Bacterial contamination of blood components

Transcription

1 CHAPTER 53 Bacterial contamination of blood components Richard J. Benjamin Cerus Corporation, Concord, CA, USA Bacterial contamination of blood components is a persistent but often overlooked problem in transfusion medicine. Although public attention has focused on transfusion-transmitted viral infection, improved methods of screening through donor questioning and testing have greatly reduced the transmission of hepatitis viruses and retroviruses. Given the reduction of viral transmission via allogeneic blood, the risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted disease. Before recent developments in skin preparation, diversion, and testing for bacterial contamination, the incidence of platelet bacterial contamination was approximately 1 in 1000 components and septic reactions were reported with 1 in ,000 transfusions. 1 This chapter provides a brief overview of risks associated with blood components. The main focus is on approaches to minimize or eliminate the risk of bacterial contamination. Transfusion-transmitted bacterial infection by red blood cells (RBCs) Sepsis associated with the transfusion of bacterially contaminated RBC components is generally regarded as a very rare event, and appears to be declining in incidence. Prospective bacterial cultures of whole blood or RBC units show that units may be culture positive, mostly with skin commensals such as Staphylococcus spp. or Propionibacterium spp., but these proliferate poorly during storage at 1 to 6 C. Organisms that grow well in the cold are more likely to be involved in septic reactions and then only after weeks of storage due to slow growth at those temperatures. An example of such a cryophilic organism is Yersinia enterocolitica where most cases of contamination occur after storage for 25 days. 2 Historical reports from New Zealand indicated a Y. enterocolitica transfusion-transmitted incidence rate of 1 in 65,000, with a fatality rate of 1 in 104,000 RBC units transfused. 3 Sepsis associated with the transfusion of Gram-negative bacterially contaminated RBCs is typically severe and rapid in onset. Patients frequently develop high fevers (temperatures as high as 109 F [42 43 C] have been observed) and chills during or immediately following transfusion. From 1987 to 1996, 20 cases of Yersinia-infected RBC units in 14 states were reported to the US Centers for Disease Control and Prevention (CDC). 4 Twelve of the 20 recipients died in 37 days or less following transfusion. The median time from transfusion to death was 25 hours. Of the seven who developed disseminated intravascular coagulation, six died. Since that time, passive reporting studies from the United States, 5 France, 3 and the United Kingdom 6 of contaminated RBCs that caused symptoms of infection show a relative paucity of Yersinia spp. cases (Table 53.1). From 1995 to 2004, 25 fatalities thought to be secondary to contamination of whole blood or RBC units were reported to the US Food and Drug Administration (FDA; Table 53.1). 20 of 25 (80%) were caused by Gram-negative organisms. 10 The risk of death from a bacterially contaminated RBC transfusion in the United States was estimated to be 0.13 per million in in one independent study and can be estimated at 1:4,800,000 (0.21 per million) from the above FDA data over the longer time period of More recently, FDA reports document only four fatalities caused by contaminated RBC between 2005 and 2013, all caused by Gramnegative bacteria, including a single case of Y. enterocolitica. This translates to an estimated incidence of 1:32,500,000 (0.031 per million). 9 Similarly, French investigators reported 25 septic reactions associated with 4.1 million RBC transfusions ( 1:141,000 transfusions), including four fatalities (1:1,025,000 transfusions) during a two-year period in All fatalities were due to Gramnegative organisms; however, Gram-positive organisms comprised 13 of 29 (45%) isolated species, with a single case of Y. enterocolitica found in an autologous unit. 8 In contrast, between 2000 and 2008, 18 million RBC transfusions were transfused and only seven septic reactions were reported (1:2,571,000 transfusions), including two deaths (1:9,000,000 transfusions), and none were linked to Grampositive units. A single fatality was caused by Y. enterocolitica. The dramatic decline in incidence of sepsis associated with RBC transfusion in the United States and France is unexplained, but may be linked to the widespread introduction of prestorage leukoreduction in both countries during the latter time periods. 7 Asymptomatic donors with transient bacteremia are presumed to be the source of most Gramnegative bacterial contamination. Leukoreduction during manufacture may decrease the risk of such contamination, as bacteria are ingested by leukocytes in the collected product and removed during processing. 11 For Y. enterocolitica, implicated donors are typically found to have elevated immunoglobulin M (IgM) antibody titers, implying blood collection during an asymptomatic bacteremia following a recent infection. 4,12,13 In one case, an outbreak of Serratia marcescens sepsis was linked to contamination of RBCs in Denmark and Sweden. 14 The contamination was thought to involve the manufacturing process, because the sterile bag sets were autoclaved and put in a clean but not sterile outer plastic package. It was thought Rossi s Principles of Transfusion Medicine, Fifth Edition. Edited by Toby L. Simon, Jeffrey McCullough, Edward L. Snyder, Bjarte G. Solheim, and Ronald G. Strauss John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. 608

2 Chapter 53: Bacterial contamination of blood components 609 Table 53.1 Bacterial species involved in septic transfusion reactions and fatalities after RBC transfusions as reported to the french national hemovigilance program and the US FDA 7 9 France Sepsis (Fatalities) United States Fatalities Total Transfusions (Millions) # 126 # Gram-positive bacteria Staphylococcus aureus 2 % Coagulase-negative staphylococci Enterococcus faecalis 1 & Streptococcus spp Bacillus cereus Propionibacterium acnes Total Gram-positive bacteria Gram-negative bacteria Enterobacter spp. 1 (1) Escherichia coli 3 & Klebsiella spp Serratia spp. 2 (1) Yersinia enterocolitica 1 1 (1) 2 1 Acinetobacter spp. 5 (1) % 1 (1) 0 0 Pseudomonas spp. 2 (1) Proteus mirabilis Pantoea agglomerans Gram-negative bacilli Total Gram-negative bacteria 16 (4) 7 (2) 20 4 TOTAL 29 (4) 7 (2) 25 4 Rate of sepsis 1:141,379 1:2,571, Rate of fatality 1:1,025,000 1:9,000,000 1:4,800,000 1:32,500,000 in two cases two isolates of Streptococcus spp. were isolated from the implicate bag. % In one case S. aureus and Acinetobacter spp. were both isolated from the implicated bag. & In one case Enterococcus spp. and E. coli were both isolated from the implicated bag. # estimated from the National Blood Collection and Utilization Surveys. 