Fatal group C streptococcal infection due to transfusion of a bacterially contaminated pooled platelet unit despite routine bacterial culture screening

BACKGROUND: An elderly man with chronic myelomonocytic leukemia developed respiratory distress and died less than 48 hours after transfusion of a pool of eight whole blood–derived platelets (PLTs). Blood cultures from the recipient and cultures of remnants from the pooled PLT bag grew group C streptococci (GCS). An investigation was conducted to identify both the infection's source and the reasons for the false‐negative screening result. STUDY DESIGN AND METHODS: Red blood cell (RBC) units (cocomponent from the eight donations) were traced, quarantined, and cultured. Specimens from the implicated donor were obtained. Isolates were identified and typed by 16S rRNA and pulsed‐field gel electrophoresis (PFGE). The blood center screening method was reviewed. RESULTS: β‐Hemolytic GCS, cultured from 1 of 8 RBC units, linked the fatal case to a single donor. The donor's throat swab collected 20 days after donation was positive for the presence of GCS, identified as Streptococcus dysgalactiae subsp. equisimilis. Isolates from the recipient, RBC unit, residual PLTs, and donor's throat swab were indistinguishable by PFGE. The donor denied any symptoms of infection before or after donation. PLT bacterial screening at the blood center was performed using a commercially available bacterial detection system (BacT/ALERT, bioMérieux) with a threshold of 15 colony‐forming units per bag. CONCLUSION: An asymptomatic donor was implicated as the source of GCS‐contaminated PLTs. Current screening methods for PLTs are not sufficient to detect all bacterial contamination. Pooled PLTs are a particular challenge because the small volume of individual units places limits on culturing strategies. Improved detection of bacterial contamination of PLTs is needed.     

Validation of a microbial detection system for use with ACD-A platelets with PAS III platelet additive solution

BACKGROUND: The BacT/ALERT microbial detection system (BTA) is used for testing leukoreduced apheresis platelets (LR‐AP) in plasma. Platelet additive solutions (PASs) such as InterSol (PAS III) may be used to reduce the amount of plasma transfused in LR‐AP. This study evaluated the performance of the two‐bottle BTA testing scheme in the recovery of seeded microorganisms from LR‐AP in InterSol‐plasma compared to a reference plate culture method. STUDY DESIGN AND METHODS: Hyperconcentrated, double LR‐AP were collected from healthy donors; InterSol was added (65% Intersol:35% plasma), equally divided into two containers, and then inoculated with an isolate of 1 of 10 clinically relevant index organisms at two levels. Aerobic (BPA) and anaerobic (BPN) BTA bottles were inoculated with 4 mL each of the inoculated LR‐AP, and blood agar plates (BAPs) for aerobic and anaerobic culture (0.5 mL each). RESULTS: Zero false‐positives from 103 bottle pairs were observed. All 400 two‐bottle BTA tests were positive within 24 hours, except for Propionibacterium acnes (maximum time‐to‐detection of 86.4 hr) and 13 of 20 pairs of Streptococcus viridans (maximum time‐to‐detection of 31.7 hr). Thirteen of 400 BAP two‐plate tests were negative for starting bacterial concentrations of 10 colony‐forming units (CFUs)/mL or less. At 40 CFUs/mL or less, BTA was 100% positive while BAP was 94% positive. CONCLUSION: Seeded organism recovery was superior in the two‐bottle BTA test system compared to the two‐plate BAP system using InterSol platelets (PLTs). This performance is comparable to previously published results for PLTs in plasma. The use of InterSol does not appear to have a detrimental effect on the performance of the two‐bottle BTA system.     

Evaluation of pooled cultures for bacterial detection in whole blood-derived platelets

