
performed fully automated and simultaneously with the
isolation of viral nucleic acids using our NAT instrument
Zelos x100. Therefore, screening of PCs for bacterial contamination by NAT is associated with little effort. Time to
result for up to 220 PCs is approximately 35 h.
In contrast, the BactiFlow method is currently validated
for individual donation testing only.
The hands-on-time for operating the semiautomated
BactiFlow is comparably high than the PCR method.
Additionally the throughput is only 25 samples (23 PCs
and two controls) per run. The time to result for these
samples is approximately 45 h.
With respect to the percentage of false positives, both
methods have shown to be comparable. The rates of initially reactive (repeatedly reactive) samples not confirmed
by retesting were 08% (009%) and 11% (011%) for 16S
PCR and BactiFlow, respectively. The sensitivities of both
methods have also shown to be in the same range.
Regarding the sensitivity, we think that a direct comparison to the culture methods is not adequate. Without
any doubt, the sensitivity of culture methods is very high.
However, due to the early sampling, these methods are
associated with the risk for a sampling error. In contrast
due to incubation of the PCs for at least 48 h before
sampling, this risk is highly reduced for the rapid detection methods. Almost all fatal septic reactions that were
reported after transfusion of platelet concentrates in Germany were associated with PCs near the end of their shelf
life (day 4 or day 5). Therefore, we are convinced that a
strategy using late sampling and testing with moderately
sensitive rapid detection methods will be highly efficient
in preventing transfusion-associated septic reactions.
Question 4
We are evaluating different pathogen reduction methods
for PCs, plasma and red cells. However, until now, these
methods are not routinely used in our blood centres. As
long as different pathogen reduction methods are needed
for different blood components, it will be difficult to
implement those systems into routine.
References
1 Schrezenmeier H, Walther-Wenke G, Muller TH, et al.: Bacterial
contamination of platelet concentrates: results of a prospective
multicenter study comparing pooled whole blood-derived
platelets and apheresis platelets. Transfusion 2007; 47:644–652
2 Schmidt M, Hourfar MK, Nicol SB, et al.: A comparison of
three rapid bacterial detection methods under simulated reallife conditions. Transfusion 2006; 46:1367–1373
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
International Forum 273
3 Schmidt M, Hourfar MK, Wahl A, et al.: Fluorescence
quencher improves SCANSYSTEM for rapid bacterial detection. Vox Sang 2006; 90:276–278
4 Sireis W, R€
uster B, Daiss C, et al.: Extension of platelet shelf
life from 4 to 5 days by implementation of a new screening
strategy in Germany. Vox Sang 2011; 101:191–199
5 Dreier J, Vollmer T, Kleesiek K: Novel flow cytometry-based
screening for bacterial contamination of donor platelet preparations compared with other rapid screening methods. Clin
Chem 2009; 55:1492–1502
E. Seifried
Medical Director
German Red Cross
Institute for Transfusion Medicine and Immunohematology
Sandhofstrasse 1
60528 Frankfurt
Germany
E-mail: [email protected]
K. Hourfar
German Red Cross
Institute for Transfusion Medicine and Immunohematology
Sandhofstrasse 1
60528 Frankfurt
Germany
E-mail: [email protected]
Fig. 1 BST bacteria ID chart.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
C. K. Lin
Question 1
PCs, whether derived from whole blood or collected by
apheresis, have a shelf life of 5 days. Whole-bloodderived PCs are prepared by the platelet-rich plasma
method and are not leucoreduced. A proportion of the
apheresis platelet concentrates are leucoreduced during
collection.
Question 2
We employ a bacterial culture technique (aerobic only)
for the detection of bacterial contamination in PCs. Five
samples (2 ml each) from each unit of whole-bloodderived PCs are pooled and inoculated into the culture
broth on day 2 for a maximum of 48 h. Results are
checked continuously.
An initial reactive result: An initial positive signal.
A false-positive result: a positive signal, but a negative
result of a subsequent culture.
A confirmed positive result: growth of the same organism in the initial and confirmatory sample.
Less than 90% of the confirmed reactives were detected
within the first 24 h [1].
Bacteria isolated were mainly Bacillus spp. and coagulase-negative staphylococci (Fig. 1).
274 International Forum
We do not have data at hand regarding the outcome of
the patients transfused with a negative-to-date PC.
Question 3
We have no experience of using rapid detection methods.
Question 4
A clinical trial of studying the safety profile and clinical
efficacy of Intercept-treated PC will be conducted.
Reference
1 Liu HW, Yuen KY, Cheng TS, et al.: Reduction in platelet
transfusion-associated sepsis by short-term bacterial culture.
Vox Sang 1999; 77:1–5
Che Kit Lin
15 King’s Park Rise
Yaumatei
Kowloon
Hong Kong SAR
E-mail: [email protected]
M. Spreafico, L. Raffaele, A. Berzuini & D. Prati
Question 1
We prepare platelet concentrates from pools of five buffy
coats (BC-PCs) according to the standards in the Council
of Europe (COE), Guide to the Preparation, Use and Quality Assurance of Blood Components, 12th eds. BC-PCs are
stored for a maximum of 5 days after preparation (6 days
after collection).
Briefly, five buffy coats from donors with the same
AB0, Rh and Kell blood groups are pooled and diluted
with a sterile platelet storage solution. After centrifugation
at 491 g for 7 min at 21°C, the resulting solution is separated from erythrocytes and leucocytes. A filtration step is
performed during the squeezing out, making the PC leucoreduced. At our centre, apheresis PCs are rarely collected,
as the number BC-PCs is sufficient for our clinical needs.
However, they are regularly used in most Italian centres,
particularly those serving large oncology and onco-haematology departments.