47 that S. marcescens present in the dust in the factory contaminated the outside of the containers, and in the presence of moisture and a nutrient (the plasticizer diethylhexylphthalate), the bacteria proliferated and gained entry into the bag. 15 Although autologous blood is generally considered a safer blood component than allogeneic blood, there have been at least six reported cases of bacterial contamination of autologous RBC units, five from Y. enterocolitica and one from Serratia liquefaciens. 16 Fortunately, all reactions were nonfatal, presumably assisted by preformed immunity. Upon retrospective questioning, all patients infected by Yersinia spp. recalled gastrointestinal symptoms in the days before donation. In the case of Serratia spp. contamination, the patient s infected toe ulcer was presumed to be the source. Transfusion-transmitted bacterial infection of plasma, cryoprecipitate, and derivatives Cell-free products such as plasma and cryoprecipitate are stored in the frozen state and thus are rarely associated with significant contamination, as documented by the lack of any reported cases to the French National Hemovigilance Program between 2000 and However, Pseudomonas cepacia and Pseudomonas aeruginosa have been cultured from cryoprecipitate and plasma thawed in contaminated waterbaths. 21,22 The increasing use of thawed plasma and the advocacy for the use of liquid, never-frozen plasma 23 that may be stored at 1 6 C for up to five days and 26 days, respectively, before transfusion imply that physicians need to be increasingly vigilant for septic reactions with these components. 24 Products derived from blood components may also be contaminated with bacteria. Human serum albumin is a good culture medium and preserves the viability of contaminants. The heating step (60 C for 10 hours) in the manufacturing of albumin is performed to inactivate certain viruses, not to ensure bacterial sterility. 25 This would require autoclaving (superheated under pressure), which would cause albumin to denature. On occasion, specific lots of albumin product have been found to be contaminated with bacteria, typically Pseudomonas spp. 26 These lots have produced endotoxic shock, transient bacteremias, and febrile reactions in recipients. Two patients in different hospitals developed Enterobacter cloacae septicemia after receiving albumin. 26,27 Cultures of unopened product grew Stenotrophomonas multophilia and Enterococcus gallinarum in addition to E. cloacae. This resulted in a worldwide recall of certain lots of 5% and 25% albumin. It is suspected that cracks in the glass bottles were responsible for the contamination. Manufacturing problems, therefore, are a source of bacterial risk from these derivatives. Transfusion-transmitted syphilis Treponema pallidum is a thin-walled, motile, spiral, Gram-negative rod or spirochete that cannot be visualized with Gram s stain and does not grow on bacteriologic media or cell culture. Although it is a bacterium, it is often treated as a distinct entity, different from other transfusion-transmitted bacterial organisms, and thus is addressed separately. Only 25% of patients with primary syphilis have a reactive serologic test for syphilis, and the test does not become routinely positive until the fourth week after the onset of symptoms; therefore, donors infected with T. pallidum may be asymptomatic with negative serology during periods of spirochetemia. 28,29 Although the organism does not survive prolonged storage at

3 610 Section V: Part I: Infectious hazards 4 C, it may live for 1 to 5 days at these cold temperatures. 30,31 Therefore, a rare infection may be associated with transfusion of a fresh RBC unit from a donor who was in the seronegative phase at the time of donation. Platelets stored at 20 to 24 C provide a more hospitable temperature for T. pallidum; however, this organism does not thrive with the high-oxygen tension in modern platelet storage bags. Since 1969, only three cases of transfusion-transmitted syphilis have been reported in the literature The extremely low rate of transfusion-transmitted syphilis infection likely results from the following: (1) donor questioning targeting high-risk behavior; (2) the cardiolipin-based assay, which, although an insensitive test in the acute post-infectious setting, does pick up a number of infected donors; (3) refrigerator storage, which results in the death of spirochetes; (4) antibiotics given to many patients at the time of platelet transfusion, which would be bactericidal for any viable organisms; and (5) donors excluded for a positive test for HIV, HCV, or HBV because of the high correlation between infection with T. pallidum and viruses such as these, even though the donors may have been in the seronegative phase of syphilis at the time of donation. 35 Because syphilis testing plays only a minor role in protecting the blood supply and is associated with a high degree of false-positive reactions, elimination of syphilis testing has been advocated. The counterargument is that such testing provides a surrogate marker for individuals at risk of other sexually transmitted diseases and, therefore, should be retained. Transfusion-transmitted bacterial infection of platelets Source of contamination Platelets are stored at room temperature (20 24 C) in oxygenpermeable bags with agitation for 5 7 days, which are excellent growth conditions for many aerobic and microaerophilic bacterial species. Bacteria most commonly enter the container at the time of phlebotomy through contamination of the needle during venipuncture with skin commensals and contaminants, or more rarely through asymptomatic donor bacteremia with oral or enteric commensals. Investigation of the donors involved in the donation of contaminated components rarely identifies a focus of infection or source of contamination. 