the growing awareness of transfusion - associated morbidity and mortality has initiated a surge of investigations into the underlying mechanisms , which may be related to storage . storage lesion of red blood cells ( rbcs ) includes alteration of the plasma composition , together with changes in morphology of rbcs resulting in reduced deformability , which together may reduce tissue oxygen availability and have pro - inflammatory and immunomodulatory effects . these changes may all reduce posttransfusion viability of red blood cells . for decades , one of the standards of blood conservation as set by the fda is more than 75  % recovery measured at 24  h posttransfusion . optimizing storage conditions may negate some of the changes occurring during storage of rbcs . to test hypotheses of storage lesion as well as interventions aimed at improving storage conditions , however , the biology of stored murine rbcs may not accurately reflect that of human rbcs . indeed , rbc storage lesion of rodents appears to occur much faster than that of human rbcs . rodent rbcs stored for 15  days in a commonly used storage solution were reported to rapidly deteriorate , with only 26  % recovery at 24  h following transfusion . however , not all reports using rodent rbcs are consistent , with also reports of normal 24-h posttransfusion survival of stored rodent rbcs . a model in which functional effects of storage can be studied using human rbcs would overcome these problems with translation to the human condition . to that end , cross species models with transfusion of human rbcs into rodents have been used to study the role of rbc storage in transfusion - related acute lung injury , in recruitment of the microcirculation , in mechanisms of storage lesion , and in rejuvenation of storage solutions [ 57 ] . however , given the differences in size of the rbcs as well as the capillaries , it is conceivable that the use of cross species may lead to enhanced clearance of human donor rbcs from the rodent circulation . in this study , we investigated the role of storage on routing of human rbcs following transfusion into rodents . the institutional animal care and use committee of the academic medical center and the medical ethical committee of sanquin blood bank foundation approved all experiments . all animals were handled in accordance with the guidelines prescribed by the dutch legislation and the international guidelines on protection , care , and handling of laboratory animals . blood of healthy volunteers was collected using a needle connected to a syringe containing 1.25  ml citrate - phosphate - dextrose . blood was handled and stored according to national standards for human blood ( sanquin blood supply foundation ) , by overnight storage at room temperature , followed by centrifugation for 10  min at 1892g and 20  c . plasma was removed , and the buffy coat was separated from the packed red blood cells . saline - adenine - glucose - mannitol was added to the red blood cells up to a hematocrit of 5560  % . samples of the rbc products were analyzed for ph , potassium , sodium , glucose , and lactate with a rapidlab 865 blood gas analyzer ( siemens medical solutions diagnostics ; breda , the netherlands ) . male sprague - dawley rats ( 275  g ) raised on a regular diet were weighed and anesthetized with isoflurane 2  % . animals were randomized to receive fresh ( 1  day storage ) or stored rbcs ( 35  days storage , n  =  6 per group ) . the tail vein was cannulated with a 24 gauge venflon ( vasofix certo ; b.braun , meisungen , germany ) , and blood was aspirated to verify intravascular placement . twenty - four hours after transfusion , rats were sedated with ketamin and medetomidin as described and bled via the inferior caval vein in citrated ( 0.109  m ) vacutainer tubes for analysis and blood culture . blood taken 1 and 24  h after transfusion were used for cell counts on the advia 2120 hematology system . analysis of the human rbc from the circulation was done by spinning down 1    10 rbcs for 15  min at 1500 rpm and subsequently removing the top layer containing the white blood cells . rbcs ( 5    10 ) were washed twice in hepes and stained with anti - human cd235a - pe ( sanquin pelicluster ref m1732 ) or control antibody ( control igg1pe ref m1628 ) and analyzed by flow cytometry ( becton dickinson lsrii ) . we were not able to collect organs and analyze them from all animals , because time on flow cytometry and personnel were limited . the organs were cut into 15  mm cubes , resuspended in hepes buffer ( 132  mm nacl , 20  mm hepes , 6  mm kcl , 1  mm mgso4 , 1.2  mm k2hpo4 ) supplemented with 2  mm cacl2 and 10  mm glucose . and homogenized using gentlemacs c - tubes ( miltenyi biotec ) and the gentlemacs dissociator ( miltenyi biotec ) . afterwards , 5    10 cells were stained with anti - cd235a or control antibody as above . results are expressed as mean    sd . results of donor rbcs are expressed as percentage human rbcs of the total amount of stained rbcs . comparison between groups at the same time period or in the same organ was done using student s t test blood of healthy volunteers was collected using a needle connected to a syringe containing 1.25  ml citrate - phosphate - dextrose . blood was handled and stored according to national standards for human blood ( sanquin blood supply foundation ) , by overnight storage at room temperature , followed by centrifugation for 10  min at 1892g and 20  c . plasma was removed , and the buffy coat was separated from the packed red blood cells . saline - adenine - glucose - mannitol was added to the red blood cells up to a hematocrit of 5560  % . samples of the rbc products were analyzed for ph , potassium , sodium , glucose , and lactate with a rapidlab 865 blood gas analyzer ( siemens medical solutions diagnostics ; breda , the netherlands ) . male