Evaluation of in vitro storage properties of prestorage pooled whole blood–derived platelets suspended in 100 percent plasma and treated with amotosalen and long-wavelength ultraviolet light

BACKGROUND: Amotosalen, a psoralen, has been utilized for photochemical treatment (PCT) of apheresis platelets (PLTs) and pooled buffy coat PLTs suspended in additive solution. In the United States, the source of many PLT transfusions is from whole blood–derived PLTs prepared by the PLT‐rich plasma (PRP) method. This study investigated the in vitro PLT properties of amotosalen‐PCT of leukoreduced pools of PLTs prepared by the PRP method and suspended in 100 percent plasma. STUDY DESIGN AND METHODS: On Day 1 of storage, 12 leukoreduced (n = 6) or 10 leukoreplete (n = 6) ABO‐identical PLT concentrates were pooled, separated into two pools of 6 or 5 units, respectively, and leukoreduced (leukoreplete pools only). Each pool of 5 or 6 units was then photochemically treated (designated “test”: amotosalen plus 3.0 J/cm² long‐wavelength ultraviolet light followed by amotosalen/photoproduct removal) while the remaining identical pool (designated “control”) was untreated. PLT in vitro assays were performed on test and control pools during 7‐day storage. RESULTS: PCT resulted in slightly reduced pH in test pools compared to that of matched control pools after 5 days of storage (5‐unit pools: test, 6.96 ± 0.12 vs. control, 7.15 ± 0.09, p = 0.0033; 6‐unit pools: test, 6.90 ± 0.10 vs. control, 7.07 ± 0.09, p < 0.0001). Test pools adequately maintained many other in vitro properties including PLT morphology, hypotonic shock response, and extent of shape change parameters during 5‐day storage, which, like pH, also differed from those of controls. The pH of test and control pools declined on Day 7, with 1 of 6 test pools (either 5 or 6 units) having a pH value of less than 6.20, while all control pools had pH values of more than 6.66. CONCLUSION: PCT of leukoreduced PLT pools of whole blood–derived PLTs in 100 percent plasma maintained adequate PLT in vitro variables through 5 days of storage.     

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.     

Evaluation of BacT/ALERT plastic culture bottles for use in testing pooled whole blood-derived leukoreduced platelet-rich plasma platelets with a single contaminated unit

BACKGROUND: In certain countries, whole blood-derived platelet (PLT)-rich plasma PLTs can only be pooled within 4 hours of transfusion. One prerequisite for prestorage pooling is the ability to detect low levels of bacteria from a single unit (approx. 10 colony-forming units [CFUs]/mL) once pooled (10/6 approx. 2 CFUs/mL). This study evaluated the BacT/ALERT (bioMérieux) for detection of bacteria in 1 unit of a 6-unit pool. STUDY DESIGN AND METHODS: Bacillus cereus, Clostridium perfringens, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus viridans, and Propionibacterium acnes were inoculated into single PLT units (target, 10 and 100 CFUs/mL; mean recovered, 5 and 92 CFUs/mL) and then pooled with 5 sterile units. Four milliliters was inoculated into both plastic aerobic and anaerobic bottles, and 0.5 mL was plated (10 sets). RESULTS: All cases were detected when the single unit had at least 6 CFUs per mL. With B. cereus (< or =2 CFUs/mL), all bottles were reactive. With K. pneumoniae and S. viridans (< or =3 CFUs/mL), all samples were detected with a two-bottle set, but not all bottles were reactive. With S. marcescens (< 2 CFUs/mL), only 7 of the 10 sets were reactive. With C. perfringens (0.2 CFUs/mL), only 3 of 10 samples were detected in the anaerobic bottles. CONCLUSIONS: This study evaluates the use of the BacT/ALERT system for detection of bacteria in PLT pools. Overall, the BacT/ALERT detected all contaminated pooled PLTs when the single inoculated unit had a calculated or recovered concentration at least 3 CFUs per mL with 10 different species of bacteria. Low recovered concentrations (< or =2 CFUs/mL) were, in some cases, only detected with a two-bottle set.     

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).     

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.     

