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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 Vox Sanguinis (2014) 106, 256–283 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 Vox Sanguinis (2014) 106, 256–283 International Forum 277 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 Vox Sanguinis (2014) 106, 256–283 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 Vox Sanguinis (2014) 106, 256–283 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 Vox Sanguinis (2014) 106, 256–283 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 © 2013 International Society of Blood Transfusion Vox Sanguinis (2014) 106, 256–283 282 International Forum 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 Vox Sanguinis (2014) 106, 256–283 International Forum 283 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 Vox Sanguinis (2014) 106, 256–283 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]