BACKGROUND: The BacT/ALERT (bioMérieux) system is highly efficient for bacterial detection in apheresis platelets (PLTs) and whole blood-derived PLTs produced by the buffy-coat method. Detection of bacterial contamination in whole blood-derived PLTs produced by the PLT-rich plasma (PRP) method, however, is problematic. Prestorage pooling of these PLTs is not permitted in some countries including Canada and the United States, and culturing individual units is costly and may significantly reduce the PLT unit content. In this study, the sensitivity and specificity of BacT/ALERT cultures performed on pools derived from PRP PLTs are reported. STUDY DESIGN AND METHODS: The sensitivity of the BacT/ALERT system was evaluated for bacterial detection in PRP PLTs with a dilution effect. Thirty PLT pools were produced with 1 PLT unit previously spiked with bacteria and then pooled with other four nonspiked PLT units. Three bacteria, usually associated with PLT contamination, were selected for spiking. The specificity of this method was evaluated in 40 nonspiked PLT pools. RESULTS: The method was found to be 100 percent specific and 97 percent sensitive. Of the five spiked pools with Streptococcus pneumoniae at levels of less than 2 colony-forming units (CFUs) per mL, four were found to be positive whereas all 25 spiked pools with greater than 9 CFUs per mL of any of the chosen bacteria gave positive results. The mean time of detection was 17 to 19 hours for Staphylococcus epidermidis and 14 to 15 hours for S. pneumoniae and Pseudomonas aeruginosa when spiked with similar bacterial inocula. CONCLUSION: The evaluated system is highly sensitive and specific and may be a feasible method for bacterial detection in PRP PLTs.     

Improving the performance of culture-based bacterial screening by increasing the sample volume from 4 mL to 8 mL in aerobic culture bottles

BACKGROUND: In the setting of bacterial detection of apheresis platelets (PLTs), the manufacturer recommended PLT inoculation volume for BacT/ALERT culture bottles (bioMérieux) ranges from 4 to 10 mL. This study compares the rate of capture of true‐positive (TP) contaminations between aerobic culture bottles inoculated with either 4 or 8 mL of sample and assesses if a larger sample volume reduces time to detection. STUDY DESIGN AND METHODS: Detection of TP samples and mean time to detection were compared for 4‐ and 8‐mL samples collected between September 1, 2003, and May 2, 2011. RESULTS: A total of 180,263 and 283,114 PLT collections were tested with an 8‐ and 4‐mL sample, respectively. Analysis of TP rates by volume sampled show an increase in the rate of detection of TP with the 8‐mL sample relative to the 4‐mL sample (139 vs. 106 per million events; odds ratio, 1.31; 95% confidence interval, 0.77‐2.23). Comparison of mean time to detection for TP shows a decrease in mean time to detection using 8 mL compared with 4 mL (12.36 ± 3.7 hr to 15.97 ± 6.3 hr, p = 0.012). CONCLUSION: Doubling the sample volume to 8 mL showed a trend in improvement for the rate of detection of TP and shortened the mean time of detection for TP by 23% when compared to a sample volume of 4 mL. The decrease in mean time to detection using a larger sample volume suggests that a shorter release time after inoculation could be achieved without significantly increasing patient risk.     

Two-year experience with aerobic culturing of apheresis and whole blood-derived platelets

BACKGROUND: Throughout its system of regional centers, Blood Systems implemented culture based bacterial testing with a standardized protocol for both apheresis and whole blood-derived platelets (PLTs). STUDY DESIGN AND METHODS: After a 24-hour hold, 4 mL of PLT product was inoculated into an aerobic bottle (BacT/ALERT, bioMérieux). Cultures were incubated for 24 hours before routine product release to prevent distribution of infected products while minimizing consignee notification, product retrievals, and hospital PLT inventory problems. Initial-positives were further tested (and bacteria identified) by performing cultures from the original component and subcultures from the BacT/ALERT bottle. Results were categorized according to AABB recommended definitions with minor modifications. RESULTS: The rate of true-positive detections from culturing 122,971 apheresis PLTs was 0.017 percent (95% confidence interval [CI], 0.011%-0.026%). All true-positive microorganisms were Gram-positive with a predominance of coagulase-negative Staphylococcus and Bacillus species. Twenty of the 21 true-positive samples (95%) were detected by 24 hours but only 14 (68%) were detected by 18 hours. The false-positive rate due to contamination was 0.1 percent with the majority of isolates being skin or environmental organisms. Results did not differ significantly for whole blood-derived versus apheresis PLTs. CONCLUSION: These data corroborate the fact that the rate of detection of truly contaminated PLT apheresis products in the United States is approximately 1 in 5000 (0.02%); this is lower than the 0.03 to 0.05 percent rates that were generally quoted in the literature before the implementation of prospective bacterial culturing programs.     

