cd64 is a high - affinity and 
 restricted isotype - specificity fcri receptor expressed on macrophages , 
 monocytes , neutrophils , and eosinophils [ 1 , 2 ] . there are several reports 
 regarding its potential utility for the diagnostic assessment of sepsis or 
 infection in adults [ 38 ] and neonates [ 912 ] , but only a few in children [ 13 , 14 ] . in an adult study , a higher intensity of cd64 expression has been found on 
 neutrophils from patients with systemic inflammatory response syndrome ( sirs ) 
 and sepsis than on neutrophils from patients with sirs only . high cd64 
 expression on monocytes accompanying high cd64 expression on neutrophils has 
 been reported in some adult studies as well [ 5 , 15 , 16 ] . cd163 is a 
 monocyte / macrophage - associated antigen which has recently been identified as a 
 haemoglobin scavenger receptor . apart from clearance of haemoglobin , it 
 has also anti - inflammatory properties and an immunoregulatory role . it has 
 been found that cd163 expression on a monocyte surface is inversely related to 
 the concentration of its soluble form ( scd163 ) in randomly selected patients 
 . during human experimental endotoxinemia , a rapid rise in plasma scd163 
 has been observed together with reduced surface cd163 expression on isolated 
 monocytes following lipopolysaccharide ( lps ) administration , suggesting that 
 lps induces shedding of cd163 from the surface of isolated monocytes . twenty - four hours after lps administration in humans , cd163 surface expression 
 is consistently increased over the baseline expression . soluble cd163 has 
 been found to be elevated in sepsis in adults [ 2123 ] , whereas surface 
 expression of membrane cd163 ( mcd163 ) in sepsis has not yet been evaluated . the first aim of the study was to evaluate the expression 
 of cd64 and cd163 on neutrophils and monocytes in critically ill neonates and 
 children with sirs and sepsis and to find out whether it can discriminate 
 between infectious and noninfectious sirs . sirs is most commonly caused by 
 infection , but other conditions such as low - cardiac output syndrome , 
 respiratory distress , and haemorrhage can trigger it in neonates and children . 
 it has been found that as many as 82% of children admitted to a paediatric 
 intensive care unit have two or more signs of sirs . the second aim of the study was to compare the 
 diagnostic accuracy of cd64 mean fluorescence intensity ( mfi ) , cd163 mfi , cd64 
 index of linearised mfi to standardised beads , and cd163 index of linearised 
 mfi to standardized beads measured on neutrophils and monocytes . with recently 
 available software for data analysis and index calculation , sources of 
 technical errors and subjectivity , which are present in manual methods , are 
 reduced and a comparison of results between different laboratories is possible 
 . this prospective observational study 
 was conducted in the level iii multidisciplinary neonatal and paediatric 
 intensive care unit between january 2006 and september 2006 . fifty - six 
 consecutive patients with sirs and clinically suspected infections were 
 eligible for enrolment . sirs was defined according to the international pediatric 
 sepsis consensus conference definitions . clinically suspected infection 
 was defined as an explicit statement by the physician in the records , 
 indicating the suspicion of an infection , combined with the initiation of 
 diagnostic workup to rule out infection and the prescription of empirical 
 antibiotic therapy . patients were not included if they had received antibiotic 
 therapy for more than 24 hours prior to admission , if they had undergone 
 surgery in the previous week , or if they had a proven viral infection . the 
 diagnosis of sepsis was confirmed by positive blood cultures or tracheal 
 aspirates when chest radiographs showed signs of pneumonia . there were no other 
 cultures ( urine , cerebrospinal , or a puncture of normally sterile body fluid ) 
 positive in our patients . the diagnosis of clinical sepsis was established in 
 patients with negative cultures , but with a strong suspicion of sepsis , who 
 received a full course of antibiotic therapy . the group without sepsis included 
 patients with a suspected infection in whom the subsequent clinical course , 
 laboratory data , and microbiological tests excluded infection , and in whom 
