Improving the bacteriological safety of platelet transfusions

Improving the Bacteriological Safety of Platelet Transfusions Morris A. Blajchman, Mindy Goldman, and Federico Baeza Despite the increased application of aseptic techniques for blood collection and the preparation of platelet concentrates, morbidity and mortality arising from the transfusion of bacterially contaminated allogeneic platelet products persist. This problem exists because stored platelet concentrates represent a nearly ideal growth medium for bacteria and because they are stored at temperatures (22° ⴞ 2°C) that facilitate bacterial growth. The presence of bacteria in blood components including platelets has been a problem for many decades and currently is the most common microbiological cause of transfusion-associated morbidity and mortality. A variety of strategies have been devised and/or proposed in an attempt to try to reduce the risk of transfusion-associated sepsis. These include pretransfusion bacterial detection, efforts to reduce the likelihood of bacterial contamination, the optimization of blood product processing and storage, reducing recipient exposure, and the introduction of pathogen inactivation methodology. With regard to doing bacterial detection, a number of

automated detection systems have become available to test for contaminated platelet components, but their utility to some extent is restricted by the time they take to indicate the presence of bacteria and/or their lack of sensitivity to detect initially low bacterial loads. A variety of other approaches has been shown to reduce the risk of bacterial contamination and include filtration to remove leukocytes and bacteria, diversion of the initial aliquot of blood during donation, and improved donor skin disinfection. Platelet pathogen inactivation methods under investigation include the addition of L-carnitine, ␥-irradiation, riboflavin plus UVA irradiation, and amotosalen HCl plus UVA irradiation. The latter process is licensed for clinical use with platelets in some countries in Europe. All of these approaches, either collectively or individually, hold considerable promise that the prevalence of adverse events associated with bacteria in platelet products will decline significantly in the very foreseeable future. © 2004 Elsevier Inc. All rights reserved.

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rial-associated transfusion risk in the United Kingdom,11 France,12-14 Belgium,15 the Netherlands,16 and the United States.17 Based on the available data, it is probable that this complication is generally underestimated or underdiagnosed.10,17,18 This review summarizes the bacterial contamination risk associated with platelet concentrates and discusses current approaches to reduce the risk of transfusion-associated sepsis.

ESPITE IMPROVEMENTS in blood donor screening procedures and heightened awareness of the risk of transmitting infections by transfusions, bacterial contamination of blood donations remains a significant risk for those receiving either allogeneic or autologous blood component transfusions, particularly platelets.1-7 Bacterial contamination of platelets can occur in a variety of ways, which include lapses in aseptic technique.8-10 Bacterial contamination of platelet products appears to represent a greater risk than that associated with other blood products. This is in large part because platelet concentrates must be kept at a temperature of 22° ⫾ 2°C during storage; therefore, unlike red blood cells bacterial growth is not inhibited by low temperature storage. This presents transfusion services and other producers of platelets with a dilemma. If platelet transfusion is delayed because of lengthy bacterial screening tests, initial nonthreatening low bacterial contamination loads may develop into potentially clinically harmful loads as the bacteria present proliferate. This is different from the static situation that exists for the viruses occasionally present in blood products. Despite the use of modern aseptic techniques, infections resulting from the infusion of blood components contaminated with bacteria are associated with appreciable morbidity and mortality. Several recent reports provide evidence for bacte-

BACTERIAL CONTAMINATION OF PLATELETS

Two factors, room temperature storage and the biological composition of platelet concentrates and their media, make them a close-to-ideal culture media for a wide variety of Gram-positive and Gram-negative organisms.19 Surveillance-based culture studies and retrospective investigations of transfusion reactions reveal that the majority of From the Departments of Pathology and Medicine, McMaster University, and Canadian Blood Services, Hamilton, Ontario, Canada; Canadian Blood Services, Ottawa, Canada; and Baxter Transfusion Therapies Europe, Madrid, Spain. Address reprint requests to Morris A. Blajchman, MD, FRCP(C), Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, HSC 2N34, Hamilton, Notario, Canada L8N 3Z5. E-mail: blajchma@ mcmaster.ca © 2004 Elsevier Inc. All rights reserved. 0887-7963/04/1801-0002$30.00/0 doi:10.1016/j.tmrv.2003.10.002