36 Skin commensal organisms such as Staphylococcus epidermidis and the anaerobic organism Propionibacterium acnes are the organisms most often detected as contaminants by culture methods Prior to the introduction of routine culture-screening processes, septic fatalities caused by platelet contamination were predominantly caused by enteric Gram-negative organisms 10 (Table 53.2). Very rarely, contamination of the collection bag, tubing, or anticoagulant with improper sterilization during manufacturing, or contamination of products after collection due to failure of the closed storage system (e.g., defects in the bags or tubing), has been documented, and these may involve environmental contaminants such as Serratia spp., Bacillus spp., and Pseudomonas spp. Table 53.2 Bacterial species involved in platelet septic transfusion reactions and fatalities as reported to the french national hemovigilance program, US FDA and american red cross hemovigilance program 7 9,39 France Sepsis (Fatalities) United States FDA Fatalities Only American Red Cross Sepsis (Fatalities) Total Transfusions (Millions) # 18 # 4.1 Gram-positive bacteria Staphylococcus aureus 0 13 (4) (3) Coagulase-negative Staphylococci (1) Enterococcus faecalis 0 2 (1) 1 Streptococcus spp Bacillus cereus Propionibacterium acnes 3 0 Clostridium perfringens 1 1 Other Gram-positive bacilli 1 Total Gram-positive bacteria (4) Gram-negative bacteria Enterobacter spp Escherichia coli 1 5 (2) 9 3 Klebsiella spp. 2 (1) 3 (3) Serratia spp. 1 (1) Yersinia enterocolitica 0 0 Acinetobacter spp Pseudomonas spp Proteus mirabilis 0 0 Salmonella spp. 2 Other Gram negative bacilli 1 Morganella spp. 1 2 Pasturella spp. 1 Eubacterium limosum 1 Total Gram-negative bacteria 6 (2) 16 (5) TOTAL 16 (2) 48 (9) Rate of sepsis 1:29,375 1:40, :106,921 Rate of fatality 1:235,000 1:216,000 1:250,000 1:642,857 1:1,015,750 # estimated from the National Blood Collection and Utilization Surveys. 47 American Red Cross data reflect apheresis platelets only, whereas French and US FDA data include both pooled WBD and apheresis platelets.

4 Chapter 53: Bacterial contamination of blood components 611 Clinical presentation The clinical sequelae resulting from transfusion of bacterially contaminated platelets range from asymptomatic to mild fever (which may be indistinguishable from a nonhemolytic transfusion reaction) to acute sepsis, hypotension, and death. The clinical picture is much more varied and often less severe than that of patients infected by transfusion of bacterially contaminated RBCs. 40 Sepsis caused by transfusion of contaminated platelets is vastly underrecognized and underreported. Indeed, Jacobs et al. found that, during periods at their institution where active culture screening was in place, contaminated platelet components and sepsis were and 10.6-fold more likely to be documented, than during a period when detection relied solely on clinician recognition and reporting. 41 Reaction severity was greater with components containing 10 5 colony-forming units/ml and with higher bacterial virulence. At lower concentrations and with less virulent organisms, patients frequently displayed no immediate symptoms. Likewise, patients on antibiotic therapy or are neutropenic; they may not display classical signs of fever and sepsis, and, when they do, these are often ascribed to other infectious causes. Patients may not react immediately after transfusion of contaminated platelets: In one well-documented outbreak of Salmonella choleraseus, seven patients were linked to one repeat donor with an occult chronic osteomyelitis. The time to the onset of illness ranged from 5 to 12 days (mean 8.6 days). In all cases, the platelet units were stored for less than one day. 42 In a similar situation, the CDC reported in 2006 a multistate outbreak of Pseudomonas fluorescens bloodstream infections. All cases could be traced to contaminated heparin flushes. 43 A total of 28 patients had delayed onset of P. fluorescens infections, ranging from 84 to 421 days after their last potential exposure. Recognizing the potential harm of misdiagnosis of septic reactions, the AABB (formerly known as the American Association of Blood Banks) recommends active steps for all patients who, within 24 hours of a platelet transfusion, display a fever 38 C (<100.4 F) with a rise of >1 C (1.8 F) plus rigors, hypotension, shock, tachycardia, dyspnea, or nausea/vomiting. Furthermore, any change in clinical condition leading to a suspicion of sepsis even in the absence of fever should lead to actions that include halting the transfusion, patient support, and investigation for possible transfusion of a contaminated component. 44 Indeed, broad-spectrum antibiotics should be considered for any patient who develops fever within six hours of platelet transfusion Incidence Sepsis resulting from transfusion of bacterially contaminated platelets is the most common transfusion-transmitted disease. Platelets are stored at room temperature (20 24 C), making them an excellent growth medium. Multiple aerobic culture surveillance studies have demonstrated that 1 in 1000 to 3000 platelet units are bacterially contaminated. 1 Based on the fact that approximately 10 million platelet units (apheresis plus whole blood derived) were transfused every year in the United States, 47 it was estimated that 2000 to 4000 bacterially contaminated platelets units were transfused every year before the advent of culture screening. Despite the estimates of contamination, the actual septic transfusion reaction rates were much lower at approximately 1 in 25,000 platelet units (range 1 in 13,000 to 100,000). 1 The French National Hemovigilance Program, utilizing active surveillance in the period , reports rates of 1:40,000 (24.7 per million) and 1:216,000 (5.14 per million) for sepsis and fatality, respectively. 8 Following the introduction of methods to limit and detect contamination in 2004 in the United States, the American Red Cross described a 70% decline in sepsis reported to their passive surveillance program, with current rates of 1:107,000 for sepsis and 1:1,016,000 for fatalities. 39 Likewise, the FDA described 60 fatalities caused by platelet transfusion in the 10-year period (average 6.0/year; 62% caused by Gram-negative bacteria; 1:250,000 transfusions) and only 28 in the nine-year period (average 3.1/year; 36.1% caused by Gram-negative organisms) 9 (Table 53.2). Even with culture screening in place, Jacobs et al. estimate that at least 550 contaminated units are still transfused in the United States, as detected by a bacterial screening test performed at the time of transfusion. 48 Prevention measures introduced in the United States The College of American Pathologists (CAP) and AABB instituted steps to require the detection of bacteria in platelet products, beginning in CAP added an item to the Laboratory Accreditation Checklist 49 regarding the detection of bacteria that was modified in December 2004 to read, Does the laboratory have a validated system to detect the presence of bacteria in platelet components? In March 2004, AABB instituted a new standard that required blood banks or transfusion services to have steps in place to limit and detect bacterial contamination in all platelet products. 50 Subsequently, this was modified to include limit, detect or inactivate bacteria in recognition of the advent of approved pathogen inactivation technologies in many countries. Since that time, blood centers and transfusion services have implemented a variety of interventions to accomplish this goal. The majority of US blood collectors implemented bacterial culture screening of all apheresis platelets using one of two FDA-approved tests, the BacT/ALERT TM system (BioMerieux inc, Durham, NC) or, less commonly, the Pall ebds TM test (Haemonetics, Braintree, MA). 