Evaluation of a rapid colorimetric assay for detection of bacterial contamination in apheresis and pooled random-donor platelet units.

BACKGROUND: Despite existing strategies, bacterial contamination of platelets (PLTs) remains a problem, and reliable testing near the time of use is needed. We evaluated the BacTx assay (Immunetics, Inc.), a rapid colorimetric assay for detection of bacterial peptidoglycan, for this purpose. STUDY DESIGN AND METHODS: Apheresis- and whole blood-derived PLT units, the latter tested in 6-unit pools, inoculated with 10 representative bacterial species (eight aerobic, two anaerobic), were tested with the BacTx assay at two sites to determine analytic sensitivity and time to detection. Specificity on sterile PLTs and reproducibility across different PLT units and assay kit lots was also determined. RESULTS: Analytical sensitivity for the 10 bacterial species ranged from 6.3 × 10(2) to 7.6 × 10(4) colony-forming units (CFUs)/mL. In time-to-detection studies after inoculation of PLTs with 0.7 to 5.3 CFUs/mL, 10 replicates of all eight aerobic species were positive when bacterial titers were above the analytic sensitivity detection limit, which occurred at 48 hours for 60 PLT units and at 72 hours for the remaining 4 units, as well as at 7 days for all units. Specificity was 99.8% and reproducibility was 100%. CONCLUSIONS: The BacTx assay had an analytical sensitivity below the 10(5) CFUs/mL threshold of clinical significance, detected all eight aerobic bacterial species 48 to 72 hours after inoculation as well as at 7 days, and had high specificity and reproducibility. These findings suggest that the BacTx assay will be a valuable test for detection of clinically relevant levels of bacterial contaminants in PLT units and pools near time of use.

Characterization of the growth dynamics and biofilm formation of Staphylococcus epidermidis strains isolated from contaminated platelet units.

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk, with Staphylococcus epidermidis being a predominant contaminant. Herein, the growth dynamics of 20 S. epidermidis strains in PCs and regular media were characterized. Strains were categorized as fast (short lag phase) or slow (long lag phase) growers in PCs. All strains were evaluated for the presence of the biofilm-associated icaAD genes by PCR, their capability to produce extracellular polysaccharide (slime) on Congo red agar plates and their ability to form surface-attached aggregates (biofilms) in glucose-supplemented trypticase soy broth (TSBg) using a crystal violet staining assay. A subset of four strains (two slow growers and two fast growers) was further examined for the ability for biofilm formation in PCs. Two of these strains carried the icAD genes, formed slime and produced biofilms in TSBg and PCs, while the other two strains, which did not carry icaAD, did not produce slime or form biofilms in TSBg. Although the two ica-negative slime-negative strains did not form biofilms in media, they displayed a biofilm-positive phenotype in PCs. Although all four strains formed biofilms in PCs, the two slow growers formed significantly more biofilms than the fast growers. Furthermore, growth experiments of the two ica-positive strains in plasma-conditioned platelet bags containing TSBg revealed that a slow grower isolate was more likely to escape culture-based screening than a fast grower strain. Therefore, this study provides novel evidence that links S. epidermidis biofilm formation with slow growth in PCs and suggests that slow-growing biofilm-positive S. epidermidis would be more likely to be missed with automate culture.

Sex, risk, and education in donor educational materials: review and critique.