Bacterial screening of outdated buffy coat platelet pools using a culture system and a rapid immunoassay

BACKGROUND: Canadian Blood Services performs bacterial screening of buffy coat platelet pools (BCPs) using aerobic BacT/ALERT cultures. This study aimed to determine the rate of detection failures during initial platelet (PLT) screening and evaluate the introduction of anaerobic cultures and immunoassay testing to assess the safety of extending PLT storage beyond 5 days. STUDY DESIGN AND METHODS: Outdated (7‐ to 10‐day‐old) BCPs that tested negative during initial screening were assayed with BacT/ALERT and the Verax PLT Pan Genera detection (PGD) test, an immunoassay that detects Gram‐positive (GP) and Gram‐negative (GN) bacteria. BacT/ALERT aerobic and anaerobic culture bottles were inoculated with 8 to 10 mL of BCP and incubated for up to 6 days. The PGD test was performed following manufacturer's instructions. Positive results were confirmed using the BacT/ALERT and PGD tests, blood agar culture, and Gram staining. Invalid PGD results were investigated. RESULTS: A total of 4002 BCPs were tested with one (0.025%) true positive (Staphylococcus epidermidis) found by both the BacT/ALERT and the PGD assays. Fifty‐four (1.35%) false‐positive BacT/ALERT cultures were obtained mainly due to instrument errors involving anaerobic cultures. Eleven (0.27%) false‐positive PGD tests were observed in the GP window of the strip. Forty‐nine (1.2%) invalid PGD results were obtained mostly before implementation of a humidity chamber. CONCLUSION: Testing of outdated BCPs suggests that introducing anaerobic cultures would result in significant PLT wastage due to a high rate of false positives. Contaminated BCPs still escape detection during initial testing; therefore, extension of PLT storage may be possible if repeat screening is performed before transfusion.     

Canadian experience with detection of bacterial contamination in apheresis platelets

BACKGROUND: In Canada, both blood suppliers, Héma-Québec (HQ) and Canadian Blood Services (CBS), implemented bacterial testing in apheresis platelets (PLTs) with an automated microbial detection system (BacT/ALERT, bioMérieux). STUDY DESIGN AND METHODS: Validation of the BacT/ALERT Classic and 3D systems involved apheresis PLT spiking with different bacteria at concentrations of 10 and 10(2) colony-forming units per mL. As of February 2006, more than 95 percent of apheresis PLTs were screened for bacterial contamination at HQ and CBS. Between 3.5 and 10 mL of PLTs is inoculated into BacT/ALERT aerobic culture bottles followed by incubation for a maximum of 7 days. RESULTS: During the validation studies, all bacteria were detected at all concentrations and volumes tested. Upon implementation of bacterial screening, the percentage of initial positive samples at CBS and HQ was 0.09 and 0.07 percent, respectively. The rate of indeterminate cultures was significantly higher at CBS than at HQ, whereas the rates for true-positive, false-positive, and false-negative results did not differ significantly. Six confirmed-positive cultures, including three coagulase-negative staphylococci and three Enterobacteriaceae species, were detected and PLT units contaminated with these bacteria were not transfused. The rate of true-positive cultures was significantly lower than that reported by other blood operators. Unfortunately, failed detection of two contaminated units resulted in septic transfusion reactions. CONCLUSION: Bacterial screening of apheresis PLTs in Canada was successfully implemented, and transfusion of contaminated units was prevented. Rapid bacterial detection systems that could be used before transfusion, however, may further reduce the risk of transfusion reactions.     

Validation of BacT/ALERT plastic culture bottles for use in testing of whole-blood-derived leukoreduced platelet-rich-plasma-derived platelets

BACKGROUND: Bacterial detection of platelet (PLT)-rich-plasma (PRP)-derived PLTs presents unique challenges for countries that do not allow pooling before storage. This study validated the BacT/ALERT for use in testing pooled PRP-derived PLTs with nine contaminating organisms. STUDY DESIGN AND METHODS: Isolates of Bacillus cereus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus viridans, and Propionibacterium acnes were inoculated into two PRP-derived PLT pools (target, 10 and 100 colony-forming units [CFUs]/mL; actual recovered concentrations, 5 and 90 CFUs/mL). Four milliliters of each postbacterial inoculation sample was inoculated into both plastic aerobic and anaerobic bottles and 0.5 mL was plated onto blood agar. RESULTS: All organisms (excluding P. acnes) were detected in 8.2 to 22.0 and 7.6 to 20.3 hours (10 and 100 CFUs/mL, respectively) and the mean time to detection was 15.0 and 13.1 hours (10 and 100 CFUs/mL, respective). P. acnes was detected with the anaerobic bottles in a mean of 74.9 and 64.3 hours (10 and 100 CFUs/mL, respectively). With E. cloacae, E. coli, K. pneumoniae, S. marcescens, and S. viridans detection with the anaerobic bottles was faster or equivalent to the detection with the aerobic bottles. This was most notable with S. viridans where the anaerobic bottle was reactive on average 21.6 and 10.8 hours (10 and 100 CFUs/mL, respectively) faster than the aerobic bottle. CONCLUSIONS: This study validates the use of the BacT/ALERT system for the detection of bacteria in PRP-derived PLTs in a pooled format. Overall, the use of the BacT/ALERT system allowed the detection of pooled PRP-derived PLTs inoculated with nine bacteria at 10 and 100 CFUs per mL in 7.6 to 22.0 hours (excluding P. acnes).     