51 These tests are not suitable for screening individual random donor platelets derived from whole blood collections (WBD), and the AABB standards allowed the use of surrogate tests validated by individual users, including ph and glucose measurements at the time of poststorage pooling and release to patients. These assays were subsequently shown to be insensitive and nonspecific, providing suboptimal protection against bacterial contamination. 52 Two subsequent innovations were the introduction of the Pall Acrodose TM (Haemonetics) prestorage pooling system that allows the pooling of WBD before storage, facilitating the use of culture testing after manufacture, and FDA-approved point-of-issue tests, including the Platelet Pan Genera Detection assay (Platelet PGD TM, Verax Biomedical, Worcester, MA) and the BacTx TM (Immunetics, Boston, MA) test. 48,53 These technologies allow bacterial testing at the time of issue. In 2010, the AABB published an interim standard effectively requiring the use of FDAapproved tests, or technologies shown to be equivalent, thereby preventing the use of surrogate tests. A survey performed by the AABB in 2011 revealed that the vast majority of platelets were derived from apheresis collections, and 89.5% of these were screened using the BacT/ALERT culture system. 54 The American Red Cross began routine aerobic cultures using the BacT/ALERT system in March 2004, and in their first 10 months of testing, 226 of 350,658 platelet collections initially tested positive. 55 Sixty-eight of these were confirmed positives for a rate of bacterial contamination of one in 5157 distributed components. Despite universal testing of all platelet products, in the two-year period from

5 612 Section V: Part I: Infectious hazards March 2004 through May 2006, the ARC reported 20 septic transfusion reactions caused by transfusion of bacterially screened units. Of the 20 septic reactions, three were fatal and involved Staphylococcus species. 37 All of the units involved with septic fatalities, and 13 of the units implicated in septic reactions, were associated with platelets transfused on the fifth day after collection. Of note, with BacT/ALERT culture screening, products are released for transfusion hours after cultures are initiated; however, blood centers continue to hold the culture until the end of the components shelf life. Therefore, an efficient communication system was put in place to recall platelet products from the hospital should a culture indicate reactivity after distribution from the blood center. Experience showed that slow-growing organisms that trigger late reactive cultures after transfusion has occurred are seldom associated with transfusion reactions. 36 Since 2006, the Red Cross further refined its approach to bacterial safety by adopting inlet-line diversion of the initial ml of blood post phlebotomy (which decreases the collection contamination rate), increased the volume screened from a 4 ml to 8 ml sample under aerobic conditions (which increases BacT/ALERT culture sensitivity), and converted from a two-step povidone iodine skin preparation technique to the use of a single step with 2% chlorohexidine isopropyl (Chloraprep, Cardinal Health, Leawood, KS) alcohol swabs. 38,56,57 The Red Cross also instituted the Acrodose prestorage pooling system with BacT/ALERT screening for WBD platelets, although these comprise only 5% of the platelet components distributed. 57 With these changes in place, the Red Cross screened 2.2 M apheresis collections between 2006 and 2011 and detected 417 confirmed positive (188 per million: 1:5320) cultures, including 22% highly pathogenic Gram-negative bacteria. 39 The majority of cultured organisms were Gram-positive (Figure 53.1), and transfusion was prevented in most cases. Nevertheless, 38 septic transfusion reactions were reported to the Red Cross hemovigilance program (Table 53.2), including four fatalities, for an overall rate of sepsis of 1:107,000 distributed platelet components and 1:1,016,000 fatalities. 39 The fatalities were caused by Staphylococcus aureus (three cases) and coagulase-negative Staphylococci (one case). Most cases of sepsis were caused by Gram-positive organisms; however, three involved Gram-negative organisms and one implicated an anaerobe, Clostridium perfringens. 58 This represents a 70% decline in the rate of sepsis and fatalities reported to the Red Cross Hemovigilance Program compared to the 10-month period in , immediately before bacterial culture was instituted. 55 During that period, 12 high-probability septic reactions, including two fatalities, were reported by passive surveillance, whereas 500,000 platelet components were distributed. A similar trend is seen in the fatality data reported to the FDA: From 1995 to 2004, 60 deaths caused by bacterially contaminated platelets were reported, averaging six deaths per year (Table 53.2) before the initiation of the AABB standard. 10 These were predominantly caused by Gram-negative organisms (37 of 60 = 62%). In the nine-year period since the Standard, 28 deaths were reported for an average of 3.1 deaths per year, with 39% (11/38) caused by Gramnegative bacteria. 9 Bacterial culture screening is therefore most effective at preventing the transfusion of platelets contaminated with rapidly growing Gram-negative bacteria. Causes of false-negative bacterial culture screening tests It is clear that there has been a significant improvement in platelet safety over the prior decade, but a substantial risk remains. All septic reactions reported to the Red Cross have been associated with negative bacterial cultures, suggesting that the sample inoculated into the culture bottles was sterile. 39 When performing bacterial culture screening, a sample is taken from the product hours after collection in order to allow bacteria at very low concentrations (e.g., as low as 1 cfu/collection) to proliferate and reach concentrations of >1 10 cfu/ml, the demonstrated average lower sensitivity limit of the BacT/ALERT system when using an 8 ml sample. 11,59,60 Klebsiella spp. 5% Escherichia coli 8% Enterococcus spp. 1% Bacillus cereus 1% Listeria spp. 1% Enterobacter spp. 2% Serratia spp. 4% Pseudomonas spp. 1% Other Gram negatives 1% Coagulase-negative staphylococci 34% Corynaebacterium spp. 1% Staphylococcus aureus 10% Streptococcus spp. 31% Figure 53.1 Distribution of bacterial species detected as confirmed positive by the BacT/ALERT culture screen in the American Red Cross between 2006 and Source: Benjamin et al. (2014). 39 Reproduced with permission.