Question 2
Italian transfusion centres are required to comply with
the COE standards. Therefore, at least 5% of PCs are
annually tested for bacterial contamination. In order to
ensure that platelet availability is not compromised, our
quality control is performed at the end of the process of
platelet production, by testing PCs by the BacT/Alert system (bioMerieux, Durham, NC, USA) culture method at
the expiry date of the PC.
During the assembly of each PC, 15 ml of product is
transferred into an integral sampling bag included in the
kit for PC production. For QC, 5 ml of PCs from this
sampling bag is inoculated into both the anaerobic and
aerobic BacT/Alert culture bottles in sterile conditions
(laminar flow hood). Samples are taken from the component at the expiry date, which is day 6 after blood collection, and independently from its issuing. PCs are thus
usually released as ‘negative to date’.
All cultures are incubated in the BacT/Alert 3D incubator for a maximum of 7 days until they are flagged as
either positive or negative. Bacterial growth is automatically and continuously monitored. When the result is negative, culture is not prolonged. When a culture is flagged
as positive, the sample is analysed for bacterial identification. If a positive component has been issued, the physician is notified to ensure appropriate patient management.
A culture is defined as initially positive when a positive result by BacT/Alert is obtained. A true positive
result requires confirmation by Gram stain and subculture
with specific culture media. A false positive result is
defined when confirmation is not obtained.
During 2010–2011, we conducted a project granted by
Regione Lombardia aimed at evaluating bacterial contamination in platelet concentrates. Testing by BacT/Alert
culture method was performed on 100% of produced PCs,
for a total of 514 PCs.
Only 1 of 514 PCs had a confirmed positive result. The
anaerobic culture bottle was flagged as initially positive
304 h after inoculation. Further analyses indicated that
the blood component contained Staphylococcus epidermidis. When the result was obtained, the BC-PCs had been
already released as negative to date and transfused the
day of production (day 1 starting from blood collection).
Clinical and laboratory follow-up of the recipient did not
show signs of infection.
Question 3
Within the same research project, we also evaluated the
performance of three different rapid detection methods,
that is, the platelet Pan Genera Detection (PGD) test system (Verax Platelets PGD Test; Verax Biomedical Incorporated, Worcester, MA, USA) [1–3], the SeptiFast kit
(SeptiFast; Roche Diagnostics, Mannheim, Germany) [4]
and a flow cytometry test (Becton Dickinson, BD, Franklin
Lakes, NJ, USA) [5, 6].
Spiking experiments with Gram-positive and Gramnegative bacteria and growth model studies were performed to evaluate the performance of these rapid tests in
BC-PCs.
BC-PCs were spiked with low level concentrations (1 CFU/
ml) of three micro-organisms (Streptococcus agalactiae,
Staphylococcus aureus and Escherichia coli) and maintained
© 2013 International Society of Blood Transfusion
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International Forum 275
at room temperature in agitation. Every 24 h, until expiry,
aliquots were tested by the three methods, and bacterial concentration was assessed by dilution plate counting.
The PGD test showed a lower sensitivity than that
declared by the manufacturer, especially with regard to
the Gram-negative species (Table 5). Therefore, despite
being easy to use and potentially applicable to perform
tests at release, it was not considered suitable for introduction in clinical practice, due to low sensitivity. The
analytical sensitivity by flow cytometry was comparable
with that obtained with the PGD test, and the test did not
discriminate between Gram-positive and Gram-negative
bacteria. The SeptiFast test resulted to be applicable to
platelets, even if originally developed for whole-blood
testing. It was able to detect, after 5 days of storage in
optimal conditions, an initial bacterial contamination of
≤1 CFU/ml. Nevertheless, it had some drawbacks including the high cost per test and the risk of contamination
from the environment (data not shown). The highest
analytical sensitivity of 1 CFU/inoculation volume was
obtained by the standard culture method BacT/Alert.
detection of bacterial contamination in platelet concentrates.
J Clin Microbiol 2010; 48:3475–3481
4 Casalta JP, Gouriet F, Roux V, et al.: Evaluation of the LightCycler SeptiFast test in the rapid etiologic diagnostic of
infectious endocarditis. Eur J Clin Microbiol Infect Dis 2009;
28:569–573
5 Schmidt M, Hourfar MK, Nicol SB, et al.: FACS technology
used in a new rapid bacterial detection method. Transfus
Med 2006; 16:355–361
6 Mohr H, Lambrecht B, Bayer A, et al.: Basics of flow cytometry–based sterility testing of platelet concentrates. Transfusion 2006; 46:41–49
Marta Spreafico, Livia Raffaele, Alessandra Berzuini &
Daniele Prati
Department of Transfusion Medicine and Haematology
Azienda Ospedaliera della Provincia di Lecco
Alessandro Manzoni Hospital
via dell’Eremo 9/11
23900 Lecco
Italy
Emails: [email protected], l.raffaele@ospedale.
lecco.it, [email protected] and d.prati@ospedale.
lecco.it
Question 4
The possible adoption of pathogen inactivation techniques
is currently debated in Italy, but it is generally not performed outside clinical trials.
References
M. Satake
Question 1
1 Yacobs MR, Smith D, Heaton WA, et al.: Detection of bacterial contamination in prestorage culture-negative apheresis
platelets on day of issue with the Pan Genera Detection test.
Transfusion 2001; 41:1331–1334
2 Dreier J, Vollmer T, Kleesiek K: Novel flow cytometry–based
screening for bacterial contamination of donor platelet preparations compared with other rapid screening methods. Clin
Chem 2009; 55:1492–1502
3 Vollmer T, Hinse D, Kleesiek K, et al.: The Pan Genera
Detection immunoassay: a novel point-of-issue method for
The Japanese Red Cross blood centres are the only facilities authorized to handle blood collection, processing,
testing and delivery in Japan.
All platelet components (PCs) are derived from apheresis. They are universally either leucoreduced by filtration
at donation sites or in-process leucoreduction by the
apheresis machine.