sprague - dawley rats ( 275  g ) raised on a regular diet were weighed and anesthetized with isoflurane 2  % . animals were randomized to receive fresh ( 1  day storage ) or stored rbcs ( 35  days storage , n  =  6 per group ) . the tail vein was cannulated with a 24 gauge venflon ( vasofix certo ; b.braun , meisungen , germany ) , and blood was aspirated to verify intravascular placement . twenty - four hours after transfusion , rats were sedated with ketamin and medetomidin as described and bled via the inferior caval vein in citrated ( 0.109  m ) vacutainer tubes for analysis and blood culture . blood taken 1 and 24  h after transfusion were used for cell counts on the advia 2120 hematology system . analysis of the human rbc from the circulation was done by spinning down 1    10 rbcs for 15  min at 1500 rpm and subsequently removing the top layer containing the white blood cells . rbcs ( 5    10 ) were washed twice in hepes and stained with anti - human cd235a - pe ( sanquin pelicluster ref m1732 ) or control antibody ( control igg1pe ref m1628 ) and analyzed by flow cytometry ( becton dickinson lsrii ) . we were not able to collect organs and analyze them from all animals , because time on flow cytometry and personnel were limited . the organs were cut into 15  mm cubes , resuspended in hepes buffer ( 132  mm nacl , 20  mm hepes , 6  mm kcl , 1  mm mgso4 , 1.2  mm k2hpo4 ) supplemented with 2  mm cacl2 and 10  mm glucose . and homogenized using gentlemacs c - tubes ( miltenyi biotec ) and the gentlemacs dissociator ( miltenyi biotec ) . afterwards , 5    10 cells were stained with anti - cd235a or control antibody as above . results of donor rbcs are expressed as percentage human rbcs of the total amount of stained rbcs . comparison between groups at the same time period or in the same organ was done using student s t test parameters of storage lesion of the human rbc products are shown in the table  1.table 1parameters of storage conditions of human rbcsrbcs stored 1  dayrbcs stored 35  daysph6.966.36lactate ( mmol / l)525potassium ( mmol / l)422glucose ( mmol / l)2413atp ( mol / g hb)432,3dpg ( mol / g hb)14.90hemolysis ( % ) 0.10.124 parameters of storage conditions of human rbcs directly following transfusion , human rbcs could be detected in the circulation of all recipients ( fig .  1 ) . of note , the percentage of human rbcs per circulating rat rbcs was higher for stored rbcs than for fresh rbcs.fig . 1percentage of human donor rbcs detected in the circulation and in organs of rat recipients , after storage for 1  day ( fresh ) or 35  days ( stored ) . f fresh , s stored percentage of human donor rbcs detected in the circulation and in organs of rat recipients , after storage for 1  day ( fresh ) or 35  days ( stored ) . f fresh , s stored in contrast , 24  h following transfusion , no donor rbcs could be detected in the circulation , regardless of storage time . donor rbcs were detected in different organs of all recipients ( fig .  1 ) . the presence of fresh donor rbcs in the liver appeared to be higher compared to stored donor rbcs . the use of human donor rbcs in a rat transfusion model resulted in clearance from cells from the circulation , associated with the presence of donor cells in all organs . a possible explanation may be the size of human rbcs , which are somewhat larger than rat rbcs ( 78 versus 6  m ) and may get stuck in the capillaries of organs . of note , 1  h following transfusion , we found that the percentage of fresh donor rbcs in the circulation was lower compared to the percentage of stored rbcs , suggesting that fresh rbcs are cleared more rapidly than stored human rbcs . in line with this , the percentage of fresh rbcs stuck in the liver following transfusion was higher compared to stored rbcs . however , this did not hold true for all organs , also not for the lungs , in which the capillaries have the smallest diameter . besides size , there may be an immunologic mechanism of clearance of human rbcs from the rat circulation . we were unable to define a precise mechanism . whether fresh rbcs may elicit a stronger immune response than stored rbcs is not known although we did not measure markers of hemolysis , visual inspection of the samples did not suggest hemolysis . this study is limited by a small sample size , in particular of the organ measurements , because we were not able to analyze organs from all recipients due to logistic reasons . although the mechanism is undefined , this study clearly shows that human rbcs get stuck in organs in a rat transfusion model . given that the standard of appropriate transfusion requires more than 75  % posttransfusion survival , the use of human rbcs in rodent recipients are not appropriate to study the efficacy of rbc transfusion . the use of human donor rbcs in a rat transfusion model is not appropriate to study the efficacy of rbc transfusion .
backgroundanimal models are valuable in transfusion research . 
 use of human red blood cells ( rbcs ) in animal models facilitates extrapolation of the impact of storage conditions to the human condition but may be hampered by the use of cross species.methodsinvestigation of clearance and posttransfusion recovery in a rat model using fresh and stored human rbcs.resultsdirectly following transfusion , human rbcs could be detected in the circulation of all recipients , with higher recovery rates for stored rbcs than for fresh rbcs . 
 after 24  h following transfusion , no donor rbcs could be detected in the circulation , but donor rbcs could be detected in all organs of all recipients.conclusionthe use of human donor rbcs in a rat transfusion model resulted in clearance from cells from the circulation . 
 donor cells were found in different organs of the recipients . 
 rat transfusion models are thus not appropriate to study the efficacy of human rbc transfusion .