Transfusion Medicine Reviews, Vol 18, No 1 (January), 2004: pp 11-24

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bacteria isolated from platelet concentrates are part of the normal skin flora. This strongly suggests that many platelet bacterial contaminants originate from the donor’s skin. Bacterial contamination of platelets can occur from donors with asymptomatic bacteremia; however, the nature of contaminating organisms suggest that this source of contamination is rare.19-27 Small bacterial loads, typically less than 10 colony-forming units (CFU) per milliliter, are unlikely to cause a transfusion reaction in a recipient.19 However, small bacterial loads can still be associated with significant morbidity and mortality. An interesting case was reported recently by Blajchman et al,28 which illustrates this important issue. It is important to note also that the absence of an immediate reaction after a transfusion does not necessarily indicate the lack of a septic transfusion event. Low levels of bacteria in platelets may not be benign. In fact, blood products containing low bacterial loads may result in recipient morbidity or mortality that may not be recognized as transfusion associated.28 Nonetheless, the storage of contaminated platelet concentrates at room temperature (20°-24°C) favors bacterial proliferation so that low levels of bacterial contamination that are undetectable and that initially may present relatively little immediate risk to the recipient can rapidly become capable of causing a serious septicemia in a recipient.19 The current risk to a recipient receiving bacterially contaminated platelets may be 10 to 1,000 times higher than the combined risk of transfusionassociated infection with human immunodeficiency virus (HIV), hepatitis C virus, hepatitis B virus, and human T-cell lymphotropic virus (HTLV).29 It has been estimated that as many as 1 in 2,000 to 1 in 3,000 units of platelet concentrates are contaminated and that the risk of a severe septic reaction after a platelet transfusion has been estimated at approximately 1 in 50,000.29,30 Higher contamination rates have been reported recently, although the degree of variation between studies is considerable.15,16,31,32 A number of strategies have been devised to try to reduce the risk of transfusion-associated bacterial infections and these are summarized in Table 1. PRETRANSFUSION BACTERIAL DETECTION

Many factors influence the efficacy of bacterial screening methods and their usefulness as routine

BLAJCHMAN, GOLDMAN, AND BAEZA

Table 1. Proposed Strategies to Reduce Platelet Transfusion-Associated Septic Risk 1. Pretransfusion bacterial detection Visual inspection of platelets before issue Alteration in metabolic parameters during storage Direct staining for bacteria Bacterial ribosomal assays Fluorescent antibiotic labeling Endotoxin detection assays Nucleic acid testing for bacterial DNA Detection of bacterial-specific peptidoglycans Measurement of CO2 production by bacteria Measurement of O2 consumption by bacteria Automated bacterial culture 2. Reducing the risk of platelet contamination Improve donor screening Improve venepuncture site disinfection Removal of the initial aliquot of donor blood 3. Optimize platelet processing and storage Optimize storage temperature Limit storage time Implement universal leukoreduction 4. Reduce recipient exposure to donor platelet products Reduce transfusion triggers for platelets Increase use of apheresis-derived platelets Optimize transfusion indications 5. Introduce pathogen inactivation methodology L-carnitine ␥-irradiation UVA or white light plus riboflavin UVA light plus amotosalen HCl

bacterial detection tools. For example, one such factor is bacterial growth kinetics. Slow-growing bacteria, such as Staphylococcus epidermidis, may be difficult to detect within a reasonable period of time (ie, before the expiry of the shelf life of a platelet concentrate). Also, the smaller the inoculum, the longer it will take for the bacteria present to grow to a level that could readily be detected.33 The initial inoculum of bacteria, if present, in a unit of red cells or a platelet concentrate has generally been thought to be very low (⬍10 CFU/ mL),19 and this level may be too low for some systems to detect. Paradoxically, it could be argued that the various aseptic techniques used to reduce the risk of contamination during phlebotomy may actually make it difficult to detect low levels of bacteria by reducing the initial bacterial load below detectable limits, in some cases.8,9 Sampling time of the platelet unit is also crucial. The earlier the sample is taken for testing, the lower the chance of detecting any contaminant. Even in experiments in which known levels of bacteria have been added to platelets, bacterial

BACTERIOLOGICAL SAFETY OF PLATELETS

contamination is not always detected if the sample is taken too soon after the inoculation.34,35 The same applies to red blood cells. Another critical factor is the sample volume. The larger the volume of the sample taken, the greater the likelihood of detecting bacterial contamination, if present.36,37 Increasing the sample volume, on the other hand, reduces the amount of component available for transfusion. When assessing the ability of a detection system to identify a contaminated unit, it is necessary to have some idea of the likelihood of the sample containing any organisms. The sample volume required should be such that there is 95% confidence that the given sample will contain at least 1 organism, given the theoretical bacteria concentration. The sensitivity of the assay used also has an impact on the ability of that assay to detect contamination. In general, the more sensitive the bacterial assay the better the detection efficacy and the fewer the number of false-negative results.33 This is relevant particularly for platelet concentrate units because of their short shelf life. Assays must be sensitive enough to detect bacterial contamination before platelet units become outdated (Fig 1). Even the most sensitive and specific test will be unsuitable if it is inconvenient to use. The assay must be easy enough to perform without significantly disrupting the flow of work through the facility providing the platelets. The test must also