We consider that early issue and use is a priority to
prevent platelet transfusion-related sepsis. Thus, the shelf
Table 5 Characteristics of the methods tested for the screening of bacterial contamination in platelet concentrates
Detection system
Type
Testing
time
BacT/Alerta
(BioMerieux)
Verax Plateletsa PGD Test
(Verax Biomedical Incorporated)
SeptiFast
(Roche Diagnostics)
Flow cytometric analysis
(Becton Dickinson)
REFERENCE: culturing method
5 days
RAPID: immunoassay
30–40 min
RAPID: nucleic acid amplification
8h
RAPID: Fluorescence-activated
cell sorting (FACS) analysis
15 min
a
FDA approved.
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
Bacteria detected
Aerobic, anaerobic,
Gram+, Gram- spp.
Gram+, Gram25 spp. among Gram+,
Gram- and Fungi
Gram+, Gram- with
absolute count
Declared
sensitivity
(CFU/ml)
Observed
sensitivity
(CFU/ml)
Cost and
execution
10–100
1–10
Low
103–105
By 105
Low
3–10–100
10–100
High
10
By 103
Low
276 International Forum
life of PCs in Japan was only 3 days (72 h) until 2007,
after which it was extended due to difficulties with logistics. The current storage limit for PCs in Japan is set at
12:00 p.m. on day 3 when blood is collected on day 0.
As the earliest PC collection will be completed around
11:00 a.m. on day 0, the actual maximal storage time is
about 85 h (35 days). With these logistics, the annual
rate of discarded PCs is 187%. This is achieved mainly
by having a wide distribution area and frequent exchange
of products between blood centres.
Due to the short shelf life of PCs, we do not perform
either release tests using culture methods or rapid tests.
During the past 6 years under these conditions, seven
patients developed a septic reaction related to PC transfusion, two and five of which were caused by PCs that had
been stored for 2 and 3 days, respectively. One fatality
related to contamination with S. aureus occurred before
the introduction of leucoreduction and initial flow diversion. In accordance with national regulations concerning
quality control for pharmaceutical facilities, we randomly
select one out of every 500 PC products and subject it to
culture testing. Among around 5500 PCs that were cultured during the past 3 years, three were contaminated by
Propionibacterium acnes.
Visual inspection of the PC product is incorporated into
the SOP at the distribution divisions in blood centres. We
have strongly recommended the visual inspection of PCs
at medical facilities. As a result, six PCs with abnormal
appearance over the past year were found to be contaminated by bacteria: two were identified before release at
blood centre distribution divisions and four were identified at medical facilities. One of them was identified on
day 2 of storage, and the other five were identified on
day 3.
Question 2
We do not perform culture tests to detect contaminated
PCs.
Questions 3 and 4
We have never used rapid detection methods to detect
bacterial contamination. We have not yet implemented
pathogen reduction measures, but we are presently evaluating that technology in vitro.
Masahiro Satake
Japanese Red Cross Central Blood Institute
2-1-67, Tatsumi
Koto-ku
Tokyo
Japan
E-mail: [email protected]
D. de Korte, P. F. van der Meer & J.-L. Kerkhoffs
Question 1
Platelet concentrates (PCs) are stored for 7 days after
collection. Storage time is the same for either wholeblood-derived PCs or apheresis PCs, for storage in
plasma as well as for storage in a mixture of plasma
and additive solution (PAS III or Intersol, Fenwal, Lake
Zurich, USA; ratio about 38:62). Whole-blood-derived
PCs are produced from pooled buffy coats, and all PCs
are leucodepleted. The majority of our supply is wholeblood-derived PCs (90–95%).
Question 2
A culture method (BacT/Alert; BioMerieux, Marcy l’Etoile, France) with two bottles (aerobic and anaerobic) is
used, with inoculation of on average 75 ml per bottle
(range 5–10 ml). Inoculation for whole-blood-derived
PCs is at 18–26 h after collection, and for apheresis PCs,
it is 4–24 h after collection (preferably within 12 h after
collection). Results of cultures are monitored continuously and transferred automatically to the blood bank
information system. In case of a positive signal, issuing
of the products is automatically prevented. Cultures are
continued for 7 days or until positive. Our definition of
initially positive is a positive signal for one or both
inoculated bottles. True (or confirmed in our definitions)
positivity is defined as an initially positive signal and
when a micro-organism can be cultured from the positively flagged bottle as opposed to false positivity when
no micro-organism can be cultured. No additional cultures are performed on samples from units with a positive signal.
After introduction of the diversion pouch [1], over the
period 2004–2011, we found an average initially positive
rate of 046%, with a mean time to a positive signal of
43 h (range 0–168 h; median 24 h) for the aerobic bottle
and 80 h (range 0–168 h; median 91 h) for the anaerobic
bottle. Positivity of the initially positively flagged bottles
could be confirmed in 80% of samples [2]. In only 4% of
the positively flagged units, both bottles were positive,
indicating a low initial load with bacteria of the contaminated units. The majority of micro-organisms identified
were diphtheroid bacteria (about 50% of confirmed cases,
mainly in the anaerobic bottle) and Staphylococcus bacteria (about 40% of confirmed cases, both in the aerobic
and anaerobic bottles). In much lower frequency, we
found Bacillus species, Peptostreptococci, Streptococci and
a variety of Gram-negative rods.
Over the years in many cases, units were issued as negative to date that became culture positive after being
issued [3]. From these units, about 70% were already
© 2013 International Society of Blood Transfusion
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transfused. Over the years 2004–2011, this involved 303
units, with follow-up for all involved patients. In none of
these cases, a transfusion reaction was reported, as also
published for part of this period [3].
Additionally, we performed a study on outdated wholeblood-derived PCs in which we recultured samples in the
BacT/Alert system on days 8–10 to detect false negatives.