Temperature cycling improves in vivo recovery of cold‐stored human platelets in a mouse model of transfusion

BACKGROUND: Platelet (PLT) storage at room temperature (RT) is limited to 5 days to prevent growth of bacteria, if present, to high levels. Storage in cold temperatures would reduce bacterial proliferation, but cold‐exposed PLTs are rapidly cleared from circulation by the hepatic Ashwell‐Morell (AM) receptor, which recognizes PLT surface carbohydrates terminated by β‐galactose. We cycled storage temperature between 4 and 37°C to preserve PLT function and reduce bacterial growth. STUDY DESIGN AND METHODS: Temperature‐cycled (TC) human PLTs were stored at 4°C for 12 hours and then incubated at 37°C for 30 minutes before returning back to cold storage. PLTs stored at RT or at 4°C (COLD) or TC for 2, 5, and 7 days were infused into SCID mice and the in vivo recovery was determined at 5, 20, and 60 minutes after transfusion. RESULTS: PLTs stored for 2 days in COLD had significantly lower in vivo recoveries than RT PLTs. TC PLTs had improved recoveries over COLD and comparable to RT PLTs. After 5‐ and 7‐day storage, TC PLTs had better recoveries than RT and COLD PLTs. PLT surface β‐galactose was increased significantly for both COLD and TC PLTs compared to RT. Blocking of the AM receptor by asialofetuin increased COLD but not TC PLT recovery. CONCLUSION: TC cold storage may be an effective method to store PLTs without loss of in vivo recovery. The increased β‐galactose exposure in TC PLTs suggests that mechanisms in addition to AM receptors may mediate clearance of cold‐stored PLTs.     

Cryopreserved buffy-coat-derived platelets reconstituted in platelet additive solution: A safe and available product with sufficient haemostatic effectiveness

BackgroundPlatelets (PLTs) stored at 20–24 °C have a short shelf life of only 5 days, which can result in their restricted availability. PLT cryopreservation extends the shelf life to 2 years.MethodsWe implemented a method of PLT freezing at ?80 °C in 5–6% dimethyl sulfoxide. Buffy-coat-derived leucodepleted fresh PLTs blood group O (FP) were used for cryopreservation. Cryopreserved pooled leucodepleted PLTs (CPP) were thawed at 37 °C, reconstituted in PLT additive solution SSP + and compared to FP regarding PLT content, PLT concentration, pH, volume, PLT loss, anti-A/B antibody titre, total protein, plasma content, and PLT swirling. Clot properties were evaluated via rotational thromboelastometry. PLT microparticle number and surface receptor phenotype were assessed via flow cytometry.ResultsCPP met the required quality parameters. The mean freeze-thaw PLT loss was 22.24 %. Anti-A/B antibody titre and plasma content were significantly lower in CPP. CPP were characterised by faster clot initiation and form stable PLT clots. The number of PLT microparticles increased 25 times in CPP and there were more particles positive for the activation marker CD62 P compared to FP.ConclusionThawing and reconstitution are easy and fast processes if platelet additive solution is used. Low anti-A/B antibody titre and plasma content make possible the use of CPP of blood group O reconstituted in SSP + as universal ABO products, including clinical situations where washed PLTs are required. Clot properties evaluated via rotational thromboelastometry demonstrated that CPP retain a significant part of their activity compare to FP and are haemostatically effective.     