6 Chapter 53: Bacterial contamination of blood components 613 Table 53.3 Results of bacterial screening assays performed late during storage or on outdate, on platelet components screened and found negative for bacterial contamination during manufacture #Tested Confirmed Positives Rate per Million Sensitivity of Day 1 Test Reference PASSPORT Study 6, (1:1,509) 25.9% Irish Blood Service Day 8 8, ,200 (1:460) 29.2% Irish Blood Service Day 4 3, ,200 (1:828) Welsh Blood Service 6, (1:1,073) 40.0% It is thought that bacteria with a prolonged lag growth phase or slow doubling rate may avoid detection simply by virtue of low concentrations at the time of sampling (e.g., sampling error). 61 Some of these bacteria may enter a rapid-growth phase later in storage and reach clinically relevant concentrations by the time of transfusion. Further evidence in support of this hypothesis is provided by cultures performed later during storage or at outdate on components screened and found negative by culture assay at the time of manufacture. Three groups (Table 53.3) now report that reculture of platelets later during storage or at outdate using the BacT/ALERT system frequently reveals residual bacterial contamination, suggesting that early cultures may only detect 25 40% of contaminated units Similarly, Jacobs et al. showed that platelets previously screened and found negative by either the BacT/ALERT or ebds system revealed bacterial contamination in 9 of 27,620 ( 1:3000) tests using the Platelet PGD assay, suggesting that less than one-half of the contaminated units were detected by early culture. 48 Overall, there is a need to further improve the safety of platelets. Strategies to reduce the risk of posttransfusion sepsis Approaches to reduce the risk of posttransfusion sepsis can be grouped into four major categories: sepsis avoidance by appropriate platelet transfusion, reducing bacterial contamination, improving bacterial detection, and eliminating contaminating bacteria. Sepsis avoidance by appropriate platelet transfusion Platelets that are transfused when not indicated can be of no benefit and only harm patients. Recently, the AABB published an extensive evidence-based analysis of the indications for platelet transfusion. 65,66 The recommendations reemphasize the 10,000/uL trigger for prophylaxis in stable nonbleeding patients and provide guidance for surgery and other situations. Strict adherence to appropriate transfusion remains the most important safety measure for prevention of bacterial sepsis and other complications of transfusion. Reduction of bacterial contamination Donor screening Donors are routinely asked whether they feel well on the day of donation and screened for a fever. Should the donor report a history of a recent infection, all antibiotic therapy must be complete before donation is permitted. The phlebotomy site is inspected to ensure a clean site without signs of inflammation or infection. Surgical wounds must be healed and dry. Unfortunately, asking donors about symptoms suggestive of infections is problematic. For example, 13% of donors have had gastrointestinal symptoms in the 30 days before donation yet are not indicative of risk. 67 Retrospective questioning of donors implicated in red cell sepsis associated with Yersinia spp. showed that only half had any gastrointestinal symptoms in the preceding 30 days. 4,13 Therefore, donor questioning about gastrointestinal symptoms does not appear to be a specific predictor of Yersinia bacteremia. Likewise, most donors implicated in septic transfusion reactions or found to have positive bacterial screening cultures have no history, symptoms, or signs of infection. Although it is known that procedures such as brushing one s teeth and straining at stool may be associated with bacteremia, this is generally asymptomatic and screening is not effective. Rarely, an investigation may reveal pathology in the donor: Bacterial culture screening that reveals Streptococcus bovis should always lead to appropriate investigation for colonic pathology, as a number of cases of colonic polyps or carcinoma have been discovered in this fashion. 36,68 Likewise, a series of Salmonella spp. infections in one institution revealed a donor with chronic osteomyelitis. 42 Skin preparation Despite excellent technique, one cannot ensure a sterile venipuncture because organisms harbored in sebaceous glands and hair follicles cannot be completely removed or killed, and skin fragments drawn up into the collection bag during the initial phase of donation can provide a source of infectious organisms Scarring or dimpling of the venipuncture site from prior donation has also been recognized as a risk factor for bacterial contamination, because these areas frequently are difficult to disinfect. 71 In one case, phlebotomy at a dimpled venipuncture site of an apheresis donor resulted in three episodes of platelet contamination with Gram-positive organisms; sepsis occurred in four recipients of those platelets. The effectiveness of skin preparation may depend on the disinfectant solution utilized, the number of applications (generally one or two), the dwell time during which the skin is exposed to the disinfectant, and the skill of the operator. 72 Blood centers have standard procedures that enforce optimal skin disinfection procedures. The FDA recognizes three principal disinfections solutions for blood donation tincture of iodine (TI), chlorhexidine, and povidone iodine each usually suspended in isopropyl alcohol. Investigations of the residual contamination after disinfection show that iodine solutions and 2% chlorhexidine are effective in reducing the donor skin bacterial burden (Table 53.4), whereas green soap and isopropyl alcohol are not. Skin of donors who are allergic to iodine is often cleansed with a chlorhexidine solution. 73 In the United States, most blood centers now use a single-step 2% chlorhexidine swab process with 30-second dwell time, and that has been shown to be as effective as TI without the risk of staining clothes and allergy. 56 Diversion Diversion of the first milliliters of whole blood from the primary container has been shown to reduce the amount of bacterial contamination from the skin, presumably by capturing skin fragments or a shower of bacteria released at the time of phlebotomy. Bruneau et al. 74 collected the first and second 15 ml aliquots of 3385 whole blood collections and cultured these under aerobic and anaerobic conditions. Seventy-three were positive in the first