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be able to detect the various strains of bacterial species that are likely to contaminate platelet concentrates and must have sufficient specificity. In addition, the incidence of false positives must be small enough to prevent wastage of a precious resource.35,38 Cost is an increasingly important consideration. A widely used assay must be affordable and should require a minimum of specialized equipment and highly trained staff.35,38 Automated Bacterial Culture Systems Several commercially available automated, continuous incubation, and detection systems have been developed. These generally depend on the detection of CO2 produced by the growing bacteria. Some of the earlier versions have been superseded by improved versions, or by newer approaches, and several are currently available. Examples include ESP (Trek Diagnostic Systems, Westlake, OH), BacT/Alert (BioMerieux, Durham, NC), BACTEC (BD Biosciences, Sparks, MD), and Isolator 10 (Vibro/DYNAMICS Corp, Broadview, IL). Such systems have been made primarily for use in the hospital environment, in which microbiology departments test large numbers of patient blood samples to detect patient bacteremia. These automated systems are generally quite convenient to use and require minimal specialized training for the operator. Typically, such systems require only that a culture medium be inoculated

Fig 1. Examples of the various methods for detecting bacterial contamination in platelets and their sensitivities. The values shown are based on data from different sources.35,62-66,70,75-78,81,84,86,87 Abbreviations used: AO, acridine orange; AP, antibiotic probe; DNA/RNA, DNA/RNA chemoluminescence; DP, dielectrophoresis; EFM, epifluorescence microscopy; EIA, enzyme-linked immunoassay; ET, endotoxin; GS, Gram stain; IFA, immunofluorescence assay; LPA, latex particle agglutination; PCR, polymerase chain reaction; pO2, PALL BDS pO2 method; RNA probe, ribosomal RNA probe; RS, reagent strips; swirling, platelet swirling patterns.

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with a blood sample, after which the detection tube is loaded into an incubator. These systems perform continuous readings of the sample tubes and provide printouts of the results obtained automatically. The concept is very valuable and has withstood the test of time. However, such systems have weaknesses, which sometimes introduce uncertainty to the result. For example, the different types of culture media used means that different systems have different abilities to detect different organisms.36 Thus, the various systems could produce different results from the same samples because of underlying differences in the assays. In parallel tests of systems using identical samples, some systems have failed to detect bacterial contamination that is detected by other systems. As many as 5% to 15% of patient samples containing different bacteria have been found to give different results in the various test systems studied.39-44 Most of the studies evaluating the efficacy of these automated bacterial testing systems report on their ability to detect samples from potentially bacteremic hospitalized patients. When considering the screening of platelet concentrates for transfusion, a false-negative result could lead to a significant problem for the recipient of that unit. Different sample sizes could be used, but there is an ongoing need to minimize product lost in screening platelet concentrates. Smaller samples are more likely to yield false negatives because of sampling error and/or longer times to detection. With some of the older systems available, sample sizes need to be at least 10 mL, some even 20 to 30 mL.36-38 Recent studies indicate that the use of 10-mL patient samples, compared with 5-mL samples, can improve detection efficiency by 7.2%.37 It therefore follows that the use of larger samples means that bacterial contamination is more likely to be detected earlier.37 Detection efficiency is low for low levels of bacterial contamination. For example, detection rates of less than 50% can be expected when the level of contamination is below 1 CFU/ mL.33,39,45,46 The available data indicate that incubation must allow bacterial proliferation to at least 10 CFU/mL before reliable detection can be expected.33,39,43,44 Thus, positive results when platelets are sampled on day 3 may not yield positive results when the same product is sampled on day 1.47 Slower-growing bacterial species, such as S epidermidis, need 2 to 3 days and perhaps as many as

