Comparison of three bacterial detection methods under routine conditions

BACKGROUND AND OBJECTIVES: Since 2004, bacterial screening of platelets has been required in the USA and is also done on a voluntary basis in many European countries. The German Red Cross blood donor services conducted a prospective multicentre study in order to investigate the prevalence of bacterially contaminated pool platelet concentrates and apheresis platelet concentrates. This substudy compares three different bacterial detection systems. STUDY DESIGN AND METHODS: Platelet concentrates were tested in parallel with BacT/ALERT, Scansystem and Pall eBDS (n = 6307) in pool platelets. Apheresis platelets were tested in parallel with BacT/ALERT and Pall eBDS (n = 4730). All initially positive results were evaluated by a standardized procedure including evaluation by a microbiology reference laboratory. RESULTS: One in 6307 pool platelets were confirmed positive by BacT/ALERT, whereas Pall eBDS and Scansystem failed to detect these samples. Only three samples were initially reactive with Pall eBDS without proof of any bacteria strains. The rate of false-positive results was substantially higher for BacT/ALERT (0.25%, 28 in 11,037 tested samples) than for eBDS (0.03%, 3 in 11 037 tested samples) or Scansystem (0.0%, 0 in 6307 tested samples). Three of 4730 apheresis platelets were confirmed positive by BacT/ALERT. These were negative with Pall eBDS. CONCLUSION: Sensitivity was best for BacT/ALERT, whereas specificity was enhanced for Pall eBDS and Scansystem. Scansystem required specially trained staff, whereas BacT/ALERT and Pall eBDS were easy, quick, user-friendly and objective methods.     

Optimal sampling time after preparation of platelet concentrates for detection of bacterial contamination by quantitative real-time polymerase chain reaction

BACKGROUND AND OBJECTIVES: A universal quantitative real-time polymerase chain reaction (PCR), based on bacterial 16S rDNA, to detect bacterial contamination of platelet concentrates (PCs), was developed previously and compared with automated culturing. In the present study, this real-time PCR method was evaluated to determine the optimal sampling time for screening of bacterial contamination in PCs. MATERIALS AND METHODS: Routinely prepared PCs were spiked with suspensions of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes to 1, 10 and 100 colony-forming units (CFU)/ml and stored at room temperature for 7 days. The presence of bacteria in these PCs was monitored by quantitative real-time PCR. As a reference method (additional control), BacT/Alert automated culturing was used. For PCR, 1-ml aliquots were drawn from all (spiked) PCs on days 0, 1, 2, 3, 6 and 7 of storage. As a control, triplicate samples (10 ml) were inoculated into aerobic and anaerobic BacT/Alert culture bottles immediately after spiking (day 0) and after storage for 1, 2, 3, 6 or 7 days. RESULTS: With quantitative real-time PCR, all bacterial species tested were reproducibly detected on day 1 after spiking at original concentrations of 10 and 100 CFU/ml. Bacteria were also detected on day 1 from PCs spiked with an initial concentration of 1 CFU/ml, except for E. coli, which was detected in only one of the three samples and P. aeruginosa, for which analysis was not performed on day 1. With the reference method, bacteria were detected in culture bottles (inoculated on day 0) within a mean time of 20.1 h, with the exception of P. acnes which was detected at a mean time of 102.3 and 49.3 h (for original spiking concentrations of 10 and 100 CFU/ml respectively). CONCLUSIONS: PCR enables the rapid detection of low initial numbers of bacteria in PCs. For reliable detection, our results support that sampling of PCs for real-time PCR screening should not be carried out earlier than 1 day after preparation (48 h after blood collection). Importantly, the real-time PCR approach has the potential to be used before the release of PCs from the blood centre or shortly before they are transfused in the hospital.     

Evaluation of the BacT/Alert automated blood culture system for detecting bacteria and measuring their growth kinetics in leucodepleted and non-leucodepleted platelet concentrates

BACKGROUND AND OBJECTIVE: To evaluate the BacT/Alert automated blood culture system for the detection of bacteria in platelet concentrates, and to determine bacterial growth kinetics in leucodepleted and non-leucodepleted units. MATERIALS AND METHODS: Apheresis (Cobe Leucocyte Reduction System [LRS]) and pooled buffy coat-derived (Optipress) platelet concentrates (PCs) were tested. Six organisms were used for spiking the PCs: Clostridium perfringens, Bacillus cereus, Group B Streptococcus, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli. Units were inoculated to give a final concentration of approximately equal to 1 and 50 colony-forming units (CFU)/ml. On days 0, 2 and 5, BacT/Alert standard aerobic and anaerobic bottles were inoculated with a 5-ml fill volume and bacteria were enumerated. RESULTS: The BacT/Alert Automated blood culture system gave rapid determination times of spiked units, with all positives detected within 48 h and 98.1% detected within 24 h. In general, as the inoculum concentration increased, the detection time decreased. Rapid growth was obtained with all organisms tested except for B. cereus, which failed to grow on four occasions. Bacterial numbers on day 2 ranged from 10(5) to 10(11) CFU/ml and on day 5 ranged from 10(4) to 10(12) CFU/ml. Growth was not significantly greater in leucodepleted units. CONCLUSIONS: The study confirmed that PCs are an excellent growth medium for bacteria. Rapid and substantial growth was obtained with all organisms under test. Leucodepletion does not appear to enhance bacterial proliferation. The BacT/Alert automated blood culture system could rapidly detect contamination of units. Bacterial screening using an automated blood culture system is therefore a potential option.     

Evaluation of a universal point‐of‐issue assay for bacterial detection in buffy coat platelet components

Bacterial contamination of platelet concentrates poses a major post‐transfusion infectious risk. This study was aimed at evaluating the efficacy of the BacTx ^® assay (Immunetics Inc.) for bacterial detection in leucocyte‐reduced buffy coat platelet pools and for its sensitivity in detecting clinical isolates, including bacteria that form surface‐attached aggregates (biofilm positives). Platelet pools were inoculated at bacterial concentrations of 0·8–13 CFU/ml. The BacTx ^® assay detected all species at concentrations ≥10³ CFU/ml within 20–69 h of platelet incubation. Detection of slow‐growing and biofilm‐forming strains was delayed in comparison with the other strains. This assay could be used as a point‐of‐issue method to increase the safety of the platelet supply.     

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection

BACKGROUND AND OBJECTIVES: The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. MATERIALS AND METHODS: A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. RESULTS: In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). DISCUSSION: The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.     

Inactivation of pathogens in platelet concentrates by using a two-step procedure

BACKGROUND AND OBJECTIVES: Platelet concentrates are contaminated with residual leucocytes and may also be infected with viruses and bacteria. We investigated whether these pathogens can be inactivated by a two-step procedure comprising photodynamic treatment in the presence of the phenothiazine dye, thionine, followed by irradiation with ultraviolet light (UV-B, wavelength range 290-330 nm). MATERIALS AND METHODS: Platelet concentrates were prepared from buffy coats. The concentrates were spiked with different viruses, bacteria and leucocytes, then illuminated with yellow light in the presence of thionine at dye concentrations between 1 and 5 microm and with UV-B at doses up to 2.4 J/cm2. The infectivity of samples and the viability of leucocytes were assayed before and after treatment. The influence of treatment on in vitro platelet function was also examined. RESULTS: The inactivation of free viruses in platelet concentrates by photodynamic treatment with thionine/light was significantly enhanced when it was followed by irradiation with UV-B. The inactivation of leucocytes and of bacteria by UV-B was improved when it was preceded by thionine/light. Sterile platelet concentrates were prepared from buffy coats infected with Staphylococcus epidermidis. Platelet function and the storage stability of platelet concentrates were only moderately influenced by the two decontamination steps. CONCLUSIONS: Photodynamic treatment in the presence of the phenothiazine dye, thionine, followed by low-dose UVB, has the potential to inactivate viruses, leucocytes and bacteria, which might contaminate platelet concentrates. Both treatments complement each other.     

Inactivation of Gram-negative and Gram-positive bacteria in red cell concentrates using INACTINE PEN110 chemistry

BACKGROUND AND OBJECTIVES: The risk of transfusion-transmitted bacterial infections as a result of the presence of bacteria in blood is one of the major concerns in transfusion medicine. The purpose of this study was to investigate whether bacteria inoculated into red blood cell concentrates can be inactivated by the INACTINE PEN110 pathogen-reduction process. Four bacterial species were chosen for the study: anaerobic Gram-positive Clostridium perfringens and Propionibacterium acnes, known to be transfusion-transmitted; and two Gram-negative species, Acinetobacter johnsonii and Acinetobacter lwoffii, recently reported to be a common cause of transfusion-associated infections in Europe. MATERIALS AND METHODS: Identical units of leucoreduced red cell concentrates were inoculated with A. johnsonii, A. lwoffii, C. perfringens, or P. acnes. The 4 degrees C control units were put on storage immediately after receiving the spike. The test units were subjected to PEN110 treatment and then stored. The bacterial titre in all units was monitored during a 6-week storage period. RESULTS: The PEN110 inactivation of all tested bacterial strains was time- and titre-dependent. For A. johnsonii and A. lwoffii, no viable bacteria were detected in the units spiked with up to 10(4) colony-forming units (CFU)/ml and treated with PEN110. For red cell units spiked with 10(4)-10(5) CFU/ml of C. perfringens and P. acnes, no viable bacteria were detected in the units treated with PEN110. In control units, there was a gradual decrease in A. johnsonii, A. lwoffii and C. perfringens titres during cold storage, while P. acnes titres remained stable. CONCLUSIONS: The PEN110 pathogen-reduction process was demonstrated to inactivate high titres of A. johnsonii, A. lwoffii, C. perfringens and P. acnes in red cell concentrates.     

血小板制品细菌污染风险的研究

目的：研究血小板在采集制备过程中细菌污染的风险,为减少血小板细菌污染,提高输血安全性并制定预防措施提供依据。方法：采用生物梅里埃公司BacT/Alert120全自动细菌培养仪及需氧瓶（BPA Culture Bottle）、厌氧瓶（BPN Culture Bottle）对10949例血小板进行细菌培养,细菌培养阳性者转种,转种阳性者进行细菌鉴定。结果：2003年3月-2005年12月共培养血小板3073例,其中19例阳性,阳性率为0.62%（手工混合血小板0.72%,单采血小板0.47%）;2006年1月-2009年5月采取预防措施后培养7876例,共发现17例细菌培养阳性,阳性率为0.22%（手工混合血小板0.24%,单采血小板0.21%）。采取预防措施后总体血小板阳性率、手工混合血小板阳性率显著低于采取预防措施前（分别为χ2=10.92,P〈0.05;χ2=4.98,P〈0.05）,而单采血小板阳性率则差异无统计学意义（χ2=2.81,P〉0.05）。结论：采取预防措施可有效减少血小板细菌污染的发生,尤其对手工混合血小板效果显著,对单采血小板效果不明显。由于血小板采集后在（22±2）℃振荡条件下保存,细菌容易生长繁殖,因此输注血小板仍存在极高的细菌污染风险,预防措施仍需持续改进。     

Evaluation of the Sensitivity and Specificity of Use of Glucose and pH for Bacterial Screening of Platelet Concentrates Compared to the Bact/Alert

Bacterial contamination of blood components is the major infectious risk in transfusion medicine. Since platelets should be stored at room temperature that makes them an excellent growth medium for bacteria; it is mentioned as a major problem in transfusion medicine. Transfusion risk of a bacterial contaminated platelet concentrate is higher than viral pathogen such as HIV, HBV, HCV and HTLV. The objective of this study was to evaluation of the sensitivity and specificity of use of glucose and pH for bacterial screening of platelet concentrates compared to the Bact/Alert. 1332 platelet concentrates were screened by the Bact/Alert system for aerobic and anaerobic bacterial contamination. Bacterial contamination was also evaluated by using urine reagent strips (Multistix10 SG Bayer) and culture methods. Moreover PH screening with a pH meter (Metrohm 744 Swiss) and glucose was also used for detection of bacterial contamination. The rate of bacterial contamination detected by the Bact/Alert system in platelet concentrates was 25 in 1332 (1.9 %). It contained 15 (1.1 %) for aerobic bacteria and 10 (.8 %) for anaerobic bacteria. 226 of 1332 were considered as containing bacteria by using urine reagent strips. Six of the 226 units were also positive by the Bact/Alert system. Three of those units were culture positive for aerobic bacteria and three for anaerobic. The result of platelet concentrates that underwent pH screening by use of pH meter and a pH portion of urine reagent strips was the same. The sensitivity and specificity of considering glucose alone for detection of bacterial contamination were 12 and 98 % respectively. For pH alone, these were 24 and 83 %. For glucose and/or pH, these were 24 and 83 %; and for combination of glucose and pH, these were 12 and 98 %. Our results showed use of glucose/pH strips would improve the safety of blood products and should be encouraged.     

Impact of donor arm skin disinfection on the bacterial contamination rate of platelet concentrates

BACKGROUND AND OBJECTIVES: Despite improved methods for detecting bacterial contamination of blood products, bacterial sepsis remains a significant risk in blood transfusion. This study was undertaken to investigate whether adopting a different skin disinfection protocol could reduce the rate of bacterial contamination of platelet concentrates. MATERIALS AND METHODS: Two skin disinfection protocols were consecutively used in the routine blood collection setting during two 10-month periods: 0.5% cetrimide/0.05% chlorhexidine solution followed by 70% isopropyl alcohol (first 10-month time-period); and 10% povidone-iodine followed by 70% isopropyl alcohol (second 10-month time-period). The rates of bacterial contamination of platelet concentrates were monitored by using a surveillance programme described previously. RESULTS: The overall bacterial contamination rate in the first time-period was 0.072%. After introduction of the povidone-iodine and isopropyl alcohol protocol, the bacterial contamination rate decreased to 0.042% (relative risk reduction: -0.42; 95% confidence interval, -0.12 to -0.61, P= 0.009). There were no differences in the types of micro-organisms identified (P = 0.7). CONCLUSIONS: Skin disinfection by povidone-iodine and isopropyl alcohol is more effective than that by cetrimide/chorhexidine and isopropyl alcohol in reducing venepuncture-associated contamination of platelet concentrates by skin flora. Our data indicate that the disinfection protocol should be used on a routine basis and such implementation should translate into a significant improvement in blood safety to patients receiving platelet transfusion.     

Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates

Background Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT‐PCR) was evaluated in an inter‐laboratory comparison in three different German blood services. Methods Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10³–4·5 × 10⁸ CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. Results The inter‐laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT‐PCR‐screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non‐detected positive samples were below the assay’s detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10⁵ and 2·10 × 10⁷ CFU/ml, respectively, K. pneumoniae: 4·79 × 10⁶ CFU/ml, S. aureus: 6·03 × 10⁵ CFU/ml). All rapid screening methods revealed no false‐positive results. Conclusions Both BactiFlow and 23S rRNA RT‐PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram‐negative strains.     

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria

Background Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion‐Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion‐Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. Material and Methods Four Bacteria References (Staphylococcus epidermidis PEI‐B‐06, Streptococcus pyogenes PEI‐B‐20, Klebsiella pneumoniae PEI‐B‐08 and Escherichia coli PEI‐B‐19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. Results Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19–1·32 × 10⁷ CFU/ml, S. pyogenes: 0·58–0·69 × 10⁷ CFU/ml, K. pneumoniae: 18·71–20·26 × 10⁷ CFU/ml and E. coli: 1·78–2·10 × 10⁷ CFU/ml. Conclusion The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low‐titre spiking of blood components, (ii) the property of donor‐independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion‐Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.     

Six years' experience of using the BacT/ALERT system to screen all platelet concentrates, and additional testing of outdated platelet concentrates to estimate the frequency of false-negative results

BACKGROUND AND OBJECTIVES: Approximately 1 in every 2000 units of platelets is contaminated with bacteria. The BacT/ALERT automated blood culture system can be used to screen platelet concentrates (PCs) for bacterial contamination. MATERIALS AND METHODS: Data were collected from May 1998 until May 2004. The number of PCs tested during this period was 36 896, most of which were produced from pools of four buffy-coats. On the day following blood collection or platelet apheresis, a 5-10 ml sample of the PC was aseptically transferred to a BacT/ALERT culture bottle for detection of aerobic bacteria. The sample was monitored for bacterial growth during the entire storage period of the PC (6.5 days). When a positive signal was generated, the culture bottle, the PC and the erythrocyte concentrates were tested for bacterial growth. In order to determine the frequency of false-negative BacT/ALERT signals, 1061 outdated PCs were tested during the period from May 2002 to May 2004. RESULTS: Eighty-eight positive signals were detected by the BacT/ALERT system, of which 12 were interpreted as truly positive. Fourteen signals were interpreted as truly false positive. Thirty-three signals were interpreted to be probably false positive. Two of 1061 outdated units tested positive, and Bacillus spp. and Staphylococcus epidermidis, respectively, were isolated from these PCs. CONCLUSIONS: Between 0.03% and 0.12% of the PCs were contaminated with bacteria. BacT/ALERT is an efficient tool for monitoring PCs for bacterial contamination; however, it is important to realize that false-negative results may occur.     

Platelet content and growth factor release in platelet-rich plasma: a comparison of four different systems

BACKGROUND: Different systems for preparation of platelet-rich plasma are commercially available, but data for comparison of these systems have not been published so far. MATERIALS AND METHODS: We investigated the performance of Vivostat PRF Preparation Kit, PCCS Platelet Concentrate Collection System, Harvest SmartPReP 2 APC 60 Process, and Fibrinet Autologous Fibrin & Platelet System. The preparations provided by these systems are platelet concentrates with high numbers of platelets in a small volume of plasma and PDGF-AB is released continuously during the 5 days after preparation. RESULTS: Vivostat PRF Preparation Kit, PCCS Platelet Concentrate Collection System, Harvest SmartPReP 2 APC 60 Process are comparable in platelet yield and total amount of released PDGF-AB after 120 h while with Fibrinet the lowest platelet yield and PDGF-AB content of supernatant was achieved. The ability of growth factor release was equal in all four systems. CONCLUSION: In conclusion, all four systems for preparation of platelet-rich plasma investigated result in considerable growth factor release. In what extent the total content of PDGF-AB as a consequence of platelet yield has an impact on wound healing has to be further investigated.     

Bacterial screening of platelet concentrates on day 2 and 3 with flow cytometry: the optimal sampling time point?

Background

There is growing concern on the residual risk of bacterial contamination of platelet concentrates in Germany, despite the reduction of the shelf-life of these concentrates and the introduction of bacterial screening. In this study, the applicability of the BactiFlow flow cytometric assay for bacterial screening of platelet concentrates on day 2 or 3 of their shelf-life was assessed in two German blood services. The results were used to evaluate currently implemented or newly discussed screening strategies.

Materials and methods

Two thousand and ten apheresis platelet concentrates were tested on day 2 or day 3 after donation using BactiFlow flow cytometry. Reactive samples were confirmed by the BacT/Alert culture system.

Results

Twenty-four of the 2,100 platelet concentrates tested were reactive in the first test by BactiFlow. Of these 24 platelet concentrates, 12 were false-positive and the other 12 were initially reactive. None of the microbiological cultures of the initially reactive samples was positive. Parallel examination of 1,026 platelet concentrates by culture revealed three positive platelet concentrates with bacteria detected only in the anaerobic culture bottle and identified as Staphylococcus species. Two platelet concentrates were confirmed positive for Staphylcoccus epidermidis by culture. Retrospective analysis of the growth kinetics of the bacteria indicated that the bacterial titres were most likely below the diagnostic sensitivity of the BactiFlow assay (<300 CFU/mL) and probably had no transfusion relevance.

Conclusions

The BactiFlow assay is very convenient for bacterial screening of platelet concentrates independently of the testing day and the screening strategy. Although the optimal screening strategy could not be defined, this study provides further data to help achieve this goal.     

Bacterial contamination of platelet concentrates prepared by different methods: results of standardized sterility testing in Germany

BACKGROUND AND OBJECTIVES: National guidelines for monitoring the bacterial contamination rate of blood components were introduced in Germany in 1997. The objective of this study was to present and evaluate the results of sterility testing of platelet concentrates (PCs) prepared by different methods. MATERIALS AND METHODS: The analysis of results of sterility testing of blood component production from transfusion medicine centres in Germany in 1998 and 2001 was based on information collected using standardized questionnaires. RESULTS: The bacterial contamination rates for single-donor PCs derived from whole blood and apheresis (0.210% vs. 0.156%) were comparable and showed no significant difference. However, pooled PCs produced from four buffy coats using the sterile docking procedure showed a significantly higher bacterial contamination rate compared with single-donor PCs derived from whole blood and apheresis (0.184% vs. 0.604%). CONCLUSIONS: Use of standardized methods for sterility monitoring is sufficient to assess collection and production processes in terms of hygiene and yields reliable data on bacterial contamination rates of blood components. The methods described are suitable for using to analyse the efficiency of newly introduced methods to reduce bacterial contamination rates of blood components (e.g. diversion, bacteria screening and pathogen inactivation).     

PEGylation prevents bacteria-induced platelet activation and biofilm formation in platelet concentrates

Bacterial contamination of platelet concentrates represents the greatest post-transfusion infectious risk. Biofilm formation in this environment resulting from platelet-bacteria interactions can lead to non-uniform contaminant distribution and thus missed detection. As formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, we examined whether shielding these events would result in reduced biofilm formation by contaminant bacteria. We introduced methoxypoly(ethylene glycol) to covalently modify the platelet surface using a process termed 'PEGylation'. In the first study of its kind, we demonstrate that PEGylated platelet concentrates inoculated with Staphylococcus epidermidis display a significant reduction in bacterial binding and biofilm formation.     

Use of the DiaMed Impact R to test platelet function in stored platelet concentrates

BACKGROUND AND OBJECTIVES: The DiaMed Impact R tests platelet function under close to physiological flow conditions using cone and plate technology. An image analyser quantifies the adhered platelets and results are expressed as percentage of well surface covered by aggregates (SC %) as an index of adhesion and average size of the aggregates (AS microm(2)) as an index of aggregation. The machine is designed to use whole blood and the aim of this study was to determine if it could be used to assess platelet function in platelet concentrates (PC). MATERIAL AND METHODS: Platelet concentrates were mixed with various ratios of platelet-poor plasma (PPP) or ABO-compatible routine leucoreduced red cells in saline-adenine-glucose-mannitol. The effects of platelet counts, haematocrit, shear rate and time of activation upon SC and AS were evaluated to identify optimized assay conditions. Routine PCs were then tested on Days 2, 5 and 7. Samples were also stored at 4 or 37 degrees C for 1 to 2 h before assay to see if function was altered. RESULTS: Platelet concentrate in PPP resulted in no detectable platelet adhesion. However, addition of red cells to PC resulted in measurable platelet adhesion and aggregation. Optimal conditions were identified as shear rate of 1800 per second, 4-min activation, platelet count between 250 and 400 x 10(6) per ml, haematocrit between 30 and 40%. When stored PCs were tested under these conditions we observed median values of 8.2% for SC and 32.7 microm(2) for AS at 2 days, which reduced to 6.9% and 25.0 microm(2), respectively, after 5-day storage and 6.8% and 21.0 microm(2) after 7 days. CONCLUSION: We were able to reconstitute PCs by adding red cells and identified conditions to allow platelet adhesion and aggregation functions of PCs to be measurable in the DiaMed Impact R. Platelet functions of adhesion and aggregation were shown to decrease during storage but improved after a 1-h treatment at 37 degrees C.     

Studies of One Invasive and Two Noninvasive Methods for Detection of Bacterial Contamination of Platelet Concentrates

Recently, a new method for noninvasive detection of bacterial contamination of platelet concentrates was described [Arpi et al: Vox Sang 1993;65:335–336]. A CO₂-sensitive label is applied on the outer surface of the plastic container of the platelet concentrate. When any contaminating bacteria have increased the pCO₂ to a certain level the label changes color, indicating bacterial growth. We have studied this technique and applied such sensitive labels both directly onto PCs and onto plastic bags with culture medium into which platelet contents have been transferred. However, in both cases the sensitivity of the technique was found insufficient for practical quality control of PCs. There seem to be two problems with this principle: (1) platelets produce CO₂ and (2) a considerable amount of the gas passes beside the detection label, in this way decreasing the sensitivity of detection by the label. This noninvasive technique was compared with an invasive method using automated bacterial culture, which was found sensitive and rapid.