Introduction: Sepsis associated with transfusion of platelet units contaminated with bacteria is the most frequent infectious complication of blood transfusion[1]. Although bacterial screening is performed for every platelet unit in Canada, missed detection of bacterial contamination has been reported, which may result in adverse reactions or even fatalities after transfusion[2]. This problem becomes more complicated knowing the fact that the majority of platelet recipients are immunocompromised, for instance cancer patients.
Risk of missed detection is higher with bacterial strains that grow slowly in platelet concentrates and/or tend to adhere to the storage bag surfaces, namely Staphylococcus epidermidis, which is one of the most common sources of platelet contamination. The hydrophobic surface of plasticized polyvinyl chloride (PVC) platelet storage bags is considered to be conducive to bacterial adhesion and biofilm formation[3]. Here we report an approach to modify platelet storage bags and reduce these effects by developing a hydrophilic polymer brush coating on medical grade plasticized PVC through surface-initiated atom transfer radical polymerization (SI-ATRP). This coating proved to be compatible with platelets.
Methods: Initially, the pPVC coupons cut from platelet storage bags were functionalized with primary amine groups via allylamine plasma modification. A water-soluble ATRP initiator, 2- chloro-N-(oxiran-2-ylmethyl) propanamide, was reacted with the amine groups on the surface. SI-ATRP was used to polymerize the hydrophilic monomer, N,N-dimethylacrylamide (DMA) in water at room temperature (Scheme 1). Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM) in wet conditions, and water contact angle measurements (Figure 1a, 1b) were used to characterize the modified surfaces. Stability of the coating in phosphate buffer solution was tested by these methods after 7 days of storage.
Polymer brush coatings were evaluated against a biofilm forming Staphylococcus epidermidis strain isolated from a contaminated platelet unit in a clinic. Colony forming unit (CFU) counting and also microscopy techniques were used to evaluate bacterial attachment on the surfaces. For evaluation of biocompatibility of the coatings with platelets, we used platelet rich plasma (PRP) prepared in the lab from fresh blood collected from healthy, unmedicated, adult volunteers.
Results and Conclusion: Optimum PDMA coating with 165 ± 22 nm wet thickness and molecular weight of 230 kDa (Mn of polymers formed in the solution initiated by the added soluble initiator) decreased the bacterial adhesion by 95% after 4 h incubation with 106 CFU/ml of S. epidermidis in tryptic Soy Broth media plus 0.5% glucose (TSBG) at 37°C. Inhibition of biofilm formation after a 24 h challenge was performed and the result was evaluated by scanning electron microscopy (SEM) and fluorescent microcopy after staining. Decrease in platelet adhesion and activation on PDMA brush coated PVC was observed by SEM after 3 h incubation of these samples with PRP at 37° and comparing the results with a bare pPVC sample (control) (Figure 1c, 1d).
A stable PDMA coating on platelet bag material decreased bacterial adhesion by 95% after 4 h challenge with high concentration of S. epidemidis. The coating was biocompatible as evident from platelet adhesion and activation analyses on bag surfaces. Preliminary SEM imaging and live-dead fluorescence microscopy imaging showed that the modified surfaces resisted S. epidermidis biofilm formation after 24 h challenge; quantification of this data is in progress.
NH is a recipient of Health Canada/Canadian Blood Services (CBS) Graduate Fellowship.; We are grateful to Dr. Sandra Ramirez, CBS, Ottawa for providing us with the clinical S. epidermidis strain.; We thank volunteers who kindly donated blood for our project and Mrs. Brankica Culibrk for collecting the blood.
References:
[1] Blajchman, M. A.; Beckers E. A. M.; Dickmeiss, E.; Lin, L.; Moore, G.; Muylle, L. Transfus Med Rev 2005, 19, 259-72.
[2] Greco, C.; Martincic, I.; Gusinjac, A.; Kalab, M.; Yang, A. F.; Ramírez-Arcos, S. Transfusion 2007, 47, 1143-53.
[3] Balazs, D. J.; Triandafillu, K.; Wood, P.; Chevolot, Y.; Delden, C. V.; Harms, H.; Hollenstein, C.; Mathieu, H. J. Biomaterials 2004, 25, 2139-51.