In 4000 units, we found 4 (01%) positives, all with coagulase-negative Staphylococci, and rapid growth in the
culture bottles (within 1 day positive). Clinically, in the
period 2008–2011, five cases of suspected transfusiontransmitted bacteraemia were reported in approximately
200 000 platelet transfusions (i.e. 005&). Only in one
out of these five cases, the final adjudication on imputability was certain; in three cases, the imputability was
probable or possible; and in one case, two different
strains were isolated from the patient blood and platelet
bag, resulting in unlikely imputability (TRIP annual
reports via website, http://www.tripnet.nl/pages/nl/publi
caties.php, last visited 26 April 2013).
a randomized controlled multicentre trial is performed
testing the haemostatic efficacy of platelets treated with
riboflavin and ultraviolet B (Mirasol; Terumo BCT,
Lakewood, CO, USA). As a site study of this trial, the
efficacy of pathogen reduction is tested. Platelet
concentrates before and after Mirasol treatment will be
screened using classic culturing techniques. Further,
the prevention of transmission of commensal viruses
by pathogen-reduced platelet concentrates will be
studied.
References
1 de Korte D, Curvers J, de Kort W, et al.: Effects of skin disinfection method, deviation bag and bacterial screening on
clinical safety of platelet transfusions in the Netherlands.
Transfusion 2006; 46:476–485
2 de Korte D: 10 Years Experience with Bacterial Screening of
Platelet Concentrates in the Netherlands. Transfus Med
Hemother 2011; 38:251–254
3 Koopman MM, van’t Ende E, Lieshout-Krikke R, et al.:
Bacterial screening of platelet concentrates: results of
2 years active surveillance of transfused positive cultured
units released as negative to date. Vox Sang 2009;
97:355–357
4 van Rhenen D, Gulliksson H, Cazenave J-P, et al.: Transfusion of pooled buffy coat platelet components prepared with
photochemical pathogen inactivation treatment: the euroSPRITE trial. Blood 2003; 101:2426–2433
5 Kerkhoffs JLH, van Putten WLJ, Novotny VMJ, et al. on
behalf of the Dutch – Belgian HOVON Cooperative Group:
Clinical effectiveness of leukoreduced, pooled donor platelet
concentrates, stored in plasma or additive solution with and
without pathogen reduction. Br J Haematol 2010; 150:209–
217
Question 3
We have no data on experience with any of the rapid
detection methods available. However, in our opinion,
the frequency of false positives with the methods
described thus far is much too high. A problem with
applying these methods in our blood bank would be that
a significant part of our stock is located in hospitals,
hampering the performance of a rapid release test shortly
before transfusion under the responsibility of our blood
bank. An interesting development is the use of PCR on
samples taken 48–72 h after collection. The late sampling
allows micro-organisms to grow in the PCs during the
first days of storage, with subsequent relatively rapid
detection (about 6-h analysis time) in a PCR method with
low sensitivity. In Germany, this method is used to
extend shelf life from 4 to 5 days. If this method can be
validated with samples taken 36 h after collection in
combination with 7 days of shelf life, it might be possible to perform this test before release of PCs from the
blood bank to the hospitals. However, because of a relatively low sensitivity, it is unknown how long PCs can be
stored safely after a negative test result in this PCR.
Dirk de Korte
Department of Blood Cell Research
Sanquin Research
Amsterdam
the Netherlands
Department of Product and Process Development
Sanquin Blood Bank
Amsterdam
the Netherlands
Question 4
In the Netherlands until now, pathogen inactivation of
platelet products has only been used in clinical trials.
Two studies tested the transfusion efficacy of platelets
treated with amotosalen HCl and ultraviolet A (Intercept; Cerus, Concord, CA, USA) [4, 5]. Both trials
showed a significantly decreased transfusion efficacy in
terms of count increments. Moreover, the last trial also
suggested decreased haemostatic efficacy [5]. Currently,
Pieter F. van der Meer
Department of Blood Cell Research
Sanquin Research
Amsterdam
the Netherlands
Center for Clinical Transfusion Research
Sanquin Research
Leiden
the Netherlands
© 2013 International Society of Blood Transfusion
Vox Sanguinis (2014) 106, 256–283
278 International Forum
Jean Louis Kerkhoffs
Center for Clinical Transfusion Research
Sanquin Research
Leiden
the Netherlands
Department of Hematology
Haga Hospital
The Hague
the Netherlands
Lydia Blanco
Centro de Hemoterapia de Castilla y Le
on
Pº Filipinos s/n
47007 Valladolid
Spain
E-mail: [email protected]
J. Kjeldsen-Kragh & A.-M. Svard-Nilsson
Question 1
L. Blanco
Question 1
We store whole-blood-derived PCs and apheresis PCs up
for 7 days.
Whole-blood-derived PCs are prepared with five buffy
coats and filtered. The 100% of PCs are inactivated with
amotosalen.
Question 2
Yes, but only for quality control in 3–5% of PCs.
•
•
•
•
•
•
The method is BacT/Alert.
We use aerobic and anaerobic culture methods.
The volume for culture is 8–10 cc.
The day of culture is day 7 after blood collection.
The cultures were evaluated every 7 days.
We do not perform tests in issued units. Our quality
control samples remain in culture for 1 week.
Initially positive: BacT/Alert positive, confirmatory test
not performed.
True positive: BacT/Alert positive, confirmatory test
positive and identification of bacteria.
False positive: BacT/Alert positive, confirmatory test
negative.
We register all positive results.
•
•
No.
No.
Question 3
No.
Question 4
Yes.
•
•
Before introducing pathogen inactivation (5 years
ago), we validated the method using a rapid bacterial
detection test before and after pathogen inactivation.
We did not find any positive result.
No.
At our institution, both whole-blood-derived PCs and
apheresis PCs are stored for 7 days, that is, a PC produced
from a donation on a Monday will be outdated the following week at midnight between Monday and Tuesday.