Food and Drug Administration guidelines prohibit men who have sex with men (MSM) from donating blood to prevent the spread of the human immunodeficiency virus (HIV/AIDS). Although the deferral criteria leave "sex" undefined, donor educational materials distributed before the health questionnaire often offer a definition. This study analyzes educational materials for their contribution to the donation process and construction of HIV/AIDS. It applies a discourse analysis approach to a sample (n = 52) of such materials obtained in summer 2009 from blood collection organizations listed in the AABB (now referred to as "Advancing Transfusion and Cellular Therapies Worldwide") Directory of Community Blood Centers and Hospital Blood Banks [AABB. Directory of Community Blood Centers and Hospital Blood Banks. Bethesda, MD: AABB; 2009]. It finds that when materials define sex, the definition is "vaginal, oral, or anal sex whether or not a condom or other protection was used," and when materials define HIV/AIDS risk behaviors, the definition is, with few exceptions, "sexual contact with an infected person or by sharing needles or syringes used for injecting drugs." Widespread use of these definitions demonstrates the influence of "Making Your Blood Donation Safe." Through analysis of this document and variations upon it, this research finds that the category MSM therefore provides one component of the construction of HIV/AIDS as the providence of MSM, together with heterosexual Africans and other risk populations, conflating group membership with individual risk. Deferring MSM therefore fails as a behavior-based deferral because it collapses multiple sexual behaviors with varying risks into a single risk category. It constructs all MSM as HIV positive and implicitly constructs non-MSM as risk-free.

Bacterial contamination of platelets.

Bacterial contamination of platelet products, both single donor apheresis platelet units and whole blood-derived platelet pools, continues to occur despite preventive measures. While some advances have been made in decreasing the rate of bacterial contamination of platelet units, particularly through diversion methods and early culture, a great deal remains to be done to eliminate the problem. Diversion methods have decreased contamination rates associated with skin commensal organisms. Culture methods are now widely used and many at-issue detection methods have been developed or are undergoing development. This article reviews the current developments and the challenges that remain to minimize and detect bacterial contamination of platelet products.

Relationship between bacterial load, species virulence, and transfusion reaction with transfusion of bacterially contaminated platelets.

BACKGROUND: Bacterial contamination is currently the major infectious hazard of platelet transfusion, but associations between bacterial species and quantity and transfusion reactions have not been characterized. METHODS: Patients receiving platelets from July 1991 through December 2006 were observed using active surveillance by quantitative culture of platelets at the time of issue or passive surveillance by investigation of clinical reactions in patients and culture of implicated units. Patient reactions were classified by type and severity and were correlated with bacterial species and number. Endotoxin content of gram-negative contaminants was determined by limulus lysate assay. RESULTS: Fifty-two bacterially contaminated platelet units were detected (50 by active and 2 by passive surveillance). Rates of bacterial contamination and septic transfusion reactions were 32.0-fold and 10.6-fold higher, respectively, as determined by active versus passive surveillance (P < .001). Including 2 index cases, bacterial contaminants included gram-negative bacilli in 4 units (3 of which were associated with fatal reactions), staphylococci in 44 units, streptococci in 4 units, and Bacillus cereus in 2 units. Endotoxin content of the 4 units that were contaminated with gram-negative bacilli ranged from 11,373 to 173,130 endotoxin units. Reaction severity was greater for units with bacterial counts of > or =10(5) colony-forming units/mL and higher bacterial virulence. A detection method with a 10(3) colony-forming units/mL threshold would detect >90% of contaminants. CONCLUSIONS: Active surveillance detected 32-fold more bacterially contaminated platelet units and 10.6-fold more septic reactions than did passive surveillance, and virulent species and bacterial counts of > or =10(5) colony-forming units/mL were associated with more-severe transfusion reactions. Improved detection methods or use of pathogen inactivation technology are needed to eliminate this problem.

Detection of bacteria in stored red cell products using a culture-based bacterial detection system.