Photochemical treatment of platelet concentrates with amotosalen and long-wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria

BACKGROUND: Bacterial contamination of platelet (PLT) concentrates can result in transfusion-transmitted sepsis. A photochemical treatment (PCT) process with amotosalen HCl and long-wavelength ultraviolet light (UVA), which cross-links nucleic acids, was developed to inactivate bacteria and other pathogens in PLT concentrates. STUDY DESIGN AND METHODS: High titers of pathogenic aerobic and anaerobic Gram-positive bacteria (10 species), aerobic Gram-negative bacteria (7 species), and spirochetes (2 species) were added to single-donor PLT concentrates containing 3.0 x 10(11) to 6.0 x 10(11) PLTs in approximately 300 mL of 35 percent plasma and 65 percent PLT additive solution (InterSol, Baxter Healthcare) or saline. After PCT with 150 micro mol per L amotosalen and 3 J per cm(2) UVA, residual bacterial levels were detected by sensitive microbiologic methods. RESULTS: The level of inactivation of viable bacteria was expressed as log reduction. Log reduction of Gram-positive bacteria for Staphylococcus epidermidis was > 6.6; for Staphylococcus aureus, 6.6; for Streptococcus pyogenes, > 6.8; for Listeria monocytogenes, > 6.3; for Corynebacterium minutissimum, > 6.3; for Bacillus cereus (vegetative), > 5.5; for Lactobacillus sp., > 6.4; for Bifidobacterium adolescentis, > 6.0; for Propionibacterium acnes, > 6.2; and for Clostridium perfringens, > 6.5. Log reduction of Gram-negative bacteria for Escherichia coli was > 6.4; for Serratia marcescens, > 6.7; for Klebsiella pneumoniae, > 5.6; for Pseudomonas aeruginosa, 4.5; for Salmonella choleraesuis, > 6.2; for Yersinia enterocolitica, > 5.9; and for Enterobacter cloacae, 5.9. Log reduction of spirochetes for Treponema pallidum was 6.8 to 7.0, and for Borrelia burgdorferi, > 6.9. CONCLUSION: PCT inactivates high levels of a broad spectrum of pathogenic bacteria. The inactivation of bacteria in PLT concentrates offers the potential to prospectively prevent PLT-transfusion-associated bacteremia.     

Maintenance of platelet in vitro properties during 7-day storage in M-sol with a 30-hour interruption of agitation

BACKGROUND: Extensive periods without agitation can occasionally occur during platelet (PLT) shipment and can affect PLT quality during 5‐ to 7‐day storage. The use of buffer‐containing PLT additive solutions (ASs) may better preserve PLT quality during storage by maintaining PLT pH and other in vitro variables. A newly described bicarbonate‐containing AS, M‐sol, was compared to plasma for preservation of whole blood–derived PLT concentrates in which a 30‐hour interruption of agitation was included. STUDY DESIGN AND METHODS: ABO‐identical PLT‐rich plasma intermediate products were pooled in sets of four, split, and centrifuged with subsequent plasma expression (n = 12). Two units were resuspended with M‐sol AS to produce a 70 percent solution/30 percent plasma PLT concentrate; 2 units were resuspended in 100 percent plasma. One M‐sol resuspended unit and 1 plasma unit were held on a laboratory bench in a standard shipping box for 30 hours between Day 2 and Day 3, while the other M‐sol and plasma unit were continuously agitated. Standard in vitro testing for PLT quality variables on each set of 4 units was performed during storage (n = 12). RESULTS: Interrupting agitation of PLTs suspended in M‐sol resulted in less of a pH decrement during storage than that of PLTs suspended in 100 percent plasma. On Days 5 and 7, the pH differences between M‐sol and plasma units were 0.56 and 0.75 pH units, respectively (p < 0.0003). In addition, PLTs suspended in M‐sol and subjected to an interruption of agitation had lesser Day 7 CD62+ cells, glucose utilization, and lactate production and greater hypotonic stress response, morphology, swirling, and aggregation response than those suspended in plasma (p ≤ 0.005). CONCLUSION: The in vitro properties of PLTs suspended in 70 percent M‐sol/30 percent plasma and subjected to a 30‐hour interruption of agitation are better maintained during 7‐day storage than those of matched units suspended in plasma.     