COMMENTARY: Relative safety of pooled whole blood–derived versus single‐donor (apheresis) platelets in the United States: a systematic review of disparate risks

BACKGROUND: Risks of transfusion‐transmitted infections (TTIs), transfusion‐associated sepsis (TAS), and transfusion‐related acute lung injury (TRALI) were compared between pooled whole blood–derived (PWBD) and single‐donor platelets (PLTs) transfused in the United States. STUDY DESIGN AND METHODS: The literature was searched for estimates of the risk of TTIs and TAS and of the effect on bacterial contamination of PLTs of process improvements, bacterial culture, and surrogate methods to detect bacteria. Seven studies published between January 2005 and December 2008 and comparing bacterial contamination frequency between PWBD and single‐donor PLTs after implementing bacterial culture testing of both components were subjected to meta‐analysis. The three retrieved studies diagnosing TRALI based on the 2004 consensus definition in settings transfusing both PWBD and single‐donor PLTs were not amenable to meta‐analysis and were assessed qualitatively. RESULTS: Under a best‐case scenario, if 100% (from the current 12.5%) of PLT doses were provided as PWBD PLTs, the number of additional transmissions of human immunodeficiency virus, hepatitis C virus, hepatitis B virus, bacteria, or a novel pathogen annually could be 1.2, 1.3, 9.0, 105.3, or 69.2 to 252.6, respectively. Compared with single‐donor PLTs, US PLT pools of five concentrates have a 5.6‐fold higher risk of bacterial contamination (summary odds ratio, 5.58; 95% confidence interval, 2.60‐11.98; p < 0.05). The three studies that diagnosed TRALI based on the consensus definition did not demonstrate a difference in risk between PWBD and single‐donor PLTs. CONCLUSIONS: TTIs and TAS determine the relative safety of PWBD versus single‐donor PLTs. The available limited data do not support a higher risk of TRALI from single‐donor (compared with PWBD) PLTs.     

Donor safety in triple plateletpheresis: results from the German and Austrian Plateletpheresis Study Group multicenter trial

BACKGROUND: The objective was to investigate potential risks for apheresis donors associated with a triple‐plateletpheresis (TP) program. STUDY DESIGN AND METHODS: Eleven hemapheresis centers randomly assigned 411 repeat donors (ratio, 1:1.2) to either double plateletpheresis (DP; 185 donors) or TP (226 donors) with a platelet (PLT) target content of at least 5.0 × 10¹¹ PLTs/DP and at least 7.5 × 10¹¹ PLTs/TP. The primary endpoint was procedure‐related postapheresis PLT count of at least 150 × 10⁹/L (probability, ≥98%). Secondary endpoints were apheresis characteristics and donor adverse reactions. RESULTS: In 6 of 1133 DPs (0.5%) in 4 of 185 donors (2.2%) and in 20 of 1020 TPs (2.0%) in 14 of 226 donors (6.2%), postapheresis PLT counts were below 150 × 10⁹/L. There were marginal but significant differences in collection efficiency (DP, 69.2 ± 9.1%; TP, 70.9 ± 9.0%; p ≤ 0.0001) and collection rate (DP, 10.4 × 10⁹ ± 2.3 × 10⁹ PLTs/min; TP, 10.8 × 10⁹ ± 2.3 × 10⁹ PLTs/min; p ≤ 0.005). The PLT yields were 5.9 × 10¹¹ ± 0.8 × 10¹¹ PLTs for DP and 8.3 × 10¹¹ ± 0.9 × 10¹¹ PLT for TP (p ≤ 0.0001) at processing times of 59 ± 13 minutes (DP) versus 80 ± 16 minutes (TP; p ≤ 0.0001). Significant PLT recruitment (1.10 ± 0.14 vs. 1.20 ± 0.23; p < 0.0001) was seen for both DP and TP. DP and TP did not differ with regard to venous access problems (VAPs) without discontinuation (3.8% for both), but DP induced fewer VAPs with discontinuation (1.1% vs. 3.0%; p < 0.01). Mild citrate toxicity (1.7% vs. 3.9%; p < 0.01) and circulatory reactions (0.4% vs. 2.2%; p < 0.01) were more often noticed in TP, but caused no increase in discontinuations. CONCLUSIONS: TP results in an increase in mild donor reactions but does not significantly impair donor safety or product quality.     

Summary of the AABB Interorganizational Task Force on Bacterial Contamination of Platelets: Fall 2004 impact survey