BLAJCHMAN, GOLDMAN, AND BAEZA

6 to 7 days of incubation before the contamination can be detected. Secondary confirmation cultures of negative samples, after 5 to 7 days, is desirable, but often not practical.16,33,48-55 Detection of some other bacteria, such as Propionibacterium acnes, could involve incubation periods even longer than the usual storage of platelet concentrates. Although confirmation of the absence of bacterial contamination may extend platelet storage life for up to 7 days, these time constraints could create logistical problems. Units of platelet concentrates thus may need to be issued for transfusion before any contamination has been detected so that the hospital to which the unit has been shipped will need to be contacted by the testing center and the unit withdrawn. The communication pathway and audit chain must therefore be very robust; however, this may not always be the case. Sometimes the bacterial contaminant is detected after the product has been issued or even after a transfusion has taken place.15 This may contribute to a loss of confidence in the transfusion service and in the blood components it produces. In addition, false-positive results can cause the unnecessary wastage of a precious resource, whereas falsenegative results are generally unacceptable. Both false-positive and false-negative results occur, depending on the test system in use and the nature of the contaminating bacteria.38,56-61 The Pall BDS Bacterial Detection System This system, developed by Pall Corporation, (Glen Cove, NY), relies on the consumption of O2 by contaminating bacteria in the absence of platelets and leukocytes.62,63 The test is carried out using a 2- to 3-mL sample of a platelet concentrate. In-line filtration of the sample is used to remove leukocytes and platelets. (This filtration step does not remove most of the bacteria present). The sample is then contained within a small non– gaspermeable plastic pouch for incubation at 35°C for 24 hours, followed by a further period of incubation at 22°C.62,63 If the percentage of oxygen measured at a single reading falls to less than 19.5% (equivalent to a pO2 less than 100 mm Hg), the test is deemed to have detected bacterial contamination.62-66 Validation tests using an inoculum containing 100 to 500 CFU/mL gave a 97% detection rate after 24 hours of incubation. This rose to 100% after 30 hours.62 However, a Streptococcus pyogenes isolate required incubation for 48 hours be-

BACTERIOLOGICAL SAFETY OF PLATELETS

fore the detection rate reached 100%, for an inoculum containing 100 CFU/mL.66 The detection rate was 80% for Streptococcus ␤ species after a 48hour incubation of samples of pooled buffy-coat platelets. To date, there are no published reports providing data about the performance of the BDS system under real transfusion service conditions. Detection efficiency data for bacteria such as Corynebacterium species or P acnes, which have only rarely been involved in cases of platelet contamination, have not been reported.67-69 It has been suggested that the shelf life of platelet concentrates could be extended if this system were to be used.62,65 This would mean that the quality of 7-day–stored platelets and their effectiveness in the clinical situation would have to be evaluated.70 This BDS system is relatively straightforward, and the assay appears to be easy to perform with a minimal amount of staff training. The O2 reading needs to be carried out immediately before the platelet transfusion (ie, in the hospital) because only 1 O2 measurement can be done with the current version of this system. This reduces the usefulness of the method because all hospital staff must be adequately trained, which may not be possible in some smaller hospitals. The method is currently licensed for use in several countries, but at this time there are insufficient validation data available to completely assess the potential role and value of this promising system. DNA/RNA-Based Detection Systems These systems rely on the detection of bacterial DNA or ribosomal RNA (rRNA). No system has yet been developed that will detect all potential bacterial contaminants through their nucleic acid. This concept is still under development, and it is not yet clear whether bacterial DNA or rRNA is the more appropriate test marker.71,72 At present, the cost and complexity of nucleic acid detection methods may render them somewhat impractical for routine testing.71 Investigations by GeneProbe (San Diego, CA) have provided interesting preliminary results using a highly conserved region of bacterial rRNA.73 Depending on the methodology used, nucleic acid-based detection systems take from 6 to 48 hours.34,35 The detection rate (sensitivity) approaches 100% when the level of bacterial contamination is above 1-5 ⫻ 105 CFU/mL (Fig 1), but it can take 4 days of storage for some bacteria to proliferate to this level in platelet con-

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centrates.74-77 This means that samples taken soon after blood donation could test as negative, whereas samples taken from the same donations after 4 or 5 days storage at room temperature would read as positive. Ideally, such a system should detect a bacterial contamination level of 102 CFU/mL to be useful as a routine tool. One recently reported DNA method allows bacterial DNA detection when the level of bacterial contamination in the sample is approximately 104 CFU/ mL.78 Detection Systems Based on Changes in Metabolic Parameters During Storage Abnormal glucose concentrations, low pH, and the cessation of platelet swirling have been reported as potential indicators of the presence of contaminating bacteria. However, these tests appear to be imperfect as markers of bacterial contamination in platelets. For example, the pH during storage has been reported to increase, decrease, or even remain unchanged, depending on the nature of contaminating bacteria.79,80 In addition, significant variations in the pH of incubated samples are seen associated with variations in platelet concentrations, especially at low concentrations or conditions of poor platelet oxygenation. From a practical point of view, high levels of bacterial contamination are needed for any correlations to be significant.81,82 Metabolic parameters, therefore, do not have sufficient sensitivity to form the basis for the routine screening of platelets for bacteria by the transfusion service. However, in the absence of other screening methods, the use of dipsticks to measure pH and glucose levels immediately before a platelet transfusion could prevent the transfusion of a heavily contaminated platelet unit. Fluorescent Antibiotic Labeling This method uses antibiotics labeled with fluorescent markers. The rationale of this approach is that antibiotics are capable of binding to specific bacteria and cause such bacteria to be detected by various methods including flow cytometry.83 Fluorescent antibiotic labeling methods can currently detect only relatively high levels of bacterial contamination, at least 105 CFU/mL (Fig 1). Such levels are often reached during the storage of platelet concentrates, but different bacteria species grow at different rates and not all bacteria will reach this level of contamination within the current 5 day