Photochemical treatment of platelet concentrates with amotosalen hydrochloride and ultraviolet A light inactivates free and latent cytomegalovirus in a murine transfusion model

BACKGROUND: A photochemical treatment (PCT) process utilizing amotosalen hydrochloride and long wavelength UVA light has been developed to inactivate pathogens in PLTs. This study investigated the effects of amotosalen/UVA treatment on free and latent murine CMV (MCMV) in PLT preparations using a murine model of transfusion-transmitted CMV (TT-CMV). STUDY DESIGN AND METHODS: In a model of latent MCMV infection, "donor" mice received 1 x 10(6) plaque-forming units (PFUs) MCMV and were rested 14 days. Subsequently harvested, pooled, and washed WBCs were PCR positive for MCMV. Murine WBC doses of 1 x 10(4), 1 x 10(5), and 1 x 10(6) were added to human apheresis PLTs in 35 percent autologous plasma and 65 percent PLT AS (PAS). The WBC-PLT products were treated with 150 micro mol/L amotosalen and 0.6 J per cm2 UVA and transfused via tail vein injection into recipient mice. Recipients were killed on Day 14. Blood and spleens were collected and assayed for MCMV by PCR. In a parallel model of active infection with free virus, human PLT in 35 percent autologous plasma and 65 percent PAS were dosed with 1 x 10(5) and 1 x 10(6) PFUs of MCMV. All other procedures were as described above. RESULTS: In the absence of amotosalen/UVA-pretreatment, transfusion of PLT latently or actively infected with MCMV produced TT-CMV in a dose-dependent fashion. In contrast, all transfusion recipients of identical PLT preparations pretreated with amotosalen/UVA were uniformly PCR negative for MCMV (abrogation of TT-CMV; p < 0.05). CONCLUSIONS: PCT of PLT preparations with the specified doses of amotosalen hydrochloride and UVA light prevents transfusion transmission of free and latent MCMV in a murine model. These results suggest that PCT of human PLTs with amotosalen/UVA should also effectively abrogate TT-CMV in the clinical setting.     

Amotosalen interactions with platelet and plasma components: absence of neoantigen formation after photochemical treatment

BACKGROUND: The INTERCEPT Blood System (Baxter Healthcare Corp., and Cerus Corp.) is a photochemical treatment (PCT) process that uses amotosalen (S-59) and ultraviolet A (UVA) illumination to inactivate a broad spectrum of pathogens. STUDY DESIGN AND METHODS: To evaluate the potential of the process to create neoantigens, the amounts of residual amotosalen and photoproducts present in PCT platelets (PLTs) and PCT plasma were quantified. The initial amount of amotosalen was 150 micromol per L. After illumination with 3 J per cm2 UVA and before transfusion, a compound adsorption device was used to substantially reduce the amounts of free amotosalen and unreactive photodegradation products. Patient serum samples from Phase III clinical trials were assayed by enzyme-linked immunosorbent assay (ELISA) for antibodies to potential amotosalen neoantigens. RESULTS: After PCT, 15 percent of the starting amount of amotosalen remains bound to PLTs, and 15 to 22 percent remains bound to plasma components. The majority of bound amotosalen is associated with lipid. Less than 1 percent of PLT-bound amotosalen and approximately 2 percent of plasma-bound amotosalen can be extracted into the water-soluble protein fraction. In seven Phase III clinical trials, 523 patients received more than 8000 units of PCT PLTs or PCT plasma. None of the patients exhibited clinical or laboratory manifestations of neoantigenicity. Furthermore, no other alteration of PLT membrane proteins was identified based on testing for lymphocytotoxic antibodies and PLT-specific alloantibodies. CONCLUSION: These results indicate that no neoantigens were detected by ELISA after PCT, suggesting that transfusion of PCT PLTs or PCT plasma does not induce adverse immunologic responses.     