BACKGROUND: New voluntary standards in the United States regarding bacterial contamination of platelets (PLTs) led to the formation of the AABB Interorganizational Task Force on Bacterial Contamination of Platelets. This article summarizes a survey conducted by the Task Force to assess the impact of bacterial detection. STUDY DESIGN AND METHODS: An Internet-based survey of AABB member institutions was conducted from September 17, 2004, to October 1, 2004. The survey was designed principally to assess PLT usage, supply, and outdating and the currently used bacteria detection methods. RESULTS: Of 900 facilities surveyed, 350 responded (38%). These facilities collected approximately 43.3 and 65.9 percent and transfused approximately 19.1 and 22.2 percent of the whole blood-derived PLT concentrates (WBPCs) and apheresis PLTs in the United States, respectively. Most facilities (64-91%) indicated that their ability to provide PLTs for transfusion had not been affected. Approximately half (50-57.1%) indicated no changes in their PLT inventory. Two-thirds (66-68%) indicated no increased PLT outdating. More than 90 percent of apheresis PLTs are tested with a culture-based method, whereas WBPCs are tested with a variety of methods (mostly non-culture-based) resulting in a 4.6-fold decrease in the confirmed positive detection rate compared with apheresis PLTs (p < 0.001). CONCLUSION: After the implementation of AABB Standard 5.1.5.1, the majority of facilities responding to this survey experienced no (or modest) impact on PLT availability or outdating. Nevertheless, a substantial portion of facilities experienced both increased outdating and decreased availability. Some facilities were greatly impacted. Based on the data gathered, it is impossible to conclude whether such shortages resulted from production or distribution problems or were due to decreased shelf life and increased outdates.     

Routine use of a rapid test to detect bacteria at the time of issue for nonleukoreduced,whole blood–derived platelets

BACKGROUND: The Pan Genera detection (PGD) test is used to screen platelet (PLT) products for bacterial contamination. We report the experience of using the PGD test on whole blood–derived PLTs (WBPs) at two large centralized transfusion services (CTS). STUDY DESIGN AND METHODS: Records of PGD test results were retrospectively reviewed. The PGD test was performed on individual WBP units or pools of WBPs ranging in size from 2 to 6 units at the time of issue. Bacterial culture was performed on PLT products with positive PGD tests, and at one CTS, the available cocomponents. RESULTS: A total of 70,561 WBP pools were screened with the PGD test. There were seven true‐positive PGD tests and 242 false‐positive tests (positive predictive value of PGD test, 2.81%). The overall contamination rate was 99 per 10⁶ WBP pools (1:10,080; 95% confidence interval [CI], 40‐204), and the false‐positive rate was 3430 per 10⁶ WBP pools (1:292; 95% CI, 3011‐3890). All seven bacterial isolates were Gram positive. The median age of the individual WBP units in the seven contaminated pools was 5 days (range, 3‐5 days) compared to 4 days (range, 1‐5 days) in the false‐positive pools (p = 0.0012). The same bacteria isolated from a positive PLT pool also grew in one red blood cell cocomponent. CONCLUSION: After testing more than 70,000 WBP pools at two large CTSs, the rate of contaminated WBP pools detected by the PGD test was 99 per 10⁶ pools (1:10,080).     

Extended storage of platelet‐rich plasma–prepared platelet concentrates in plasma or Plasmalyte

BACKGROUND: Using bacterial detection or pathogen reduction, extended platelet (PLT) storage may be licensed if PLT viability is maintained. The Food and Drug Administration (FDA)'s poststorage PLT acceptance guidelines are that autologous stored PLT recoveries and survivals should be 66 and 58% or greater, respectively, of each donor's fresh PLT data. STUDY DESIGN AND METHODS: Nonleukoreduced PLT concentrates were prepared from whole blood donations. Autologous PLT concentrates from 62 subjects were stored in 100% plasma (n = 44) or 20% plasma/80% Plasmalyte (n = 18), an acetate‐based, non–glucose‐containing crystalloid solution previously used for PLT storage. Fresh PLTs were obtained on the day the donor's stored PLTs were to be transfused. The fresh and stored PLTs were alternately radiolabeled with either ⁵¹chromium or ¹¹¹indium, and in vitro measurements were performed on the stored PLTs. RESULTS: The FDA's PLT recovery criteria were met for 7 days of plasma storage, but PLT survivals maintained viability for only 6 days. Plasmalyte‐stored PLTs did not meet either acceptance criteria after 6 days of storage. After 7 days of storage, PLT recoveries averaged 43 ± 4 and 30 ± 4% and survivals 4.1 ± 0.4 and 2.0 ± 0.2 days for plasma‐ and Plasmalyte‐stored PLTs, respectively (p = 0.03 for recoveries and p < 0.001 for survivals). Poststorage PLT recoveries correlated with the commonly used in vitro PLT quality measurements of hypotonic shock response and annexin V binding, while survivals correlated with extent of shape change, morphology score, and pH. CONCLUSION: There is a progressive decrease in recoveries and survivals of plasma‐stored PLTs over time. PLT viability is better maintained in plasma than Plasmalyte.     