BACKGROUND: The Pall eBDS uses oxygen consumption as a surrogate marker for bacterial detection in platelet (PLT) products. This article describes the evaluation of eBDS to detect bacterial contamination in inoculated fresh and stored leukoreduced red cell (RBC) units. STUDY DESIGN AND METHODS: Field studies were conducted at three sites to establish eBDS pouch incubation time and the pass/fail threshold. RBC units were inoculated with each of 12 bacterial species known to cause sepsis at target inocula of either 1 to 15 or 100 colony-forming units (CFUs) per mL. Units were mixed and stored at 2 to 6 degrees C. Samples were taken for culture and eBDS testing weekly from 0 hour to 42 days and incubated for 35 degrees C for 24 to 30, 48, and 72 hours, followed by measurement of percent oxygen content. RESULTS: The studies showed growth of five bacterial species (including Yersinia enterocolitica) in RBC units, while seven bacterial species showed no growth or autosterilized. A pass/fail oxygen threshold of 14.4 percent was determined based on results from noninoculated controls (n = 633) and from inoculated samples (n = 884) after 48 hours of incubation. Detection was 100 percent at all sampling times during refrigerated storage with both 48 and 72 hours of pouch incubation. CONCLUSION: With incubation of eBDS pouches for 48 and 72 hours, 100 percent detection was obtained in 884 samples with bacterial levels of at least 1 CFU per mL, and no false-positive samples were obtained. Based on the bacterial growth patterns, RBC units may be sampled 1 to 3 days after collection for optimal efficacy and read after 48 to 72 hours of incubation of the eBDS sample pouches at 35 degrees C. The Pall eBDS is suited for detection of typical bacterial contaminants in fresh and stored RBCs.

Development of proficiency testing for detection of bacterial contamination of platelet products.

BACKGROUND: Accrediting agencies now require quality control for minimizing platelet (PLT) bacterial contamination (PBC). A proficiency testing (PT) strategy for PBC testing was developed. STUDY DESIGN AND METHODS: During Phase 1, validation of the pH meter and an enhanced bacteria detection system (Pall eBDS)--methods used in our blood bank--was accomplished with two aliquots of an apheresis PLT unit inoculated with Escherichia coli (ATCC 25922, 70 CFU/mL) or Staphylococcus aureus (ATCC 27217, 46 CFU/mL) with an uninoculated aliquot serving as a negative control. Quantitative plate culture was the reference method. PLTs were stored on a rotator at 22 degrees C. Units were sampled in duplicate at 0, 24, and 48 hours. pH testing was considered positive when the pH value was less than 6.6. eBDS was positive when the oxygen concentration was less than 9.4 percent. During Phase 2, synchronized PT of pH and eBDS was performed at four independent sites. PLT samples were inoculated and incubated as above, and aliquots were removed at Time 0 for eBDS testing and at 48 hours for pH testing. RESULTS: In Phase 1, on inoculated bags eBDS was positive at all time periods but pH was positive only at 48 hours. In Phase 2, synchronized results showed positive eBDS at Time 0 and positive pH at 48 hours on inoculated bags with agreement between paired sites. CONCLUSIONS: This strategy may serve as a useful model for developing PT materials for PBC detection. eBDS was able to identify low levels of PBC, and pH testing, only much higher levels. It is important to carefully coordinate and standardize handling of PT materials and reporting of results.

Detecting bacterial contamination in platelet products.

Bacterial contamination of platelets is an important cause of transfusion-associated morbidity and mortality. It is currently the most frequent infectious complication of transfusion therapy, with between 1 in 1,000 and 1 in 3,000 platelet units being bacterially contaminated at time of transfusion. Several factors have contributed to the persistence of this problem including lack of sensitive detection methods, lack of recognition of the frequency of the problem, inadequate recognition of septic reactions by clinicians treating patients receiving platelet transfusions, differences in transfusion reactions between bacterial species and bacterial inocula transfused, and differing methodologies and time of testing for detection of bacteria in platelet units. There are also important correlations between the receipt of bacterially contaminated platelet units and the development of transfusion reactions and bacteremia. In the last few years the recognition of the importance of platelet bacterial contamination prompted the College of American Pathologists (CAP) and the American Association of Blood Banks (AABB) to set new standards requiring the screening of platelets for bacterial contamination. In the wake of these standards, an increasing number of approaches have been and are being developed to deal with this problem. The clinical sensitivity, specificity and predictive value of these detection methods vary considerably and need to be defined for routine laboratory practice. In this review, we focus on the practical aspects and feasibility of implementing FDA-cleared detection methods for identifying bacterially contaminated platelet units. We also present details of a number of methods under development for at-issue use.