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BLAJCHMAN, GOLDMAN, AND BAEZA

shelf life of platelets.35 It is also unlikely that a single antibiotic can be found that will bind to all bacteria that might be present in platelet concentrates. Moreover, such antibiotics would need to bind to all potentially contaminating bacteria with similar affinity, which is unlikely. As a general bacterial screening method then, such methods are unlikely to be very useful. Detection of Specific Peptidoglycan Components of Bacterial Cell Walls The peptidoglycan component of bacterial cell walls is an attractive potential target for the detection of bacterial contamination. Two peptide markers have been described that enabled the detection of 11 bacterial species (3 Gram-positive and 8 Gram-negative species) spiked into platelet concentrates. The level of contamination detected in these studies was 103 to 104 CFU/mL. The tests took 25 minutes to perform and were carried out using leukoreduced platelet samples.84 This approach shows considerable promise, but the preliminary report does not describe the volume of sample needed and it is not clear yet whether the 2 peptides will detect all relevant species of bacteria. One potential significant advantage of this approach is its rapidity. Dielectrophoretic Method The basis of this technique is that bacteria move in an electric field, whereas platelets do not. This method uses 2 electrodes to provide an electric field and image analysis to identify those elements that migrate in the electric field. The test requires only 1-mL samples and takes just 1 hour to perform.85 The reported detection limits for a range of test bacteria are shown in Table 2, meaning that ⱖ105 CFU/mL should be the minimal threshold (Fig 1). This is a promising method but it is still in its early experimental stage. A full understanding Table 2. Detection Limits for the Dielectrophoretic Method for Detecting Different Species of Bacteria Contaminating Platelets85 Bacterial Species

Detection limit (CFU/mL)

Escherichia coli Staphylococcus aureus Staphylococcus epidermidis Bacillus cereus Clostridium perfringens Group B Streptococci

103 104

105

of the details of this method will be needed before its usefulness for routine use can be fully evaluated. Moreover, additional data are required about specificity, sensitivity, and reliability. Microscopic Examination of Stained Samples Two stains have been investigated: acridine orange and the Gram stain.35 For acridine orange, a contaminating bacterial concentration of at least 106 CFU/mL is needed (Fig 1). This relatively high level of contamination means that most platelet units must be stored for a considerable period of time (⬎3 days) to allow the bacteria within the samples with initially low levels of contamination to proliferate to this level. The Gram stain method is even less sensitive than the acridine orange method. This approach was reported to have failed to detect Pseudomonas aeruginosa at a concentration of ⬎107 CFU/mL.35 The time needed for such levels of bacterial contamination to be achieved makes the Gram stain somewhat impractical for routine use for detecting the bacterial contaminants in platelets. Thus, direct staining-based methods appear not to be sensitive enough for routine use, but more promising direct staining methods are under development. Among these are tests that depend on the selective fluorescent staining of bacterial nucleic acids and their detection using automated epifluorescence microscopy86 or the ScanSystem (Hemosystem, Marseille, France), which has a sensitivity of 103 CFU/mL. It should be pointed out that this latter result comes from the assessment of only 3 types of bacteria.87 Endotoxin Detection Gram-negative bacteria produce endotoxin. However, considerable levels of contaminating bacteria would need to be present for enough endotoxin to be produced for it to be reliably detected. Another drawback of endotoxin-based methods is that only Gram-negative species produce endotoxin. This fact and a lack of consistency of the available assays means that this method is probably inadequate for routine use.35 REDUCING THE RISK OF PLATELET CONTAMINATION

Improve Donor Screening The approach of these methods is to exclude blood donations from individuals with clinically

BACTERIOLOGICAL SAFETY OF PLATELETS

unapparent bacteremia. Such screening is usually based on either donor questionnaire about symptoms of infection and the determination of a donor’s temperature or the detection of antibodies to suspected contaminating bacteria. One major problem that will need to be overcome before such techniques can be routinely used is the diversity of serotypes to even a single species.88 A major weakness of these approaches to bacterial screening is that it cannot detect bacteria that enter the donation from sources other than the donor’s blood. Improve Venepuncture Site Disinfection As indicated previously, most of the bacteria isolated from contaminated blood products are normal skin flora. Thus, optimal disinfection of donor venesection sites may significantly reduce the bacterial contamination of blood products.9,89,90 Recent studies exploring blood culture contamination rates have indicated that both the quality of the antiseptic used and the mode of application of the antiseptic can influence the efficacy of skin disinfection method used. The reduction in transfusionassociated septic risk by this means will not completely solve the problem because many clinically evident transfusion-associated septic episodes are not caused by bacteria species that are generally classified as skin flora.90 Removal of the Initial Aliquot of Donor Blood This approach is based on the assumption that contaminating bacteria originating from the donor’s skin will be contained mainly in the initial flow of blood. However, the available data suggest that even under ideal experimental conditions, diverting the initial aliquot reduces the bacterial load by only 90%.91 Diversion of the first 13.5 mL has been reported to reduce the contamination by 72% (3.1-fold). Bacteria not removed in the initial diverted sample could still be detected in the second diverted sample or even in the component unit.92 Recent data provide evidence for a reduction of approximately 40% in the bacteria contamination rate of whole blood using this approach.67 Sequential culture studies suggest that at least 30 mL of blood should be diverted. This approach has been shown to reduce the contamination rate of whole blood and buffy coat platelet pools. However, thus far, the ability of initial aliquot diversion to prevent clinical cases of transfusion-associated septic reactions has not yet been reported. Similar to im-