Acquired cytochrome C oxidase impairment in apheresis platelets during storage: a possible mechanism for depletion of metabolic adenosine triphosphate

BACKGROUND: Intracellular adenosine triphosphate (ATP) levels decline significantly during storage of platelet (PLT) products, in part due to PLT degranulation. However, metabolic ATP stores also become depleted during storage through an unclear mechanism. Since both anaerobic glycolysis and oxidative phosphorylation are important for PLT ATP production, it is possible that the reduction in metabolic ATP reflects impaired oxidative phosphorylation. To assess this, we evaluated the kinetic activity and protein expression of cytochrome C oxidase (CcOX) in stored apheresis PLTs. STUDY DESIGN AND METHODS: Apheresis PLTs were collected and stored with agitation at 22 ± 2°C for 7 days. In vitro measurements of PLT metabolic state, function, and activation were performed on Days 0, 2, 4, and 7 of storage. Total PLT ATP content, steady‐state CcOX kinetic activity, and protein immunoblotting for CcOX Subunits I and IV were also performed using isolated PLT mitochondria from simultaneously collected samples. RESULTS: Intra‐PLT ATP and steady‐state PLT CcOX activity declined significantly and in a progressive manner throughout storage while steady‐state levels of CcOX I and IV protein remained unchanged. Time‐dependent decline in CcOX activity correlated with progressive ATP depletion over time. CONCLUSION: During storage of apheresis PLTs for 7 days, the parallel decline in CcOX function and intra‐PLT ATP suggests development of an acquired impairment in PLT oxidative phosphorylation associated with perturbed ATP homeostasis in stored PLTs.     

Calcium is a key constituent for maintaining the in vitro properties of platelets suspended in the bicarbonate‐containing additive solution M‐sol with low plasma levels

BACKGROUND: Commercially available additive solutions (ASs) require 30% to 35% plasma for optimal storage of platelets (PLTs). PLTs suspended in M‐sol, a bicarbonate‐based experimental platelet additive solution (PAS), maintain in vitro PLT properties during storage with low levels of plasma (≤5%). STUDY DESIGN AND METHODS: Four different formulations of M‐sol were prepared at the optimal pH (6.1): M‐sol, M‐sol without calcium, M‐sol without citric acid, and M‐sol without calcium and citric acid. Apheresis PLT units (100% plasma) were equally divided into five 50‐mL aliquots in PL732 containers, centrifuged, and resuspended to prepare units suspended in the four different PASs (95%) with 5% plasma and 1 unit in 100% plasma. Units (n = 10) were stored under standard conditions and assayed for in vitro properties on Days 1, 5, and 7. The data were analyzed by analysis of variance for repeated measures (n = 10, p < 0.001). RESULTS: On Day 5 of storage, PLTs suspended in the M‐sol formulation containing calcium but lacking citric acid had similar pH, extent of shape change (ESC) values, and percentage of CD62‐positive PLTs and greater hypotonic shock response (HSR) and percentage of discoid PLTs compared to those of PLTs suspended in 100% plasma. In contrast, PLTs suspended in the M‐sol formulation lacking calcium had lesser ESC values, greater percentage of CD62‐positive PLTs, and similar HSR values and percentage of discoid PLTs compared to those of PLTs suspended in 100% plasma on Day 5 (p < 0.001). CONCLUSIONS: Calcium plays an important role in maintaining CD62‐negative PLTs and relatively high ESC in 5% plasma. The removal of citric acid from M‐sol may improve PLT storage properties with low plasma levels.     

Prevention of transfusion of platelet components contaminated with low levels of bacteria: a comparison of bacteria culture and pathogen inactivation methods