7 614 Section V: Part I: Infectious hazards Table 53.4 Proportion of donors with bacterial growth after skin disinfection Bacterial Colonies per Plate Povidone Iodine Isopropyl Alcohol + Tincture of Iodine Chlorhexidine Gluconate Green Soap and Isopropyl Alcohol % 63% 60% 0% % 34% 25% 17% % 2% 12% 47% > % 1% 3% 36% p value compared to povidone iodine <0.001 >0.3 <0.001 Source: Goldman et al. (1997). 72 Reproduced with permission of Wiley. 15 ml and 21 in the second, including four species not detected in the initial 15 ml. Overall contamination rate was 2.2%, mainly with Gram-positive Staphylococcus spp. and Bacillus spp. The residual risk of contamination in the collection was 0.6%, showing that diversion could significantly reduce the overall risk. A study from the Netherlands compared the bacterial contamination rates of whole blood collections with and without the removal of the first 10 ml. The diversion of the first 10 ml showed a significant decrease in bacterial contamination (18,263 collections with 0.39% contamination without diversion compared with 7115 collections with 0.21% contamination with diversion, p < 0.05). 75 Diversion is most effective at decreasing contamination with skin flora. A majority of bacteria-related fatalities involve Gram-negative organisms, which are likely not interdicted by diversion. Similarly, the ARC data reported data between 2004 and 2006 where it is possible to compare the confirmed-positive bacterial culture rates with and without diversion (Table 53.5). 38 The authors report on the contamination rate of one-arm collections (which incorporated inlet-line diversion) and two-arm collections (which did not) on the same apheresis equipment. There was a 2.2-fold higher rate of skin contaminants with the two-arm procedure compared with the one-arm procedure, and this difference was apparent only for skin contaminants. Apheresis versus whole blood derived platelet concentrates Therapeutic doses of platelets can be obtained from a single donor through an apheresis procedure, or from whole blood donations. Four to six platelets concentrates from whole blood donations are pooled to make a therapeutic dose; therefore, it would be expected that pooled platelets obtained from multiple donors would be at higher risk of bacterial contamination. From 1986 to 1998, Johns Hopkins Hospital increased the use of apheresis platelets from 51.7% to 99.4% and saw a threefold reduction in septic transfusion reaction involving platelets, from one in 4818 transfusions to 1 in 15,098 transfusions. 76 With the introduction of AABB Standard in 2004, most apheresis platelets were screened using culture methods, whereas WBD were screened by the transfusion service after pooling using surrogate markers (e.g., ph or glucose), as many institutions felt that culturing of individual WBD platelets was impractical. The Acrodose PL system is now available for prestorage pooling of WBD platelets, which allows institutions to pool WBD platelets and then store them for up to five days after collection. 57 By using prestorage pooling, the volume of the product is acceptable for culturing with either the BacT/ALERT or ebds. The Red Cross reported that BacT/ALERT cultures performed on pools of five WBD were fivefold more likely to be contaminated than apheresis platelets, using identical screening processes, in keeping with the concept that pooled platelets are more likely to be contaminated than single-donor apheresis platelets. 57 Table 53.5 Comparison of one- and two-arm apheresis platelet collections Prepooled WBD platelets prepared by the buffy coat method have been available in Europe since the early 1990s. Recently, Canada transitioned from the platelet-rich plasma method to the buffy coat method. The conventional wisdom in Europe is that the confirmed positive culture and sepsis rate of prepooled buffy coat platelets is equivalent to those of apheresis platelets, 77 perhaps due to the overnight incubation of whole blood in the presence of white cells before preparation of buffy coats and leukoreduction of the pooled product. Bacterial culture screening results, however, show wide variability, with some investigators showing a 2 5-fold increased confirmed positive culture rate with pooled products 62,75,78 80 and others showing no difference (Figure 53.2). 81 In the United States, there has been a gradual move away from pooled platelets to apheresis platelets. A survey of AABB-accredited blood centers, hospital blood banks, and transfusion services in late Confirmed Positive Rate (per 10 6 ) Rate per 10 5 Cultures 1,200 1, Two-Arm Procedures One-Arm Procedures OR (95% CI) Skin contaminants ( ) Nonskin organisms ( ) Total confirmed positives ( ) An inlet-line diversion pouch was in place on the one-arm procedures only. Source: Eder et al., 2008 [ 57 ]. Reproduced with permission of Wiley Apheresis Buffy coat Platelet-rich plasma Figure 53.2 Rate of confirmed positive BacT/ALERT TM screening cultures broken out by platelet type, with data derived from 16 international studies. The x-axis in each panel represents the number of products tested varying from on a logarithmic scale. Solid symbols represent the use of both aerobic anaerobic cultures conditions. Used with permission. Source: Benjamin and McDonald (2014). 78 Reproduced with permission of Elsevier.