Evolution of surveillance methods for detection of bacterial contamination of platelets in a university hospital, 1991 through 2004.

BACKGROUND: Platelet (PLT) bacterial contamination (PBC) is the most common transfusion-associated infection. It is important to understand the impact of interventions addressing this problem. STUDY DESIGN AND METHODS: PBC was studied by prospective (active) and transfusion-reaction triggered (passive) surveillance from July 1991 to December 2004. Active surveillance, utilized for 10 years, included bacterial culture of all or 4- and 5-day-old PLTs at issue and intermittent use of Gram stain, pH measurements, and early (24-hr) culture of single-donor plateletpheresis (SDP) units. RESULTS: Active surveillance detected 38 instances of PBC, 7 in SDP units (1:2213) and 31 in random-donor PLT units (1:2090 units, p = 0.89; or 1:418 pools of 5 units, p < 0.001). Contaminants were coagulase-negative staphylococci (CONS; n = 27), Staphylococcus aureus (4), Bacillus cereus (1), Serratia marcescens (2), streptococci (2 S. bovis, 1 S. uberis), and CONS with viridans group streptococcus (1). Only one instance of contamination, caused by Pseudomonas aeruginosa, was detected by passive surveillance, with fatal outcome. Colony counts of contaminants ranged from 0.5 x 10(2) to 4 x 10(11) colony-forming units per mL at time of issue. PBC was interdicted before transfusion in 6 cases through Gram stain screening. Transfusion reactions occurred in 13 of 32 recipients (41%), with 9 severe reactions (28%) and 3 deaths (9%). pH testing failed to detect 5 contaminated units and resulted in discard of nearly 2 percent of units, whereas culture of SDP units at 24 hours failed to identify a contaminated unit. CONCLUSION: Improved active surveillance methods for detecting PBC are needed to improve the safety of PLT transfusions.

Acute hemolytic transfusion reaction in a pediatric patient following transfusion of apheresis platelets.

The practice of transfusing ABO-incompatible platelets, driven primarily by concerns about inventory management, has been considered generally safe because the accompanying plasma is usually diluted in the recipient's total blood volume. However, if the platelet product contains a large volume of plasma or a high concentration of incompatible isoagglutinin, there may be hemolysis of the recipient's red cells. Patients with a small blood volume, such as babies and children, are considered to be at particular risk for such a complication. We describe the case of a baby who suffered massive hemolysis of her group A red cells after transfusion of group O Apheresis Platelets containing a high-titered anti-A isoagglutinin. We also offer a review of the literature on this subject and recommendations to avoid acute hemolytic reactions as a result of platelet transfusion.

Enhancement of a culture-based bacterial detection system (eBDS) for platelet products based on measurement of oxygen consumption.

BACKGROUND: An enhanced bacterial detection system (Pall eBDS) was developed that distinguishes itself from its predecessor (Pall BDS) by removal of the platelet (PLT)-retaining filter allowing for optimal bacterial transfer, modification of the culture tablet to reduce the confounding effects of respiring PLTs while enhancing bacterial growth, and facilitation of nutrients and gas exchange by agitating the sample pouch during incubation at 35 degrees C. The objective was to evaluate the performance of the new eBDS. STUDY DESIGN AND METHODS: Leukoreduced whole blood-derived PLT concentrates (LR-PCs) and LR single-donor PLTs (LR-SDPs) were inoculated with 1 to 15 colony-forming units (CFUs) of bacteria per mL in studies of each of 10 bacterial species associated with fatal transfusion-transmitted bacterial infection. Immediately after inoculation and after 24 hours of storage at 22 degrees C, samples of inoculated LR-PCs were aseptically transferred into the eBDS pouches. Pouches were then incubated for 24 hours at 35 degrees C with agitation and oxygen concentration was then measured. RESULTS: Median inoculation levels ranged from 5 to 13 CFUs per mL for each species studied. No significant differences in oxygen concentration were found when comparing LR-PCs with LR-SDPs. When sampling occurred from the PLTs 24 hours after inoculation, all 280 cases (24-33 replicates of each species) were detected as contaminated by the device (100% sensitivity). No false-positives were obtained with 713 uninoculated PLT units. CONCLUSIONS: The eBDS demonstrated improved detection sensitivity in the range of 1 to 15 CFUs per mL with no observed false-positives compared to the original BDS (detection range 100 to 500 CFUs/mL) with no false-positives.