BACKGROUND: This study compared the efficacy of bacterial detection with inactivation for reducing the risk associated with transfusion of platelet (PLT) components contaminated with low levels of bacteria. STUDY DESIGN AND METHODS: Twenty-one double-dose PLTs were spiked with seven species of bacteria at three levels (0.003-0.03, 0.03-0.3, 0.3-3 colony-forming units [CFUs]/mL). After split, each PLT unit contained 1 to 10, 10 to 100, and 100 to 1000 CFUs. One unit was photochemically treated (PCT; 150 micromol/L amotosalen and 3 J/cm(2) ultraviolet A). The other unit was untreated. All units were stored and sampled on Days 1, 2, and 5 of storage for aerobic and anaerobic culture in the BacT/ALERT system (bioMérieux). PLTs were classified as sterile when no bacterial growth was detected after 120 hours of culture. RESULTS: In all PCT PLTs, no bacteria were detected throughout 5 days of storage regardless of species, level of contamination, and sampling time. In untreated PLTs, Staphylococcus aureus was consistently detected by culturing. Growth of 1 to 10 CFUs per unit Staphylococcus epidermidis, 1 to 100 CFUs per unit of Klebsiella pneumoniae, and 1 to 1000 CFUs per unit Propionibacterium acnes was delayed and only detectable after 5, 2, and 5 days of storage, respectively. Low levels of Streptococcus agalactiae (1-10 CFUs/unit), Escherichia coli (1-100 CFUs/unit), and Clostridium perfringens (1-100 CFUs/unit) were not detected during 5 days of storage, although bacterial outgrowth was detected at higher levels of contamination. CONCLUSIONS: For the seven bacterial species examined, contaminated PLTs may be released for transfusion on test-negative-to-date status. In contrast, bacterial inactivation by PCT could reduce the risk associated with transfusion of PLTs contaminated with low levels of these bacteria.     

Longitudinal management with crossmatch‐compatible platelets for refractory patients: alloimmunization,response to transfusion,and clinical outcomes (CME)

BACKGROUND: The use of crossmatch‐compatible platelets (PLTs) improves posttransfusion corrected count increments (CCIs) in patients with alloimmune PLT refractoriness. However, few reports address the efficacy of utilizing this strategy for patients requiring intensive PLT transfusion therapy lasting several weeks to months. STUDY DESIGN AND METHODS: Medical records of patients with two or more PLT crossmatch assays performed between 2002 and 2010 were reviewed. All patients were refractory to random single‐donor apheresis PLT units, defined as two consecutive 1‐hour posttransfusion CCIs of less than 7500. A commercial solid‐phase adherence assay was used for crossmatching. RESULTS: Seventy‐one patients were included. A median of four crossmatch assays were performed per patient (range, 2‐17). Mean percent reactivity in initial (58.6%) versus last (55.3%) crossmatch assay for each patient demonstrated no trend toward progressive alloimmunization (p = NS). A total of 738 crossmatched PLT units were administered with a mean ± standard deviation CCI of 7000 ± 7900 (n = 443 units with adequate 1‐hr posttransfusion counts), a significant improvement over random PLTs (p < 0.001). Patients with an initial crossmatch reactivity of greater than 66% were significantly more likely to demonstrate at least one panreactive crossmatch assay, impacting the availability of compatible PLTs for optimum transfusion support. One patient (1.4%) developed WHO Grade IV bleeding. CONCLUSIONS: Progressive alloimmunization to mismatched antigens does not impact medium‐term transfusion support with crossmatched PLTs. Increased reactivity in the initial crossmatch assay can serve as a trigger to initiate workup for HLA‐matched PLTs as a second‐line approach. However, for most patients, medium‐term transfusion support with crossmatched PLTs offers an effective and rapid first‐line approach to management of PLT transfusion refractoriness.     

Pasteurella multocida bacteremia in asymptomatic plateletpheresis donors: a tale of two cats

BACKGROUND: Bacterial contamination of platelet (PLT) concentrates occurs in 1 in 1000 to 1 in 3000 components and has been a leading cause of transfusion-associated morbidity and mortality. Two cases of Pasteurella multocida bacteremia in asymptomatic plateletpheresis donors are reported. Clinical outcomes were profoundly different, emphasizing the importance of robust methods to detect bacterial contamination. CASE REPORTS: The first case occurred before the implementation of bacterial testing of PLTs. A plateletpheresis component was collected from a 70-year-old man and transfused to an 88-year-old man, who developed rigors, tachycardia, and hypotension within 15 minutes of the start of the transfusion. Cardiopulmonary arrest ensued and he expired 6 hours after transfusion. Blood cultures collected after transfusion and cultures of the PLT component were positive for the presence of P. multocida. Investigation revealed that a feral cat had bitten the donor 100 minutes before his donation. He had not reported the event to the donor room staff. The second case involved a 74-year-old woman who developed a flulike syndrome 2 days after plateletpheresis donation. P. multocida was isolated in routine bacterial culture of her PLT component. The donor had several feral cats, and although there was no history of bite or scratch, one cat liked to lick her hands, which were chapped from gardening. CONCLUSION: Occult bacteremia with P. multocida transmitted by feral cats was the source of PLT contamination in two cases over 3 years. Bacterial testing of PLTs is critical in the prevention of transfusion-acquired sepsis and allows the identification and treatment of asymptomatic bacteremic donors.