The use of a bacteria detection system to evaluate bacterial contamination in PLT concentrates

BACKGROUND: Random-donor PLTs (RDPs) are functional at 7 days. Nevertheless, since the mid-1980s, concern for bacterial contamination has caused the storage period to be reduced to 5 days. The ability of a bacteria detection system (BDS, Pall) to determine bacterial contamination and permit extension of the PLT shelf life to 7 days was assessed. STUDY DESIGN AND METHODS: Blood was collected into CP2D and leukoreduced RDPs were prepared. Upon arrival at the hospital, a 2- to 3-mL aliquot was removed from each RDP and introduced into the Pall BDS pouch with a sterile docking device. The pouch was incubated at 37 degrees C for 24 hours and then the oxygen content was measured to determine bacterial contamination. Additionally, the RDPs were pooled and an aliquot was removed for culture with standard manual techniques. CCIs were calculated 1 hour after infusion. RESULTS: A total of 12,062 individual RDPs were tested. The Pall BDS detected bacteria in 5 units. All of these were positive on repeat sampling. Propionibacterium acnes, coagulase-negative Staphylococcus, and Bacillus species were confirmed by manual technique in 3 units, one could not be identified, and one was negative. Aliquots from PLT pools were positive in 80 of 2201 pools when tested by manual methods. Of these, 79 were false-positives and 1 unit contained coagulase-negative Staphylococcus. The Pall BDS was easy to use and required less than 5 minutes for all manipulations. After 7 days of storage, the PLTs gave an average CCI of 16 x 10(11)+/- 3.39 x 10(11) 1 hour after transfusion (n = 9). CONCLUSIONS: The Pall BDS permits evaluation of RDPs for bacterial contamination. Culture-negative PLTs were successfully transfused in our institution up to and including 7 days after storage with good CCIs.     

Functional characteristics of buffy-coat PLTs photochemically treated with amotosalen-HCl for pathogen inactivation

BACKGROUND: One blood system for PLTs (INTERCEPT, Baxter Transfusion Therapies) is based on photochemical treatment (PCT) with small molecules that target cross-link nucleic acids (Helinx technology, Cerus Corp.) with amotosalen-HCl (S-59) and UVA light (320-400 nm) to inactivate pathogens and WBCs. STUDY DESIGN AND METHODS: A two-arm in vitro study was conducted to compare pooled buffy-coat-derived PLT concentrates (PCs) treated with the INTERCEPT blood system, resuspended in PLT additive solution (PAS) III (InterSol, Baxter Transfusion Therapies), and stored for up to 7 days (test units, n = 20) with unpaired, nontreated PCs, resuspended in PAS II (T-Sol, Baxter Transfusion Therapies), and prepared at the same center in the same manner (control units, n = 18). RESULTS: PLT dose (x 1011/unit +/- SD) on Day 1 immediately following PCT was 3.0 +/- 0.4 for test units and 3.2 +/- 0.4 for control units. After 7 days of storage, the pH of all test units was maintained above 6.8. No marked trend was observed in the hypotonic shock response (HSR). Values among study groups were similar at the end of observation period: 68 +/- 11 percent for control unites versus 67 +/- 8 percent for test units (p > 0.05). Aggregation response to ristocetin was slightly lower in test units: at Day 7, 65 +/- 10 percent versus 76 +/- 6 percent (p < 0.05). Significantly higher (p < 0.001) glucose consumption, lactate production, and CD62P expression were observed in test units. CONCLUSION: Compared to nontreated PLTs, the PCT process was associated with a variety of differences of in vitro analyses. Although significant, these changes were relatively small in most cases. Characteristics correlated with survival in vivo such as HSR and swirling were comparable between both study groups, indicating that the viability of the majority of cells appears to have persisted throughout 7 days of storage. The impact of this finding, however, remains to be investigated in clinical trials performed with 7-day stored PLTs.     