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proved skin disinfection, this approach will only reduce the level of skin contaminants.90 OPTIMIZING PLATELET PROCESSING AND STORAGE

Optimizing Storage Temperature The temperature at which platelets are stored is a significant factor influencing bacterial proliferation in blood products. For example, there is an interesting report suggesting that red blood cell storage at 0°C may be associated with decreased bacterial growth compared with that seen at 4°C.93 Moreover, the development of nutrient solutions and conditions permitting platelet storage at 4°C would decrease the potential for the growth of many species of bacteria that contaminate platelet concentrates. However, the hemostatic activity of such platelets would have to be retained, if storage at 4°C were to be used.94,95 Current data suggest that platelets stored at 4°C do not retain their hemostatic function.96 Limit Storage Time Most platelet transfusion-associated septic events occur with units that have been stored for 3 days or more because this is the time required for most organisms to multiply and achieve a bacterial level that produces clinically apparent signs and symptoms.56 It is interesting that before 1986, the Food and Drug Administration in the United States permitted the storage of platelet units for up to 7 days. In 1986, however, the US Food and Drug Administration reduced the then acceptable storage time to 5 days after several reports of platelet-associated sepsis with units transfused 6 or 7 days after the initial blood donation. Thus, it is reasonable to presume that a further reduction in storage time might reduce the prevalence of septic events even further. However, a reduction in platelet storage time to less than 5 days would result in important supply problems in most jurisdictions. In contrast, the recent use of automated culture systems for bacteria detection has enabled some blood centers in Sweden and Norway to store platelets for up to 7 days primarily to improve supply needs. However, a recent report recommends against extending platelet storage beyond 5 days, based on data that show a significant risk of slow growing bacteria 5 and 7 days after collection.97

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Universal Leukoreduction

Optimizing Transfusion Indications

One way of reducing the risk of bacterial contamination in platelet concentrates is to reduce the level of leukocytes present. Filtration to remove leukocytes does not prevent bacterial growth, although it does reduce the level of bacterial contamination.98-101 However, this process is not effective against some species of bacteria (eg, S epidermidis).101 This method, therefore, does not provide full protection against the risk of bacterial contamination but has been shown to significantly reduce the risk of transfusion-associated sepsis.14 Further investigations are required to fully evaluate this approach in the prevention of transfusion-associated bacterial sepsis.

Finally, attention should be paid to optimizing transfusion indications. Audits of blood component use have indicated regularly that blood products, both cellular and plasma products, are often inappropriately transfused to patients. The reduction of the inappropriate use of platelets would significantly reduce the incidence of platelet transfusionassociated sepsis, as well as other adverse events relating to allogeneic transfusions.

REDUCED RECIPIENT EXPOSURE TO DONOR PLATELET PRODUCTS

Reducing Transfusion Triggers Reducing recipient exposure to donor blood products has not been widely used in an attempt to prevent transfusion-associated bacteremia and septicemia. In this regard, it should be possible to reduce the prevalence of such events by reducing platelet transfusion triggers for recipients. Recent publications recommending reduced transfusion triggers for allogeneic red blood cells as well as for prophylactic platelet transfusions have appeared that suggest that this approach reduces recipient exposure without endangering recipient health.102,103 This approach would not only reduce the risk of transfusion-associated sepsis but would reduce the risk of all of the other transfusion-associated adverse events. Using Apheresis-Derived Platelets The use of apheresis-derived platelets in preference to pools of whole-blood– derived platelets has recently been suggested in an attempt to reduce the risk of transfusion-associated sepsis.104 Thus, the universal use of apheresis-derived platelets would significantly reduce recipient exposure to contaminated pooled platelets by a factor of 4 to 6. The latter represent the usual numbers of whole-blood– derived platelet units used per platelet transfusion episode in an adult recipient. Again, this approach, although useful, represents only a partial step in reducing the risk of platelet transfusion-associated sepsis.