8 Chapter 53: Bacterial contamination of blood components examined platelet usage, supply, and testing methods. 51 Between 2003 and 2004, there was an 11.3% reduction in the use of WBD platelets, and 77.2% of platelets transfused by the institutions surveyed were apheresis platelets. By 2011, the National Blood Collection and Utilization survey revealed that 91.1% of platelets were from apheresis collections. 82 Reducing storage duration Longer platelet storage time is associated with an increased probability of clinically significant contamination, as low initial concentrations of bacteria may proliferate over time. In 1983, in the United States, platelet storage for WBD platelets was transiently approved for seven days based on acceptable in vitro function, in vivo recovery, and survival data. However, because of anecdotal reports of bacterial proliferation over the extended storage time, the shelf life was returned in 1986 to five days. With the introduction of prestorage bacterial culture screening, the FDA again allowed the investigation of seven-day storage under the Post Approval Surveillance Study of Platelet Outcomes, Release Tested (PASSPORT) protocol, where each apheresis collection was screened under aerobic and anaerobic culture conditions. 64,83 This protocol successfully demonstrated improved platelet logistics and decreased wastage; however, poststorage culture revealed a substantial rate of residual contamination, and the protocol was closed. Even with five days of storage, reports of septic transfusion reactions and fatalities persist, especially on days 4 and 5 of storage (Table 53.2). Only by severely reducing the storage time of platelets would one significantly impact the risk of bacterial overgrowth. However, operational changes affect the storage age of available platelets. With increased complexity of disease-marker testing, the availability of one-day-old platelets has decreased. For example, in the United States in 1982 the mean age of distributed platelets was 1.6 days, in 1983 (after extension of the dating period to five days) it was 2.0 days, and in 1992 (after addition of increased laboratory testing) it was 2.5 days. In 1983, only 5% of issued platelets were greater than three days old. In 1992, just 10% were older than three days. But with the introduction of centralized testing and bacterial screening, the mean age of issued platelets increased to 2.7 days, with 20% older than three days. With the addition of nucleic acid testing for HIV and hepatitis C virus (HCV), additional delays occurred. Not only can this decrease available shelf life of an already precariously limited supply of platelets, but also it can decrease the availability of fresh platelets, which are the most hemostatic and the least likely to be bacterially contaminated. One US center that collects its own whole blood and apheresis platelets has focused on early transfusion as a means of ensuring bacterial safety: 84 All products are sampled and cultured for bacteria, but the components are transfused as soon as possible after sampling. Apheresis platelets are transfused within 48 hours, and pooled WBD platelets within 72 hours of collection. The authors report that after 23,199 transfusions, 71 products were shown to be contaminated, all with Gram-positive Staphylococci spp. or Streptococci spp., except for a single Gram-negative Enterobacter cloacae isolate. Only a single mild septic reaction was reported to a product contaminated by a coagulase-negative Staphylococcus spp. isolate. The authors argue for the safety of fresh products despite contamination by bacteria, especially in patient groups that are usually on prophylactic antibiotic therapy for other clinical indications. Other countries limit the shelf life of platelets in order to improve safety: Japan limits platelets to a <3.5-day shelf life, and in 2009 the German Paul Erlich Institute limited storage to four days following a series of fatal sepsis cases. 85 Neither country employs bacterial culture screening. Germany allows transfusion on day 5 after collection if the product is screened using a culture system or point-of-release screen on day 4 or 5 (see below). In December 2012, the FDA Blood Products Advisory Committee suggested that the US centers similarly reduce shelf life to four days unless rescreened for bacterial contamination on day 4 or 5. At the time of writing, the FDA had not converted this recommendation into guidance, and US blood centers continue to label platelets for five days of storage. Bacteria detection Bacterial contamination of platelet products is thought to initially involve minimal numbers of bacteria, some of which may proliferate to high levels during storage. In vitro inoculation experiments confirm that the growth dynamics differ by bacterial species. In one study, bacterial growth characteristics were reported for 165 platelet units, each inoculated on the day of collection with 1 5 CFU/ml of one of the following organisms: Bacillus cereus, P. aeruginosa, Klebsiella pneumoniae, S. marcescens, S. aureus, and S. epidermidis. 86 All examples of B. cereus, P. aeruginosa, K. pneumoniae, S. marcescens, and S. aureus had concentrations >10 2 CFU/mL by Day 3 following inoculation. By Day 4, all units with these organisms contained >10 5 CFU/mL. Units contaminated with S. epidermidis showed slower and more varied growth. This study concluded that an assay capable of detecting 10 2 CFU/mL on Day 3 of storage would detect a vast majority of bacterially contaminated platelet units. Based on these and similar observations, sensitive culture techniques that can detect 1 10 CFU/ml are used soon after manufacture, and rapid, less sensitive detection methods are used closer to the time of transfusion. Examples of screening systems with high sensitivity that may be used soon after manufacture are two culturebased systems that are FDA approved in the United States, the BacT/ALERT and ebds systems mentioned in this chapter. These can theoretically detect as few as a single viable bacterium in a 4 10 ml sample. In practice, they are validated to detect 1 10 CFU/ ml of a range of transfusion-relevant bacterial strains. In contrast, there are a number of detection systems that detect bacteria directly, such as the FDA-approved PGD assay (Verax Biomedical, Worcester, MA), BacTx (Immunetics, Boston, MA) assay, and other tests in development especially in Europe, including NAT assays and flow cytometry based assays (BactiFlow TM ALS, BioMerieux, Durham, NC). These assays typically have a lower analytical sensitivity than culture methods ( cfu/ml), but may be performed rapidly (1 4 hours) and so are suitable for use close to the time of issue when bacterial concentrations are likely to be higher. Bacterial culture The BacT/ALERT system uses an automated liquid culture system that includes culture broth under aerobic or anaerobic conditions. Each bottle contains a sensor that changes color as a consequence of increasing CO 2 produced by bacterial proliferation. The system monitors both the absolute color change and rate of change of the colorimetric sensor. The bottles are inoculated with a needle through a rubber stopper, rendering the system incompletely closed and susceptible to contamination by introducing bacteria into the bottle. The method reliably detects contamination of platelets inoculated to 10 CFU/mL and in many cases <5CFU/mL (e.g., B. cereus, S. marcescens, C. perfringens, S. epidermidis, S. pyogenes, E. coli, K. pneumoniae, S. aureus, and viridans streptococci) in hours. 60