Evaluation of antimicrobial peptides as novel bactericidal agents for room temperature–stored platelets

BACKGROUND: A single cost-effective pathogen inactivation approach would help to improve the safety of our nation's blood supply. Several methods and technologies are currently being studied to help reduce bacterial contamination of blood components. There is clearly need for simple and easy-to-use pathogen inactivation techniques specific to plasma, platelets (PLTs), and red blood cells.
STUDY DESIGN AND METHODS: In this report, we introduce a novel proof of concept: using known therapeutic antimicrobial peptides (AMPs) as bactericidal agents for room temperature–stored PLT concentrates (PCs). Nine synthetic AMPs, four from PLT microbicidal protein-derived peptides (PD1-4) and five Arg-Trp (RW) repeat peptides containing one to five repeats, were tested for bactericidal activity in plasma and PC samples spiked with Staphylococcus aureus , S. epidermidis , Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumoniae, and Bacillus cereus . A 3-log reduction of viable bacteria was considered as the bactericidal activity of a given peptide.
RESULTS: In both plasma alone and PCs, RW3 peptide demonstrated bactericidal activity against S. aureus , S. epidermidis , E. coli , P. aeruginosa, and K. pneumoniae ; PD4 and RW2 against P. aeruginosa ; and RW4 against K. pneumoniae . The activity of each of these four peptides against the remaining bacterial species in the test panel resulted in less than a 3-log reduction in the number of viable bacteria and hence considered ineffective.
CONCLUSIONS: These findings suggest a new approach to improving the safety of blood components, demonstrating the potential usefulness of screening therapeutic AMPs against selected bacteria to identify suitable bactericidal agents for stored plasma, PCs, and other blood products.     

Photochemical inactivation with amotosalen and long-wavelength ultraviolet light of Plasmodium and Babesia in platelet and plasma components

BACKGROUND: Transfusion-transmitted cases of malaria and babesiosis have been well documented. Current efforts to screen out contaminated blood products result in component wastage due to the lack of specific detection methods while donor deferral does not always guarantee safe blood products. This study evaluated the efficacy of a photochemical treatment (PCT) method with amotosalen and long-wavelength ultraviolet light (UVA) to inactivate these agents in red blood cells (RBCs) contaminating platelet (PLT) and plasma components. STUDY DESIGN AND METHODS: Plasmodium falciparum- and Babesia microti-contaminated RBCs seeded into PLT and plasma components were treated with 150 micromol per L amotosalen and 3 J per cm2 UVA. The viability of both pathogens before and after treatment was measured with infectivity assays. Treatment with 150 micromol per L amotosalen and 1 J per cm2 UVA was used to assess the robustness of the PCT system. RESULTS: No viable B. microti was detected in PLTs or plasma after treatment with 150 mol per L amotosalen and 3 J per cm2 UVA, demonstrating a mean inactivation of greater than 5.3 log in PLTs and greater than 5.3 log in plasma. After the same treatment, viable P. falciparum was either absent or below the limit of quantification in three of four replicate experiments both in PLTs and in plasma demonstrating a mean inactivation of at least 6.0 log in PLTs and at least 6.9 log in plasma. Reducing UVA dose to 1 J per cm2 did not significantly affect the level of inactivation. CONCLUSION: P. falciparum and B. microti were highly sensitive to inactivation by PCT. Pathogen inactivation approaches could reduce the risk of transfusion-transmitted parasitic infections and avoid unnecessary donor exclusions.     

Comparison of bacteria growth in single and pooled platelet concentrates after deliberate inoculation and storage

BACKGROUND: The ability to store pools of platelet concentrates (PCs) for extended periods would provide logistical flexibility. However, reports of severe adverse reactions due to the transfusion of contaminated PCs led to an examination of whether the total bacteria levels after storage of pools containing a deliberately inoculated platelet unit would be significantly different than the levels in paired unpooled concentrates. STUDY DESIGN AND METHODS: A single PC was deliberately inoculated on Day 0 with one of three bacterial species (0.1–8.0 colony-forming units/mL). On Day 1, the deliberately inoculated PC was divided into three equal parts and either 1) pooled with 5 half-volume, ABO- and Rh-identical PCs; 2) similarly pooled and white cell reduced; or 3) kept as a control. Sterile connections were used during pooling; modified storage containers were used to ensure the correct surface-to-volume ratio of the single unit. RESULTS: Between Day 2 and Day 5 of storage, in 26 of 36 paired samples, nonfiltered pools containing Escherichia coli had greater total numbers of bacteria than did the paired single PCs. Day 2 pools had total bacteria levels approximately five times higher (colony-forming units/mL × container volume) than those in single units (p < 0.05). There was rapid growth of Staphylococcus aureus by Day 2 in pooled and unpooled PCs; by Day 3, total bacteria levels were approximately five times higher in pools than in single units (p < 0.05). Between Days 3 and 5 of storage, in 23 of 27 paired samples, nonfiltered pools containing S. aureus had greater total bacteria levels than the single PCs. By Day 5, 15 of 16 non-white-cell reduced pools had total levels of Staphylococcus epidermidis bacteria approximately five times those in the paired single PCs. Greater total bacteria levels in pooled units than in single units generally occurred when bacteria in pools reached the stationary phase of growth (when bacteria concentration became constant), and they were well correlated with the sixfold volume of pooled units. White cell reduction did not substantially affect the time required to attain stationary phase. CONCLUSION: The potential during storage for greater total bacteria levels in pools than in single PCs is a consequence of the greater volume of the pool.     