PATHOGEN INACTIVATION METHODOLOGY

A caveat for all of the approaches discussed earlier to reduce recipient exposure to blood products, although capable of reducing the prevalence of transfusion-associated septic reactions, will not prevent such events from occurring. None of the previously mentioned approaches will ensure the absolute bacteriological safety of platelet concentrates. The only approach to achieve the latter would be the introduction of approaches to inactivate the bacteria and other pathogens that regularly contaminate platelet concentrates. A variety of approaches have been used to inactivate the bacteria and viruses that might be present in blood components. Some of these approaches are likely to become available soon and in some parts of the world they are already available. L-Carnitine Adding L-carnitine to the platelet storage medium has been preliminarily reported to only slightly inhibit the growth of coagulase-negative Staphylococcus species.105 However, the level of bacterial inactivation achieved by L-carnitine appears to be much too small for this method to offer any realistic protection against most bacterial species that might contaminate platelet concentrates.

␥-Irradiation ␥-Irradiation has been shown to inactivate T lymphocytes, and it has been reasoned that ␥-irradiation could also be effective in abrogating bacterial growth.106 However, relatively high doses of ␥-irradiation are required to inactivate bacteria (100-150 Gy). Such doses could well compromise platelet function. The available data indicate that doses of 75 Gy do not prevent the growth of contaminating bacteria to any useful extent.106 The

BACTERIOLOGICAL SAFETY OF PLATELETS

dose of ␥-irradiation that is generally used to prevent graft-versus-host disease is much lower (25-30 Gy) and represents a dose that does not affect bacterial growth. Although this dose is insufficient to kill bacteria, this level of ␥-irradiation does not appear to affect platelet function. Riboflavin Plus Ultraviolet A Storage of platelet concentrates in a solution containing riboflavin has been reported to reduce the rate of deterioration of the platelets. In addition, illumination of riboflavin-containing media (20 mmol/L) with UVA (or visible light) has been shown to inactivate pathogens.108 This method being investigated by Navigant Biotechnologies (Lakewood, CO), using 80 J/cm2/min of visible light (419 nm) for approximately 23 minutes, has been reported to inactivate 4 species of bacteria in culture media with starting contamination levels of 102 CFU/mL (total of 3 ⫻ 103 CFUs).109 Three strains of coagulase-negative Staphylococcus species were effectively inactivated in these experiments, as was 1 strain of Staphylococcus aureus, 1 strain of Pseudomonas aeruginosa, and 1 strain of Bacillus species. However, in these studies, the bacteria were added to the culture media rather than to platelet concentrates. Doses of 50 ␮moL/L riboflavin with 5 J/cm2 light in 90% plasma were able to inactivate S epidermidis, S aureus, Klebsiella pneumoniae, Escherichia coli, and Bacillus cereus with efficiencies greater than 5 log10.108,109 This method has also been reported to inactivate intra- and extracellular HIV, as well as the model viruses Bovine viral diarrhea virus, Pseudorabies virus, Porcine parvovirus, and Vesicular stomatitis virus.110,111 More details and further studies are needed before the value of this approach can be fully assessed. This method, if it works as well in a routine setting as in the experimental setting, appears to offer acceptable bacterial inactivation efficacy. Nevertheless, there is much still to be done as neither clinical trials or toxicological studies appear to have been done yet. Amotosalen HCl Plus UVA Light The basis of this approach is the photochemical inactivation of bacteria using the synthetic psoralen, amotosalen-HCl, followed by the illumination with UVA light. In the absence of light, amotosalen intercalates into the helices of DNA and

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RNA, without forming covalent bonds. In the presence of long-wavelength UVA light (320-400 nm), however, covalent bonds are formed between the amotosalen and the DNA or RNA that results in the permanent inactivation of the nucleic acids. Once the photochemical inactivation has been carried out, the residual amotosalen-HCl and their photodegradation products are removed by adsorption onto a special adsorption device.112 The amotosalen/UVA method has been tested for its capability for bacterial inactivation in platelet concentrates spiked with 17 different species of bacteria, including Gram-positive and Gram-negative organisms, aerobes and anaerobes, as well as sporulated bacteria (Table 3).113,114 In addition, 3 phase III randomized clinical trials with this system have been completed. Based on these data, this method known as the INTERCEPT Blood System for platelets (Cerus Corporation, Concorde, CA and Baxter Healthcare, Deerfield, IL) was licensed for use in Europe in 2002.115-117 The system has also been tested for potential pathogen inactivation of plasma.118 The levels of bacterial inactivation shown in Table 3 were achieved in samples of apheresis platelet concentrates taken from unselected donors Table 3. Reduction in Bacterial Load by the Amotosalen/UVA System for Apheresis Platelet Concentrates Spiked With Different Species of Bacteria111,112 Bacterial Species

