Bacterial survival in whole blood depends on plasma sensitivity and resistance to neutrophil killing.

BACKGROUND: Whole blood (WB) is held at room temperature for not more than 24 hours before blood component manufacturing. The ability of several culture collection, skin-derived, and transfusion-related bacteria to survive in WB stored at 22 ± 2°C for 24 hours was investigated in this study. STUDY DESIGN AND METHODS: Twenty-one bacteria of the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Streptococcus agalactiae, Serratia liquefaciens, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, and Yersinia enterocolitica were inoculated into 7-mL aliquots of WB at a concentration of 500 colony-forming units (CFU)/mL. Spiked WB was stored aerobically at 22 ± 2°C, and bacterial viability and growth were monitored at 3, 8, and 24 hours during WB storage. Bacteria that showed decreased viability during WB incubation were further characterized for their sensitivity to plasma factors and neutrophil killing. RESULTS: There were three different scenarios for bacterial behavior during the hold of WB at 22 ± 2°C. Five bacteria proliferated (p < 0.03), 11 remained viable or showed low proliferation, and a third group of five bacteria had decreased or lost viability (p < 0.01). Three of the latter five bacteria were plasma-sensitive while the other two were plasma-resistant but susceptible to neutrophil killing (p = 0.01). CONCLUSIONS: The bactericidal activity of WB can be the result of plasma sensitivity or neutrophil killing. Bacteria with a starting inoculum of 500 CFU/mL, and able to resist WB immune factors, can proliferate to clinically significant levels posing a potential safety risk to transfusion patients. Results of this pilot study should be validated under standard WB collection and storage conditions.

Effects of methoxypoly (Ethylene glycol) mediated immunocamouflage on leukocyte surface marker detection, cell conjugation, activation and alloproliferation.

Tissue rejection occurs subsequent to the recognition of foreign antigens via receptor-ligand contacts between APC (antigen presenting cells) and T cells, resulting in initialization of signaling cascades and T cell proliferation. Bioengineering of donor cells by the covalent attachment of methoxypolyethylene glycol (mPEG) to membrane proteins (PEGylation) provides a novel means to attenuate these interactions consequent to mPEG-induced charge and steric camouflage. While previous studies demonstrated that polymer-mediated immunocamouflage decreased immune recognition both in vitro and in vivo, these studies monitored late events in immune recognition and activation such as T cell proliferation. Consequently little information has been provided concerning the early cellular events governing this response. Therefore, the effect of PEGylation was assessed by examining initial cell-cell interactions, changes to activation pathways, and apoptosis to understand the role that each may play in the decreased proliferative response observed in modified cells during the course of a mixed lymphocyte reaction (MLR). The mPEG-modified T cells resulted in significant immunocamouflage of lymphocyte surface proteins and decreased interactions with APC. Furthermore, mPEG-MLR exhibited decreased NFκB pathway activation, while exhibiting no significant differences in degree of cell death compared to the control MLR. These results suggest that PEGylation may prevent the direct recognition of foreign alloantigens by decreasing the stability and duration of initial cell-cell interactions.

Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline.

The grafting of low-immunogenic polymers to cells dramatically reduces antigenic recognition and immunogenicity of allogeneic donor cells consequent to steric and charge camouflage (i.e., immunocamouflage). While methoxypoly(ethylene glycol) [mPEG] has historically been utilized for the immunocamouflage of cells, other low-immunogenic polymers such as polyethyloxazoline propionic acid (PEOZ) may also be capable of conferring immunoprotection. Moreover, PEOZ may have attributes that could have enhanced pharmacological and biological utility relative to mPEG. To evaluate the immunocamouflage efficacy of PEOZ relative to mPEG, human red blood cells (RBC) and leukocytes were modified with mPEG or PEOZ. The differential effects of mPEG and PEOZ was assessed via grafting efficacy, cell morphology and viability, immunocamouflage of surface antigens, and the prevention of in vitro immune recognition (RhD and HLA). Although membrane grafting of mPEG and PEOZ were similar, mPEG demonstrated superior immunocamouflage efficacy as measured by antibody binding and phagocytosis of opsonized RBC while PEOZ showed improved RBC morphology. While mPEG appears to be superior to PEOZ in the immunocamouflage of cells, PEOZ may still be a valuable addition to our repertoire of immunomodulatory polymers. Moreover, our results demonstrate the importance of indirect immunocamouflage of antigens found in membrane protein complexes.

Microfluidic analysis of cellular deformability of normal and oxidatively damaged red blood cells.

Microfluidic analysis of blood has potential clinical value for determining normal and abnormal erythrocyte deformability. To determine if a microfluidic device could reliably measure intra- and inter-personal variations of normal and oxidized human red blood cell (RBC), venous blood samples were collected from repeat donors over time. RBC deformability was defined by the cortical tension (pN/µm), as determined from the threshold pressure required to deform RBC through 2-2.5 µm funnel-shaped constrictions. Oxidized RBC were prepared by treatment with phenazine methosulphate (PMS; 50 µM). Analysis of the control and oxidized RBC demonstrated that the microfluidic device could clearly differentiate between normal and mildly oxidized (20.13 ± 1.47 versus 27.51 ± 3.64 pN/µm) RBC. In vivo murine studies further established that the PMS-mediated loss of deformability correlated with premature clearance. Deformability variation within an individual over three independent samplings (over 21 days) demonstrated minimal changes in the mean pN/µm. Moreover, inter-individual variation in mean control RBC deformability was similarly small (range: 19.37-21.40 pN/µm). In contrast, PMS-oxidized cells demonstrated a greater inter-individual range (range: 25.97-29.90 pN/µm) reflecting the differential oxidant sensitivity of an individual's RBC. Importantly, similar deformability profiles (mean and distribution width; 20.49 ± 1.67 pN/µm) were obtained from whole blood via finger prick sampling. These studies demonstrated that a low cost microfluidic device could be used to reproducibly discriminate between normal and oxidized RBC. Advanced microfluidic devices could be of clinical value in analyzing populations for hemoglobinopathies or in evaluating donor RBC products post-storage to assess transfusion suitability.