Clinical safety of platelets photochemically treated with amotosalen HCl and ultraviolet A light for pathogen inactivation: the SPRINT trial

BACKGROUND: A photochemical treatment (PCT) method utilizing a novel psoralen, amotosalen HCl, with ultraviolet A illumination has been developed to inactivate viruses, bacteria, protozoa, and white blood cells in platelet (PLT) concentrates. A randomized, controlled, double-blind, Phase III trial (SPRINT) evaluated hemostatic efficacy and safety of PCT apheresis PLTs compared to untreated conventional (control) apheresis PLTs in 645 thrombocytopenic oncology patients requiring PLT transfusion support. Hemostatic equivalency was demonstrated. The proportion of patients with Grade 2 bleeding was not inferior for PCT PLTs. STUDY DESIGN AND METHODS: To further assess the safety of PCT PLTs, the adverse event (AE) profile of PCT PLTs transfused in the SPRINT trial is reported. Safety assessments included transfusion reactions, AEs, and deaths in patients treated with PCT or control PLTs in the SPRINT trial. RESULTS: A total of 4719 study PLT transfusions were given (2678 PCT and 2041 control). Transfusion reactions were significantly fewer following transfusion of PCT than control PLTs (3.0% vs. 4.1%; p = 0.02). Overall AEs (99.7% PCT vs. 98.2% control), Grade 3 or 4 AEs (79% PCT vs. 79% control), thrombotic AEs (3.8% PCT vs. 3.7% control), and deaths (3.5% PCT vs. 5.2% control) were comparable between treatment groups. Minor hemorrhagic AEs (petechiae [39% PCT vs. 29% control; p < 0.01] and fecal occult blood [33% PCT vs. 25% control; p = 0.03]) and skin rashes (56% PCT vs. 42% control; p < 0.001) were significantly more frequent in the PCT group. CONCLUSION: The overall safety profile of PCT PLTs was comparable to untreated PLTs.     

Microcalorimetry: a novel method for detection of microbial contamination in platelet products

BACKGROUND: Measuring heat from replicating microorganisms in culture may be a rapid, accurate, and simple screening method for platelets (PLTs). Microcalorimetry for detection of microorganisms in in vitro contaminated PLT products was evaluated. STUDY DESIGN AND METHODS: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus sanguinis, Escherichia coli, Propionibacterium acnes, and Candida albicans were inoculated in single-donor apheresis PLTs to achieve target concentrations of 10(5), 10(3), 10, or 1 colony-forming units (CFU) per mL of PLTs. Contaminated PLTs in growth medium were incubated at 37 degrees C for 5 days in a calorimeter. Positivity was defined as heat flow of at least 10 microW above the lowest value of the power-time curve. RESULTS: With microcalorimetry, inocula of 10 CFUs per mL PLTs could be detected with the following detection times: S. epidermidis (31.65 hr), S. aureus (24.24 hr), S. sanguinis (7.82 hr), E. coli (7.53 hr), P. acnes (73.57 hr), and C. albicans (43.77 hr). The detection time was less than 4 hr at 10(5) CFUs per mL PLTs for S. aureus, S. sanguinis, and E. coli. Noncontaminated PLTs remained negative. The total heat ranged from 2.8 (S. sanguinis) to 8.3 J (E. coli).The shape of the power-time curve was species-specific and independent from the initial concentration of microorganisms. CONCLUSION: The detection limit of microcalorimetry was 1 to 10 CFUs per mL PLTs. Microcalorimetry is a promising novel method for detection of contaminated PLTs. Applying this method to all PLT products could reduce the frequency of transfusion-related sepsis and prolong the shelf life of PLTs.     