Evaluation of the Scansystem method for detection of bacterially contaminated platelets.

BACKGROUND: Platelet (PLT) bacterial contamination occurs in approximately 1 in 2000 PLT units. The College of American Pathologists recommends and AABB requires procedures to detect PLT bacterial contamination. Although two methods, BacT/ALERT (bioMerieux) and Pall BDS (Pall Corporation), have FDA approval for quality control testing, additional methods are in development. One such method was evaluated, the Scansystem (Hemosystem), which has been developed for use on leukoreduced PLT components between 30 and 72 hours after collection. STUDY DESIGN AND METHODS: Leukoreduced, single-donor apheresis PLT units (LR-SDPs) were inoculated with 10 bacterial species (low and high inocula) associated with PLT contamination. Bacterial detection was compared with the Scansystem and BacT/ALERT. Testing was initiated (10 replicates performed) when LR-SDPs were experimentally inoculated with bacteria. The Scansystem was evaluated 30 hours later, the shortest manufacturer recommended time after PLT collection. RESULTS: All replicates were positive with the Scansystem at 30 hours and with the BacT/ALERT, at 9.3 to 24.0 hours after inoculation. The Scansystem detected bacteria in 83 of 200 replicates (42%) at the time of inoculation indicating a potential for earlier application. CONCLUSIONS: The Scansystem, used to test LR-SDPs 30 hours after bacterial inoculation, detected all 20 replicates with a sensitivity equal to the BacT/ALERT system. Based on use of Scansystem with LR-SDPs 30 hours after collection and the BacT/ALERT being inoculated 24 hours after collection and incubated for an additional 24 hours before being determined to be negative, the Scansystem will potentially provide results at an earlier time point (32 hr) than provided by the BacT/ALERT system (48 hr).

Bacterial contamination of blood products.

The occurrence of a septic reaction resulting from bacterial contamination of blood products, particularly with room-temperature stored platelets, is the most common transfusion-associated infectious risk in the United States. Bacterial contamination of blood products was first identified more than 60 years ago; yet, strategies to resolve this problem have proved daunting despite ongoing awareness and increasing concern especially in the last few years. With the recent US Food and Drug Administration (FDA) approval of culture methods for quality control testing of platelet units and the promulgation of accreditation standards by the College of American Pathologists and American Association of Blood Banks to detect bacterially contaminated platelet units and to prevent transfusion of these units, blood banks and transfusion services have finally started to address this problem, in a more standardized manner. Furthermore, as new methods of interdicting, inactivating and detecting bacterially contaminated blood products emerge, it is hoped that the problem of bacterial contamination of blood products will be overcome.

Relapsed thrombotic thrombocytopenic purpura presenting as an acute cerebrovascular accident.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon but severe disorder that classically presents with microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and fluctuating neurological changes. Previously, it was impossible to make a diagnosis of TTP in the absence of thrombocytopenia or microangiopathic hemolysis (MAHA). We describe two cases of relapsing TTP that presented with acute cerebrovascular accident (CVA) without concurrent thrombocytopenia or MAHA after initial classical presentation of TTP. In both cases, the diagnosis of TTP as the cause of the CVA was attributed to severe deficiency of the von Willebrand factor cleaving protease, ADAMTS13 in plasma (11 and 12%, normal 79-127%). Each patient had a dramatic clinical improvement in response to therapeutic plasma exchange. The experience in these two cases suggests that TTP should be considered as a potential cause among patients presenting with a CVA, particularly if the patients have a history of TTP.