Whole-blood-derived PCs are produced from four buffy
coats using the OrbiSac System. All PCs are leucoreduced.
Question 2
The Bact/Alert 3D microbial detection system is used for
surveillance of all produced PCs. On day 1, that is, the
day after donation, a 10-ml sample is transferred to a
culture bottle (BacT/Alert FA Plus). We do not use culture bottles specifically optimized for the detection of
anaerobic bacteria. The results of the cultures are not
linked to the blood bank information system, but an
alarm from Bact/Alert system will immediately notify the
staff on duty at the immunohaematology laboratory. In
case of an alarm, the implicated PC will immediately be
removed from the platelet inventory. The cultures are
continued until the PCs are outdated. In case of an alarm
after a PC has been issued to a patient, the blood bank
physician on duty will call the clinical department and
investigate the BacT/Alert alarm.
An alarm from the BacT/Alert system is defined as an
initial positive signal, and if bacteria are detected in the
PC, it is redefined as a true positive alarm. We categorize
an alarm as false positive if no bacteria are detected in
the culture bottle or the PC after an initial positive alarm.
As the current routine in our institution was recently
implemented, we cannot provide annual data regarding
type and number of positive results.
Questions 3 and 4
We do not have experience with any methods for rapid
detection of bacterial contamination or any of the pathogen reduction technologies.
Jens Kjeldsen-Kragh & Ann-Margret Svard-Nilsson
Department of Clinical Immunology and Transfusion Medicine
University and Regional Laboratories Region
Skane Akutgatan 8
© 2013 International Society of Blood Transfusion
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International Forum 279
Question 2
221 85 Lund
Sweden
Email: [email protected]
C. P. McDonald, I. Symonds, R. Moule & S. Brailsford
Question 1
The shelf life of National Health Service Blood and Transplant platelet components is now 7 days since the introduction of bacterial screening. The previous shelf life was
5 days. There is no difference between whole-bloodderived platelet components and apheresis components.
The preparation method for whole-blood-derived platelet components is collection in bottom and top (BAT)
packs. These are then processed either on day 0 (80–90%)
or early day 1 into buffy coats. The buffy coats are then
stored for a minimum of 4 h at 18–22°C postprocessing
before being pooled on day 1 (a.m. or p.m., dependent on
workload). The pools are of four buffy coats and the
plasma from one (male) donation from the four.
All NHSBT platelet components are leucodepleted.
Table 6 Initial reactive and confirmed positive rates
Platelet
components
Apheresis
Pooled
Total
Number
Initial
reactive
rate (%)
Confirmed
positive
rate (%)
265 157
47 220
312 377
060
040
057
002
008
003
Screening of apheresis platelets began in February 2011
with screening of pooled platelets following in June 2011.
Table 7 Detection times of confirmed positives
Time to
detection (h)
Gram positives
Propionibacterium spp. (42)
Staphylococcus spp. (18)
Streptococcus spp. (14)
Lactobacillus sp. (1)
Lactococcus sp. (1)
Corynebacterium sp. (1)
Listeria monocytogenes sp. (1)
Peptostreptococcus sp. (1)
Granulicatella sp. (1)
Actinomyces sp. (1)
Aggregatibacter sp. (1)
Gram negatives
Escherichia spp. (2)
Klebsiella spp. (2)
© 2013 International Society of Blood Transfusion
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74–132
15–69
7–41
37
21
71
14
60
20
41
50
4
4–11
(1) The BacT/Alert automated microbial detection system
is used for the bacterial screening of all NHSBT platelet
components.
(2) Aerobic and anaerobic cultures are performed.
(3) The culture volume taken from the platelet bag is
16 ml (8 ml into an aerobic and 8 ml into an anaerobic
culture bottle). All adult splits are screened, as we have
detected contamination in one, but not the other units
from the same donation.
(4) The culture bottles are inoculated 36–48 h post
donation.
(5) The BacT/Alert system sends results to our computer
system every 10 min. A positive result will automatically
hold all associated components.
(6) The culture bottles are incubated for the shelf life of
the component (7 days of shelf life).
(7) These are our definitions for automated blood culture
bacterial screening:
Initial reactive: Positive bottle or bottles on initial
screen.
False positive: A positive signal is obtained for a culture bottle, but no bacteria are detected on subculture.
Repeat reactive: Positive bottle or bottles from repeat
testing of the index unit.
False negative: Initial screen test negative, but component associated with a confirmed bacterial transfusion
reaction or subsequently untransfused found to be
contaminated with bacteria, that is, rejected by visual
inspection.
Confirmed positive: Both the initial screen bottle and
the index and/or associated pack(s) are positive, and
the same organism is identified in both.
Indeterminate positive: Bacteria detected in only the
initial screen or repeat tests, but not both, or bacteria
detected in the initial screen or repeat tests, which
cannot be matched at species level, or no index or
associated units available for retest.
Indeterminate negative: There was no growth from the
initial reactive bottle, but a negative result could not
be confirmed because the index pack was not available.
Confirmed negative: No organism was isolated from
the initial reactive bottle and the index pack.
We collect and report weekly, monthly, quarterly and
cumulative data for bacterial screening, which are formally reviewed by NHSBT. Data are given below
for the period February 2011 to June 2012 (Tables 6
and 7).
(8) We have observed cases when the culture has become
positive after the platelet component has been issued to
hospitals. If not transfused, the unit is recalled together
280 International Forum
with any associated units. In the event of the unit or
associated units having been transfused, the clinicians
caring for the recipient are asked to complete a form
which asks whether the recipient experienced an acute
transfusion reaction and, if so, the details of this reaction.
Since the introduction of bacterial screening in February
2011, no adverse reactions due to bacterial contamination
have been observed in recipients from units that have
subsequently been detected as positive post-transfusion.