Pathogen inactivation of platelets with a photochemical treatment with amotosalen HCl and ultraviolet light: process used in the SPRINT trial

BACKGROUND: A photochemical treatment (PCT) system has been developed to inactivate a broad spectrum of pathogens and white blood cells in platelet (PLT) products. The system comprises PLT additive solution (PAS III), amotosalen HCl, a compound adsorption device (CAD), a microprocessor-controlled ultraviolet A light source, and a commercially assembled system of interconnected plastic containers. STUDY DESIGN AND METHODS: A clinical prototype of the PCT system was used in a large, randomized, controlled, double-blind, Phase III clinical trial (SPRINT) that compared the efficacy and safety of PCT apheresis PLTs to untreated apheresis PLTs. The ability of multiple users was assessed in a blood center setting to perform the PCT and meet target process specifications. RESULTS: Each parameter was evaluated for 2237 to 2855 PCT PLT products. PCT requirements with respect to mean PLT dose, volume, and plasma content were met. Transfused PCT PLT products contained a mean of 3.6 x 10(11) +/- 0.7 x 10(11) PLTs. The clinical process, which included trial-specific samples, resulted in a mean PLT loss of 0.8 x 10(11) +/- 0.6 x 10(11) PLTs per product. CAD treatment effectively reduced the amotosalen concentration from a mean of 31.9 +/- 5.3 micromol per L after illumination to a mean of 0.41 +/- 0.56 micromol per L after CAD. In general, there was little variation between sites for any parameter. CONCLUSIONS: The PCT process was successfully implemented by 12 blood centers in the United States to produce PCT PLTs used in a prospective, randomized trial where therapeutic efficacy of PCT PLTs was demonstrated. Process control was achieved under blood bank operating conditions.     

Leukoreduced buffy coat-derived platelet concentrates photochemically treated with amotosalen HCl and ultraviolet A light stored up to 7 days: assessment of hemostatic function under flow conditions

BACKGROUND: Amotosalen plus ultraviolet A light photochemical treatment (PCT) inactivates high titers of bacteria, and other pathogens, in platelet concentrates (PCs) potentially allowing the storage of platelets (PLTs) for up to 7 days. Adhesion and aggregation of PLTs to injured vascular surfaces are critical aspects of PLT hemostatic function. STUDY DESIGN AND METHODS: Two ABO-identical leukoreduced buffy coat-derived PCs in additive solution were mixed and divided: one-half underwent PCT (PCT-PCs) and the other was kept as a control (C-PCs); both were stored under standard conditions. The total number of paired PCs studied was nine. Samples were taken on Day 1 (before PCT) and after 5 and 7 days of storage. The adhesion and aggregation capacities were evaluated under flow conditions in a ex vivo perfusion model. RESULTS: Compared to control, PCT resulted in a decrease in PLT count of 6.5 percent (p = 0.004) and 10.2 percent (p = 0.008) after 5 and 7 days' storage, respectively (n = 9). PLT interaction with subendothelium was mainly in form of adhesion. The surface covered by PCT PLTs on Day 1 was 26.0 +/- 4.2 percent (mean +/- SEM). On Day 5, PCT-PCs showed a covered surface of 20.9 +/- 2.2 percent, and the C-PCs, 20.6 +/- 1.6 percent. After 7 days, PCT-PCs produced a nonsignificant higher PLT deposition compared to control (27.1 +/- 2.9% vs. 21.2 +/- 2.8%, p = 0.06). CONCLUSION: PCT of PCs and storage up to 7 days was associated with a 10.2 percent decrease in PLT count due to processing losses compared to C-PC. PLT adhesive and aggregating capacities under flow conditions of PCT-PCs were similar to C-PCs and remained well preserved for up to 7 days of storage.     

