endothelial cells ( huvec passage 46 ; clonetics , walkersville , md ) were grown to confluence , then stimulated with plasminogen activation inhibitor-1 ( pai-1 , 10 ng / ml ; american diagnostica inc . , supernatant was collected 3 hours after stimulation and centrifuged ( 145 g , 8 minutes ) to remove cell debris . the supernatant was subsequently ultracentrifuged ( 100,000 g , 6 minutes , 4 degrees ) and pelleted . emps were re - suspended in phosphate buffered saline ( pbs ) and stored at 80c . flow cytometric analysis was used to characterize and quantify emps based on cd31- and annexin v - positivity occurring in a size gate less than 1 m . this size gate was set on forward versus side scatter using latex standard beads measuring 0.84 m ( spherotech fp-0856 - 2 ) . enumeration was completed against a known concentration of 7.6 m polystyrene beads placed within the sample ( spherotech pps-4 ) . the flow rate used for analysis was 12 l / min ( figs . 1 and 2 ) . 7.6 m latex beads are shown in gate p1 and allowed for enumeration of emps . within gate p2 , events positive for annexin v ( p3 ) and cd31 ( p4 ) were enumerated against p1 to quantify emp levels . the same settings were used to quantify emps in perfusate samples . as a separate and confirmatory measure , transmission electron microscopy ( tem ) . a 25-l drop of emp in solution was pipetted onto a formvar plastic coated grid ( ted pella , inc . ) and allowed to air dry for 1 hour . the microparticle grid was examined and photographed by tem with a jeol 100 cx microscope ( fig . 3 ) . emp size distribution as measured by tem ( 10,000magnification ) accompanied by a representative micrograph . emps ( 30010 ) were introduced to an iv bag ( model patient ) containing 300 ml of 0.9 ns ( model patient ) and manually agitated for 3 minutes . the iv bag was connected to a primed cvvh circuit containing 170 ml of 0.9 ns and mixed ( ~65010 emp / ml final mixed perfusate concentration ) as shown in fig . , the perfusate was circulated through the cvvh filter ( nxstage purema , 200 m pore size , 35 m wall thickness , rate 250 ml / hour ) for a period of 70 minutes . 0.5 ml aliquots were removed for flow cytometric analysis at 510 minute intervals for 70 minutes of dialysis . emp levels were recorded and expressed as a fraction of starting concentration after mixing occurred . three runs were completed and the fraction of emps versus time was plotted with standard error of the mean ( sem ) . at the end of 4 hours filtration , . a 300 ml iv bag of 0.9 ns served as the model patient and was spiked with emps . the circuit contained 170 ml 0.9 ns and ran counter - current against 1 l of 0.9 ns dialysate . after mixing , 0.5 ml aliquots were drawn from the  patient  for analysis as dialysis was completed via the filter . endothelial cells ( huvec passage 46 ; clonetics , walkersville , md ) were grown to confluence , then stimulated with plasminogen activation inhibitor-1 ( pai-1 , 10 ng / ml ; american diagnostica inc . , supernatant was collected 3 hours after stimulation and centrifuged ( 145 g , 8 minutes ) to remove cell debris . the supernatant was subsequently ultracentrifuged ( 100,000 g , 6 minutes , 4 degrees ) and pelleted . emps were re - suspended in phosphate buffered saline ( pbs ) and stored at 80c . flow cytometric analysis was used to characterize and quantify emps based on cd31- and annexin v - positivity occurring in a size gate less than 1 m . this size gate was set on forward versus side scatter using latex standard beads measuring 0.84 m ( spherotech fp-0856 - 2 ) . enumeration was completed against a known concentration of 7.6 m polystyrene beads placed within the sample ( spherotech pps-4 ) . the flow rate used for analysis was 12 l / min ( figs . 1 and 2 ) . 7.6 m latex beads are shown in gate p1 and allowed for enumeration of emps . within gate p2 , events positive for annexin v ( p3 ) and cd31 ( p4 ) were enumerated against p1 to quantify emp levels . the same settings were used to quantify emps in perfusate samples . as a separate and confirmatory measure , transmission electron microscopy ( tem ) . a 25-l drop of emp in solution was pipetted onto a formvar plastic coated grid ( ted pella , inc . ) and allowed to air dry for 1 hour . the microparticle grid was examined and photographed by tem with a jeol 100 cx microscope ( fig . 3 ) . emp size distribution as measured by tem ( 10,000magnification ) accompanied by a representative micrograph . emps ( 30010 ) were introduced to an iv bag ( model patient ) containing 300 ml of 0.9 ns ( model patient ) and manually agitated for 3 minutes . the iv bag was connected to a primed cvvh circuit containing 170 ml of 0.9 ns and mixed ( ~65010 emp / ml final mixed perfusate concentration ) as shown in fig . , the perfusate was circulated through the cvvh filter ( nxstage purema , 200 m pore size , 35 m wall thickness , rate 250 ml / hour ) for a period of 70 minutes . 