Gram-positive species Staphylococcus epidermidis Staphylococcus aureus Staphylococcus pyogenes Listeria monocytogenes Bacillus cereus (vegetative form) Corynebacterium minutessimum Bacillus cereus (including spores) Gram-negative aerobes Escherichia coli Serratia marcescens Salmonella choleraesius Klebsiella pneumoniae Yersinia enterocolitica Enterobacter cloacae Pseudomonas aeroginosa Anaerobes Lactobacillus species Bifidobacterium adolescentis Propiobacterium acnes Clostridium perfringens Treponema pallidum

Level of Load Reduction (log10)

⬎6.0

3.6-3.9

⬎6.0

⬎5.0 4.5

⬎6.5

8.0

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BLAJCHMAN, GOLDMAN, AND BAEZA

and which had been stored for up to 5 days. An important feature of the amotosalen/UVA system for platelets is that it also inactivates a range of viruses (eg, HIV-1, HIV-2, hepatitis C virus, hepatitis B virus, HTLV-1, HTLV-2, cytomegalovirus, Parvovirus B-19) and parasites (P falciparum, T cruzi, and L mexicana) as well as bacteria.113,114,120,121 In addition, the system appears to be active for leukocytes and so potentially it can also prevent graft-versus-host disease,122-124 so that gamma irradiation may no longer be necessary for this purpose.125 The data from the available randomized clinical trials (Table 4) indicate that the amotosalen/UVA treatment of platelets results in a preparation that has normal hemostatic function when transfused into thrombocytopenic patients. One significant problem that is associated with the amotosalen/UVA approach is the reduced platelet recovery and platelet survival compared with control platelets.115-117 Whether this problem is clinically relevant to recipients remains to be established. As safety of the inactivating agent is a critical requirement for potential licensing, amotosalen HCl has been subjected to a range of toxicological testing in rats, dogs, and rabbits, the results of which formed part of the submission to the European regulatory bodies that led to this process being granted the European license.112,126 CONCLUDING REMARKS

Septic transfusion reactions associated with the transfusion of platelets contaminated with bacteria is a potentially very serious adverse event to recipients. In fact, the presence of bacteria in platelet concentrates is a more frequent adverse event than that caused by most other microbiological agents that affect the blood supply together. However, such adverse clinical events often go undiagnosed because health care workers do not think of blood products as the source of the bacterial infections in their patients. Moreover, the patients who usually

receive platelets are often also granulocytopenic, or otherwise immunosuppressed, and thus susceptible to a variety of sources of bacterial infections. A large array of measures (Table 1) have been proposed in an attempt to try to reduce the frequency of transfusion-associated septic reactions. Bacterial detection systems intended to screen out bacterially contaminated units of platelets could play an important role in this regard and the American Association of Blood Banks has recently distributed an Association Bulletin advising its membership how they might proceed in this regard.127 The bulletin concludes by indicating that “. . . the ultimate approach to interdicting platelet bacterial contamination is not yet clearly defined.” Thus, the optimal method for detecting bacterial contamination has not yet been devised. All of those listed in Table 1 are associated with both theoretical and practical limitations. Despite such limitations, cases of transfusion-associated bacterial sepsis can be prevented by the application of such methods, as well as some of the other strategies proposed in Table 1. Finally, some recent data indicate that even with routine short-term bacterial culture, a significant risk of bacterial contamination remains at both 5 and 7 days after collection.97 It is the view of the authors of this present article that the best prospect for prevention of transfusionassociated bacterial sepsis lies with the potential widespread availability and adoption of effective and safe methods for inactivating bacteria (as well as other microbial agents) that are likely to become available soon for platelet concentrates. Such systems would need to be highly effective in inactivating a wide range of bacterial species, including those that are spore forming. Moreover, such pathogen inactivation systems should not adversely affect the biological function of platelets (recovery, survival, and hemostatic ability) and most importantly should be free of potential toxicological problems (short and long term) to the recipient.

Table 4. Phase 3 Clinical Trials That Have Reported on the Clinical Efficacy of Platelets Treated With the Amotosalen/UVA System of Pathogen Inactivation Number of Patients Study Authors

Platelet Type

Controls

Treated

Total

Van Rhenen et al (2003)115 Kluter et al (2002)116 McCullough et al (2001)117

Buffy-coat poor Apheresis Apheresis Total

51 21 327 399

52 22 318 392

103 43 645 791

BACTERIOLOGICAL SAFETY OF PLATELETS

21

Such pathogen inactivation systems are actively being developed and some “first-generation” systems are already clinically available in Europe and could become available soon in other

parts of the world. Thus, the potential reduction in the risk of transfusion-associated septic reactions is a very realistic possibility in the foreseeable future.

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