Staphylococcus epidermidis forms biofilms under simulated platelet storage conditions

BACKGROUND: Staphylococcus epidermidis grows slowly in platelet (PLT) preparations compared to other bacteria, presenting the possibility of missed detection by routine screening. S. epidermidis is a leading cause of nosocomial sepsis, with virulence residing in its ability to establish chronic infections through production of slime layers, or biofilms, on biomedical devices. This study aims to establish biofilm formation (BF) as a mode of growth by S. epidermidis in PLT preparations. STUDY DESIGN AND METHODS: Biofilm-positive (BFpos) and -negative (BFneg) S. epidermidis strains were grown in whole blood-derived PLTs (WBDPs) and in glucose-rich medium (TSBg). An assay for BF was adapted for cultures grown in WBDPs or filtered WBDPs in polystyrene culture plates. Bacterial attachment to polyvinylchloride PLT bags and PLTs was examined by scanning electron microscopy. RESULTS: Both strains display similar growth profiles in WBDPs and TSBg. Unexpectedly, evidence of BF was observed on PLT bags and on PLTs directly, not only by the BFpos strain but also by the BFneg strain. The BFpos strain displayed greater plastic adherence than the BFneg strain in WBDPs (p < 0.05). BF by the BFneg strain was approximately 10-fold greater in WBDPs compared to TSBg (p < 0.05), likely by use of PLTs as a scaffold. Furthermore, BF by S. epidermidis was significantly decreased when PLT concentration was reduced 1000-fold. CONCLUSIONS: S. epidermidis forms biofilms on PLT aggregates and on PLT bags under PLT storage conditions. Our results demonstrate that the PLT storage environment can promote a BF growth mechanism for contaminant bacteria.     

Photochemical treatment of plasma with amotosalen and long-wavelength ultraviolet light inactivates pathogens while retaining coagulation function

BACKGROUND: The INTERCEPT Blood System, a photochemical treatment (PCT) process, has been developed to inactivate pathogens in platelet concentrates. These studies evaluated the efficacy of PCT to inactivate pathogens in plasma and the effect of PCT on plasma function. STUDY DESIGN AND METHODS: Jumbo (600 mL) plasma units were inoculated with high titers of test pathogens and treated with 150 micromol per L amotosalen and 3 J per cm(2) long-wavelength ultraviolet light. The viability of each pathogen before and after treatment was measured with biological assays. Plasma function was evaluated through measurement of coagulation factors and antithrombotic protein activities. RESULTS: The levels of inactivation expressed as log-reduction were as follows: cell-free human immunodeficiency virus-1 (HIV-1), greater than 6.8; cell-associated HIV-1, greater than 6.4; human T-lymphotropic virus-I (HTLV-I), 4.5; HTLV-II, greater than 5.7; hepatitis B virus (HBV) and hepatitis C virus, greater than 4.5; duck HBV, 4.4 to 4.5; bovine viral diarrhea virus, 6.0; severe acute respiratory syndrome coronavirus, 5.5; West Nile virus, 6.8; bluetongue virus, 5.1; human adenovirus 5, 6.8; Klebsiella pneumoniae, greater than 7.4; Staphylococcus epidermidis and Yersinia enterocolitica, greater than 7.3; Treponema pallidum, greater than 5.9; Borrelia burgdorferi, greater than 10.6; Plasmodium falciparum, 6.9; Trypanosoma cruzi, greater than 5.0; and Babesia microti, greater than 5.3. Retention of coagulation factor activity after PCT was expressed as the proportion of pretreatment (baseline) activity. Retention was 72 to 73 percent of baseline fibrinogen and Factor (F)VIII activity and 78 to 98 percent for FII, FV, FVII, F IX, FX, FXI, FXIII, protein C, protein S, antithrombin, and alpha2-antiplasmin. CONCLUSION: PCT of plasma inactivated high levels of a wide range of pathogens while maintaining adequate coagulation function. PCT has the potential to reduce the risk of transfusion-transmitted diseases in patients requiring plasma transfusion support.