0.5 ml aliquots were removed for flow cytometric analysis at 510 minute intervals for 70 minutes of dialysis . emp levels were recorded and expressed as a fraction of starting concentration after mixing occurred . three runs were completed and the fraction of emps versus time was plotted with standard error of the mean ( sem ) . at the end of 4 hours filtration , a 300 ml iv bag of 0.9 ns served as the model patient and was spiked with emps . the circuit contained 170 ml 0.9 ns and ran counter - current against 1 l of 0.9 ns dialysate . after mixing , 0.5 ml aliquots were drawn from the  patient  for analysis as dialysis was completed via the filter . the decline in emp levels due to dialysis is rapid with 50% of clearance being seen in the first 30 minutes . within 40 minutes , the circulating emp levels were reduced to 29% of starting concentration and this level stayed relatively constant over the duration of the study . to determine the fate of the cleared emps , we measured emp levels in the dialysate . the dialysate contained 5.7% of the original emp concentration ( ~65010 emp / ml ) at the end of 4 hours of uninterrupted dialysis . emps levels expressed as a fraction of initial concentration by time and averaged over 3 experiments . the data presented here demonstrate that counter - current dialysis using a standard clinical cvvh filter and under clinical conditions removes emps from a modelled patient 's circulation . given our previous evidence of emp - induced ali , these findings support the idea of cvvh as a meaningful therapeutic intervention in ali due to clearance of emps from the patient circulation . the experimental conditions achieve the same plasma level of emps as seen in human clinical disease ( 110 per ml ) . while this work demonstrates that a standard 200-m cvvh circuit and filter entraps emps , there are significant limitations to the model presented . the observed emp clearance is likely a saturable phenomenon , which would explain the relatively constant emp concentration from 40 to 70 minutes and low dialysate emp levels . the relative volume of human circulation ( ~5 l ) to cvvh circuit ( ~200 ml ) is much higher in the clinical condition as compared to our model ( 300 and 170 ml , respectively ) . while this would not affect a steady state of dialysis , a saturable filter emp - binding additionally , emps may continue to be generated in a patient and a non - saturable clearance method would be required to achieve a lower steady state emp concentration . convection and pressure gradient forces were not modelled , as these interventions require replacement fluid and this would confound emp measurement . however , if emp charge and size are amenable , forced hindered diffusion via the application of convective and pressure forces coupled with replacement by emp - free fluid would likely sustain clearance . in conjunction with particle trapping by the filter , the first order kinetics of emp clearance could be prolonged at a minimum . additionally , emp interactions in vivo with plasma oncotic elements ( circulating cells , albumin , etc . ) we do not feel that such elements have a major impact upon the steady state of emps in vivo , since characterized emps in human disease states using flow cytometry have identified the 0.510 per ml threshold injury value using flow cytometry of submicron events occurring in platelet - free plasma ( 54 ) . thus , the absence of these elements in our model is representative of the steady state of unbound plasma emps present in human disease . the low dialysate emp level and plateauing circulation emp level beyond 40 minutes suggest that emp sequestration occurs within the cvvh filter or tubing and that this is a saturable phenomenon . frequent changing of the cvvh filter is a logical step that may overcome this limitation . additionally , more efficient and selective emps clearance can be realized by conjugating endothelium - specific antibodies to commercially available specialty cvvh filters ( aethlon adapt ) . this approach would provide more selective binding sites and higher affinity for circulating emps . by combining a selective filter with convection and pressure gradients , emp clearance could likely be substantially prolonged . clinical data generally support the protective role of cvvh in ali management . a clinical randomized trial and several animal trials demonstrate that the achievement of negative fluid balance is associated with improved outcomes from ali ( 6265)(67,68 ) . in addition to its salutary effects on emp levels , early cvvh may prevent or minimize volume overload that is commonly observed with the resuscitation phase of critical illness , particularly in persons suffering concomitant acute kidney injury ( aki ) . thus , cvvh could be used as an effective tool targeting both the molecular machinery and iatrogenic components of ali . as a translatable next step , we plan to assay patients pre- and post - dialysis to observe emp concentration effects . ultimately , the application of dialysis to rodent models of emp - induced ali would examine efficacy in on - going diseases and emp thresholds associated with worse injuries . while the utility of ultrafiltration can be tested in an animal model , diffusion appears to be enough to remove emps due to their small size : standard dialysis alone was effective in this in vitro model . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .