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 .
 backgroundendothelium - derived microparticles ( emps ) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury , apoptosis or activation . 
 we have previously demonstrated emp - induced acute lung injury ( ali ) in animal models and endothelial barrier dysfunction in vitro . 
 current treatment options for ali are limited and consist of supportive therapies . 
 we hypothesize that standard clinical continuous venovenous hemofiltration ( cvvh ) reduces serum emp levels and may be adapted as a potential therapeutic intervention.materials and methodsemps were generated from plasminogen activation inhibitor-1 ( pai-1)-stimulated human umbilical vein endothelial cells ( huvecs ) . 
 flow cytometric analysis was used to characterize emps as cd31- and annexin v - positive events in a submicron size gate . 
 enumeration was completed against a known concentration of latex beads . 
 ultimately , a concentration of ~650,000 emp / ml perfusate fluid ( total 470 ml ) was circulated through a standard cvvh filter ( pore size 200 m , flow rate 250 ml / hr ) for a period of 70 minutes . 
 0.5 ml aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis . 
 emp concentration in the dialysate was measured at the end of 4 hours to better understand the fate of emps.resultsa progressive decrease in circulating emp concentration was noted using standard cvvh at 250 ml / hr ( a clinical standard rate ) from a 470 ml volume modelling a patient 's circulation . 
 a 50% reduction was noted within the first 30 minutes . 
 emps entering the dialysate after 4 hours were 5.7% of the emp original concentration.conclusionthese data demonstrate that standard cvvh can remove emps from circulation in a circuit modelling a patient . 
 an animal model of hemofiltration with induction of emp release is required to test the therapeutic potential of this finding and potential of application in early treatment of ali . 
Methods
EMP generation, identification and enumeration
CVVH dialysis
Results
Discussion
Conflict of interest and funding