(9) No confirmed reported cases of bacterial transfusiontransmitted infection have been identified since 2009.
Question 3
(1) We have experience with rapid detection methods, but
these have not been used routinely to screen platelet
components. The methods we have evaluated are the
ScanSystem [1], a dielectropheresis system [2], Verax
PGD and an in-house PCR method [3]. The ScanSystem
and dielectropheresis system are no longer available, so
we will not discuss these further.
(2) The Verax PGD (test time approximately 30 min) and
the in-house 16S PCR (test time approximately 4 h): the
Verax assay is easy to perform, with the PCR assay being
technically more demanding. We have no specificity data
for the Verax PGD assay. Data with regard to PCR assay
performance are presented in references [3, 4].
(3) We have shown culture methods to have a sensitivity
of 1–10 CFU/ml [5]. The sensitivity of the Verax PGD system is claimed by the manufacturer to be 103–105 CFU/
ml, but we found the assay to be less sensitive with
regard to certain organisms, as have other workers
[6, 7].
The sensitivity of the PCR assay was shown to be in
the order of 1–15 CFU/ml [3] in a model system, but may
be slightly reduced when used for screening platelet concentrates [4]. Overall, the sensitivity of PCR is less than
that of BacT/Alert culture, although we have observed
instances in which the PCR assay is able to detect bacterial contamination in platelet concentrates before the
BacT/Alert signal is generated (e.g. 4 h by PCR vs 10 h
by BacT/Alert).
Question 4
NHSBT does not use a pathogen reduction technology to
reduce the transmission of bacteria by platelet component
transfusion.
References
1 McDonald CP, Colvin J, Robbins S, et al.: Use of a solidphase fluorescent cytometric technique for the detection of
bacteria in platelet concentrates. Transfus Med 2005;
15:175–183
2 McDonald CP, Smith R, Colvin J, et al.: Evaluation of a novel
Dielectrophoresis system for the rapid detection of bacteria in
platelet concentrates (abstract). Transfusion 2001; 41(Suppl):
34S
3 Patel P, Garson JA, Tettmar KI, et al.: Development of an
ethidium monoazide-enhanced internally controlled universal
16S rDNA real-time polymerase chain reaction assay for
detection of bacterial contamination in platelet concentrates.
Transfusion 2012; 52:1423–1432
4 Garson JA, Patel P, McDonald C, et al.: Evaluation of an ethidium monoazide- enhanced 16S rDNA real-time PCR assay for
bacterial screening of platelet concentrates and comparison
with automated culture. Transfusion 2013. doi: 10.1111/
trf.12256 [Epub ahead of print].
5 McDonald CP, Pearce S, Wilkins K, et al.: Pall eBDS: an
enhanced bacterial detection system for screening platelet
concentrates. Transfus Med 2005; 15:259–268
6 Ramirez-Arcos S, Kou Y, Mastronardi C, et al.: Bacterial
screening of outdated buffy coat platelet pools using a culture system and a rapid immunoassay. Transfusion 2011;
51:2566–2572
7 Vollmer T, Hinse D, Kleesiek K, et al.: The Pan Genera Detection immunoassay: a novel point-of-issue method for detection of bacterial contamination in platelet concentrates.
J Clin Microbiol 2010; 48:3475–3481
C. P. McDonald, I. Symonds, & S. Brailsford
National Health Service Blood and Transplant
Charcot Road,
London NW9 5BG
UK
Email: [email protected]
R. Moule
National Health Service Blood and Transplant
Bridle Path,
Leeds LS15 7TW
UK
R. Yomtovaian & M. R. Jacobs
Question 1
Platelet concentrates (PCs) are prepared by two regional
blood centres supplying our 947-bed academic medical
centre transfusion service. These are prepared by either
apheresis or the pool and store method (Acrodose; PALL
Medical, Port Washington, NY, USA). The whole-bloodderived (WBD) platelets, used for the pool and store
method, are made from platelet-rich plasma without the
use of an additive solution; the buffy coat production
method is not used [1]. WBD platelet units are leucocyte
reduced at the time of their production such that at one of
the blood centres, greater than or equal to 95% of a 1%
© 2013 International Society of Blood Transfusion
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International Forum 281
sample of each WBD platelet unit contains <83 9 105 leucocytes/mm3. At the other blood centre, four WBD units
are sampled per week (16–20 per month) and 95% must be
below 83 9 105 leucocytes/mm3. The leucocyte content is
not measured in the completed pools. Apheresis platelets
are leucocyte reduced during the collection process.
Platelet storage is generally limited to 5 days following
collection, with the day of collection considered day 0
and midnight on the fifth storage day the time of outdate.
Occasionally, at the discretion of the transfusion service,
the platelet storage time, as permitted by the FDA for
medical need, may be extended up to 7 days. This would
be the case, for example, during a severe inventory shortage or for a difficult-to-obtain specially typed, usually
HLA-matched, platelet unit critical to a patient need.
Question 2
Samples of the apheresis and pool and store platelets,
prepared by both blood centres, are sampled for bacterial
contamination and tested by one of the blood centres
using the BacT/Alert 3D culture system (bioMerieux, Inc,
Durham, NC, USA) and by the other blood centre using
the Haemonetics (PALL) eBDS Enhanced System for the
Detection of Bacteria (Haemonetics Corp, Braintree, MA,
USA). These methods are representative of methods typically used in the USA [2]. With the use of the BacT/Alert
3D culture system, a single aerobic culture bottle per
apheresis collection or pool is inoculated using a sample
volume of 8–10 ml. For apheresis platelets, the culture
sample is procured 24 h from the start of the apheresis
platelet collection, while for WBD platelets, the sample is
obtained 24–25 h from the start of the WBD platelet unit
collections used for the pool and store products. With the
BacT/Alert system, the incubated culture bottles are monitored for up to 5 days for bacterial growth, with a reading performed every 10 min and a loud audible alarm
using the Plexxium system (Mack Information Systems,
Inc, Wyncote, PA, USA) sounding when positive. Bottles
with positive signals are subcultured to determine
whether signals are associated with true or false positives,
and bacterial isolates are identified to determine the bacterial species involved. Platelets are held in the BacT/Alert
system by the blood centre for their entire shelf life of
5 days, but released for transfusion after 12 h of incubation if negative, with continued monitoring of BacT/Alert
bottles for the shelf life of platelet products. When a positive signal occurs in the BacT/Alert system before a unit
is released for transfusion, the unit and its associated
components are removed from inventory and the culture
is repeated. If the repeat culture is positive for the same
organism, it is considered a true positive. If the repeat
culture is negative, the initial result is considered a false
positive. Released units that become positive after release
are recalled if not yet transfused and culture repeated as
above. For those instances where repeat culture is not
possible because the unit has been transfused, it is considered an indeterminate result. For the year 2012, there
was a 016% incidence (n = 21) of initial positives and a
005% incidence (n = 6) of true positives with the BacT/
Alert system, with 12 false positives and 3 indeterminate
cases. The identification of the six true-positive results
was as follows: Staphylococcus epidermidis (n = 3), Streptococcus group C (n = 1), Pseudomonas aeruginosa (n = 1)
and Proteus mirabilis (n = 1).
The blood centre using the eBDS system, whether for
WBD or apheresis units, holds the platelets for a minimum of 24 h following collection, at which time 5 ml of
the PC product is diverted into an integrally attached
eBDS pouch, which is filled by 3–4 ml of the product.
The sampled platelets are incubated in this pouch in a
platelet rocker at 35°C for 18 h, at which time a single
manual reading is made to assess the oxygen content of
the head space. If the oxygen level is <94% threshold,
the test signals that the test has failed and the platelet
unit is considered positive for bacterial contamination.
According to the AABB definition (Association Bulletin
04–07), when the eBDS signals a positive result and the
subsequent culture from the associated unit is positive,
the result is considered a true positive, while a negative
culture from the associated unit indicates a false-positive
result. For the year 2012, there was a 025% incidence
(n = 3) of initial positives with the eBDS system, all true
positives. The identification of the three true-positive
results was as follows: coagulase-negative Staphylococcus
(n = 1), Streptococcus group G (n = 1) and Bacillus species (n = 1).
Table 8 Bacterial contamination detected in whole-blood-derived (WBD) pools and apheresis units at the time of issue from 2004 through 2011
Platelet type
Time period
WBD
Pooled at issue
2004–2006
Pooled at production
2007–2011
Apheresis
2004–2006
2007–2011
Number tested
Number bacterially contaminated
Bacterial contamination rate per million platelet units
4241
10
2538
9608
4
416
11 589
8
690
28 978
17
587
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No cases where the culture performed by the collecting
centre using the BacT/Alert system became positive after
PCs had been released to our institution have occurred.
In addition to the testing performed to detect platelet
bacterial contamination at our regional blood centres, all
platelets released from our transfusion service undergo an
additional surveillance culture, with an aliquot of each
PC obtained at the time of issue for transfusion [3–5].
This culture is performed by the plate culture method,
with inoculation of 01 ml of product on a blood agar
plate that is incubated aerobically at 35°C for up to 48 h.
If a plate culture is positive, a quantitative culture is performed on the PC aliquot retained at 4°C to confirm the
culture result and determine the bacterial load. These
results are available following transfusion and are used to
monitor ‘breakthrough’ cases of platelet bacterial contamination missed by early culture sampling by blood centres, manage instances in which bacterially contaminated
units were infused to patients and correlate the occurrence of transfusion reactions with bacterial species and
load [3]. More extensive cultures, including anaerobic
plate culture and culture in broth, are performed on
platelets administered to patients in whom a septic transfusion reaction is reported.
These surveillance cultures, over a longitudinal time
period of now over 20 years, have provided valuable
data on the incidence of platelet bacterial contamination
over time [3–6]. The findings of this surveillance programme for the years 2004–2011 are shown in Table 8.
Since the introduction of early culture of apheresis collections at blood centres in 2004, 25 bacterially contaminated apheresis units were detected out of 40 567
platelet units transfused (616 per million apheresis
units), with little difference between 2004–2006 and
2007–2011. During 2004–2006, WBD units were pooled
at the time of issue, with 10 bacterially contaminated
pools detected out of 4241 pools transfused (2358 per
million pools). During 2007–2011, WBD units were
pooled at the time of production and early culture performed at blood centres; 4 bacterially contaminated
pools were detected out of 9608 pools transfused (416
per million pools). These findings document the continuing occurrence of ‘breakthrough’ cases of bacterial contamination despite early culture in both apheresis and
pooled WBD platelets and the beneficial effect of prepooling and early culture of WBD platelets, which reduced
bacterial contamination rates to levels comparable with
those of apheresis units.
Question 3
Our institution has experience with the PGD test (Verax
Biomedical, Wooster, MA, USA), which was performed
as part of a multicentre study from September 2009
through November 2010 in a research setting, with 7671
apheresis and 2046 pooled WBD units tested at the time
of issue [7]. Testing was performed on batches of six
units, and initial reactive tests were repeated in duplicate
and regarded as true positives if one or both of the
repeat tests were positive. Six bacterially contaminated
units were detected by plate culture during this period,
with five of these units being apheresis units. Three of
these bacterially contaminated units were detected by
the PGD test, with bacterial species (with loads in CFU/
ml) being two coagulase-negative Staphylococcus species
(106 and 107) and the third Bacillus species (107). Two
of the three false negatives were contaminated with
coagulase-negative Staphylococcus species (4 9 102 and
105), and the third with Streptococcus oralis (107), a species not covered by the reagents included in the PGD
test. Five of the contaminated units were transfused,
with reactions occurring in the three recipients of units
with bacterial loads of >105 CFU/ml. Specificity was
993%.
Testing was continued in the transfusion service from
December 2010 until December 2011 [8]. Based on workflow analysis, personnel availability and evaluation to
optimize testing strategy, the PGD test could not be integrated into the current workstation/task structure at the
time of issue because a dedicated individual to perform
testing was not logistically feasible as this would affect
timely provision of blood components when urgently
required. The ‘receipt’ workstation was selected to perform
testing at the time of receipt of units from blood centres,
with testing repeated on units that were not transfused
within 24 h. Testing was performed on batches of six
units, and results were recorded manually, with a little
over an hour needed to complete testing and paperwork
per batch of six units tested. Initial reactive tests were not
repeated.
Four bacterially contaminated units were detected by
plate culture during this testing period, with three of the
units being apheresis units. Bacterial species (with loads
at the time of issue in CFU/ml) were all coagulase-negative Staphylococcus species (5 9 106, 4 9 107, 4 9 102
and 5 9 104). None of these bacterially contaminated
units were detected by the PGD test at the time of
receipt, although the two units with the highest bacterial
loads, which had been derived from the same apheresis
collection, were detected retrospectively at the time of
issue for one (day 5) and at outdate for the other. Three
of the contaminated units were transfused, with a reaction occurring in the recipient of the unit with the bacterial load of 5 9 106 CFU/ml. A positive plate culture
result from this unit prevented transfusion of the other
unit prepared from the same collection. Specificity was
994%.
© 2013 International Society of Blood Transfusion
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Based on the sensitivity not being as high in service
versus research laboratory experience, likely related to
testing on receipt in the former versus at-issue in the latter, the effect of false positives when inventory was low
or HLA-matched platelets were tested, the impact of false
positives on blood centre notification and recall and competing resources in a service environment, PGD testing
was discontinued in December 2011.
Question 4
Pathogen inactivation strategies to reduce the risk of
platelet bacterial contamination are currently not available in the USA.
Acknowledgements
We thank Dr. James Westra, MD, Medical Director; Ms.
Darlene Morris, BS, SBB (ASCP), MBA, Manufacturing
Manager, Quality Control Laboratory, Northern Ohio
American Red Cross Blood Services; and, Mr. R. Michael
Dash, MS, MT (ASCP), Vice President, Operations; and
Ms. Barbara Hallenburg, MT (ASCP), CQA (ASQ), Director
of Quality Assurance, LifeShare Community Blood Services, Elyria, Ohio, for providing information on platelet
collection, processing and testing methods.
Disclosures
Roslyn Yomtovian has received research support to investigate bacterial contamination of platelets from Gambro,
Hemosystem, Immunetics, Pall Corporation, GenPrime,
Verax and Fenwal. She also serves as a consultant to Verax and Immunetics and is a member of the AABB Bacterial Contamination Subcommittee.
Michael R. Jacobs has received research support and/or
honoraria from Verax, Pall, Gambro, Hemosystem, Immunetics, Genprime, Fenwal and Charles River Labs and has
been a consultant for BioSense Technologies and Lynntech, Inc. He is also a member of the Bacterial Contamination Task Force of the AABB and ISBT.
References
1 Devine DV, Serrano K: The platelet storage lesion. Clin Lab
Med 2010; 30:475–487
2 Brecher ME, Jacobs MR, Katz LM, et al.: Survey of methods
used to detect bacterial contamination of platelet products in
the United States in 2011. Transfusion 2013; 53:911–918
3 Jacobs MR, Good CE, Lazarus HM, et al.: Relationship
between bacterial load, species virulence, and transfusion
reaction with transfusion of bacterially contaminated platelets. Clin Infect Dis 2008; 46:1214–1220
© 2013 International Society of Blood Transfusion
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4 Yomtovian R, Lazarus HM, Goodnough LT, et al.: A prospective microbiologic surveillance program to detect and prevent
the transfusion of bacterially contaminated platelets. Transfusion 1993; 33:902–909
5 Yomtovian RA, Palavecino EL, Dysktra AH, et al.: Evolution
of surveillance methods for detection of bacterial contamination of platelets in a university hospital, 1991 through 2004.
Transfusion 2006; 46:719–730
6 Jacobs MR. Microbiology of Platelets for Transfusion. Presented September 21, 2012, Rockville, MD, at the meeting of
the US Food and Drug Administration Blood Products
Advisory Committee on considerations for strategies to further reduce the risk of bacterial contamination in platelets.
Available at: http://www.fda.gov/AdvisoryCommittees/Calendar/ucm313863.htm
7 Jacobs MR, Smith D, Heaton WA, et al.: Detection of bacterial contamination in prestorage culture-negative apheresis
platelets on day of issue with the Pan Genera Detection test.
Transfusion 2011; 51:2573–2582
8 Downes KA, Jacobs MR. Point of Care Testing: Transfusion
Service Experience: University Hospitals Case Medical Center.
Presented at Public Conference: Secondary Bacterial Screening of Platelet Components, July 17, 2012, Bethesda, MD,
sponsored by AABB. Available at: http://www.aabb.org/even
ts/misc/Pages/public-conference.aspx
Roslyn Yomtovian
Clinical Professor
Department of Transfusion Medicine
Case Western Reserve University
Cleveland, OH, USA
Department of Pathology
Louis Stokes Veterans Administration Medical Center
10701 East Boulevard, Cleveland, OH, USA
Department of Pathology and Laboratory Medicine
113W, Cleveland, OH, 44106 USA
Email: [email protected] and [email protected]
Michael R. Jacobs
Professor
Department of Pathology
Case Western Reserve University
Cleveland, OH, USA
Director
Clinical Microbiology
University Hospitals Case Medical Center
11100 Euclid Ave
Cleveland, OH, 44106 USA
Email: [email protected]