9 616 Section V: Part I: Infectious hazards The BacT/ALERT system is widely implemented in the United States and around the world. There are several variables that may affect culture sensitivity, including the delay between collection and sampling; the volume of product inoculated into the culture bottle; and the use of both aerobic and anaerobic culture conditions. 78 Wagner and Robinette 88 used a model system of platelets contaminated in vitro with S. epidermidis or E. coli at 10, 1 or 0.1 cfu/ml and then sampled 0, 6, 24, or 48 hours later. 0.5, 1, or 2 ml were inoculated and incubated in 12 BacT/ALERT aerobic bottles for each data point. Their results clearly showed that higher initial concentrations, larger sample volumes, and longer delay before sampling were associated with a larger proportion of inoculated bottles signaling positive at any given time point. This work was subsequently replicated for a wider range of organisms. 60 Various laboratories around the world have implemented the test in different ways, as can be seen in Table No definitive data yet support the superiority of any given conformation. Assuming that the initial number of bacteria contaminating a clinical collection is very low, an increased time delay before sampling would make it more likely that a higher concentration of bacteria are available in any given sample. In the United States and Canada, most blood centers therefore wait >24 hours after collection before taking the sample; however, many European countries, including Ireland, Wales, and Holland, use shorter periods in order to facilitate workflow in the center. 54,78 Conversely, the English National Blood Service delays sampling until hours after collection to maximize the probability of detecting contamination. 80 The shelf life of platelets is extended to seven days to increase the time available for transfusion in the hospitals. In a similar fashion, increasing the volume of sample inoculated might be expected to increase culture sensitivity. Theoretical calculations suggest that doubling the sample volume may increase the proportion of contaminated products detected by as much as 25%, and Eder et al. found that BacT/ALERT culture was 1.54 ( ) fold more likely to detect bacteria using an 8 ml sample versus a 4 ml sample. 38,89 Further theoretical calculations have been provided by Tomasulo and Wagner, who advocate the use of a sample size that is a constant proportion of the product volume, in order to standardize the sensitivity of the assay for single, double, and triple collections. 90 Despite the theoretical consideration that larger volume products may be less safe if all collections are sampled at the same volume, Eder et al. report no difference in the septic transfusion reaction rates between single, double, and triple collections. 91 The downside of increased volume inoculation is the loss of product volume for transfusion and decreased splits rates for multiple component collections. Many centers inoculate both aerobic and anaerobic culture bottles in order to maximize culture sensitivity. Most clinically relevant bacteria found contaminating platelets grow under both conditions, although specific strains may grow faster in one condition than the other. This was best demonstrated for a single strain of Streptococcus lugdenensis implicated in a fatal transfusion reaction. 92 A small number of species are obligate aerobes (e. g., Pseudomonas fluorescens) or obligate anaerobes (e.g., P. acnes and Clostridium spp.). Utilizing both aerobic and anaerobic conditions allows the detection of a complete range of species, speeds up detection of some strains, and doubles the volume of product cultured, thereby increasing the sensitivity of the overall detection system. Nevertheless, most US blood centers use only aerobic culture conditions, as anaerobic conditions mostly detect P. acnes, a species that does not grow well under the aerobic conditions of platelet storage; are rarely implicated in transfusion reactions; and are detected after a median of 120 hours of incubation, when most products are already transfused. Anaerobic conditions are necessary for the detection of Clostridium spp., a rare but potentially lethal contaminant of platelets. 58,93 Even under optimized culture conditions that include both aerobic and anaerobic culture conditions, false-negative screening cultures continue to be detected. As outlined in Table 53.3, outdate cultures suggest that only 25 40% of contaminated products are intercepted and septic reactions continue to be reported Under the most sensitive conditions used by the English National Blood Service, no septic reactions have been reported despite >500,000 platelet transfusions; however, this finding is difficult to interpret as zero septic reactions were reported to the UK SHOT hemovigilance system for the two years prior to the introduction of culture screening. 80 The consensus view is that although culture screening has significantly reduced the risk of bacterial sepsis, especially for Gram-negative organisms, no iteration can ensure the absolute safety of platelets from contamination. Bacterial screening creates substantial logistic burdens due to delay of product release into inventory; product loss due to sampling, especially when large volumes are used; and the need for product recalls when culture screenings turn reactive after components are distributed to Table 53.6 International approaches to assuring bacterial safety of platelet products utilizing bacterial culture (BacT/ALERT TM ), flow cytometry (BactiFlow TM ), or pathogen inactivation (Intercept TM ) technologies Bacterial Culture Country Storage Duration (d) Flow Cytometry Pathogen Inactivation Aerobic (A)/Anaerobic (An) Volume (ml) Delay (Hours) Ref. US 5 A Canada 5 A England 7 A/An Ireland 5 A/An >12 Wales 5 A/An >16 Australia 5 A/An 16 Holland 7 A/An Hong Kong 5 A 10 Switzerland 5 X Germany 4 X France 5 $ Japan < Allows extension to five-day storage. $ One regional blood center and all overseas departments use pathogen inactivation routinely.