 antibiotic therapy was discontinued after a few days . patients were classified 
 into two groups : sirs with sepsis and sirs without sepsis . the classification 
 was carried out by the attending physician unaware of the results of flow 
 cytometry . we analyzed data separately for two age groups : neonates aged less 
 than 28 days , and children older than 28 days , and for gram - positive and gram - negative 
 sepsis . pathogens isolated 
 from blood cultures were staphylococcus epidermidis(n = 3 ) , streptococcus 
 agalactiae(n = 2 ) , neisseria meningitidis(n = 1 ) , streptococcus 
 pneumoniae(n = 1 ) and streptococcus mitis(n = 1 ) , while pathogens 
 isolated from tracheal aspirates were haemophilus influenzae(n = 5 ) , streptococcus 
 pneumoniae ( n = 3 ) , moraxella catarrhalis ( n = 2 ) , escherichia 
 coli ( n = 1 ) , staphylococcus aureus ( n = 1 ) , and pseudomonas 
 aeruginosa ( n = 1 ) . the two groups were similar regarding gender (  test , p = 0.288 ) , pediatric risk of mortality score ( prism ) iii score ( anova , p = 0.835 ) , age ( anova , p = 0.212 
 and p = 0.278 for neonates and children , resp . ) , gestational age 
 ( anova , p = 0.839 ) and birth weight ( anova , p = 0.720 ) . the study was approved by the 
 national medical ethics committee of the ministry of health , republic of slovenia , 
 and written consent was obtained from parents before blood sampling . blood 
 samples ( 0.5 ml ) were obtained at the time of suspected sepsis ( day 0 ) and 24 
 hours later ( day 1 ) together with samples for routine laboratory tests . whole 
 blood edta - anticoagulated samples were immediately transported to the flow 
 cytometry laboratory during working hours or stored refrigerated ( 4c ) during 
 the night or at weekends ( up to 36 hours ) . expressions of cd64 and cd163 on 
 neutrophils , monocytes , and lymphocytes were measured by quantitative flow 
 cytometry with a facscalibur flow cytometer ( becton dickinson , ny , usa ) using the leuko64 assay ( trillium 
 diagnostics , llc , me , usa ) . the assay is for research use only and is composed of three antibodies with 
 specificities to cd64 ( clones 22 and 32.2 , both fluorescein isothiocyanate 
 ( fitc ) conjugated ) and to cd163 ( clone mac2 - 148 , phycoeritrin ( pe ) conjugated ) , 
 and a fluorescence bead suspension with three fluorescence signals ( green 
 fluorescence due to fitc , orange fluorescence similar to pe , and red 
 fluorescence of starfire red ) for unique identification of beads , and used for 
 instrument calibration and standardization of leukocyte cd64 and cd163 
 expression in human blood . briefly , 50 l of whole blood , or 
 diluted whole blood to adjust leukocyte concentration to less than 25  10/l , was incubated for 15 minutes in the dark at room temperature with a 
 mixture of murine monoclonal antibodies followed by red cell lysis with an 
 ammonium - chloride - based red cell lysis solution ( trillium lyse ) . fluorescence 
 beads were then added and flow cytometer analysis was performed on a minimum of 
 50 000 leukocytes . data analysis for fluorescence intensity was performed by 
 cellquest software ( becton dickinson , 
  calif , usa ) 
 ( table 2 ) . mfi was measured as a linearized value of log scale on lymphocytes ( red , 
 negative control , measuring cd64 expression ) , monocytes ( green , positive 
 control , measuring cd64 , and cd163 expression ) , neutrophils ( blue , measuring 
 cd64 expression ) , and beads ( aqua blue , measuring fitc , and pe expression ) ( figure 1 ) . index calculation was performed by leuko64 quanticalc software ( trillium 
 diagnostics , me , usa ) . index measurements were derived by the ratio of linearized mfi of the cell 
 population to the fitc signal from the beads . an internal negative control of 
 the assay was provided by the automated measurement of the lymphocyte cd64 
 index , which had to be less than 1.0 , and an internal positive control of the 
 assay was provided by automated measurement of the monocyte cd64 index , which 
 had to be more than 3.0 . flow cytometry was performed up to 36 hours after blood sampling . before the beginning 
 of the study the influence of delayed sample analysis was done and no 
 significant difference in levels of cd64 and cd163 expression was detected in 
 the first 36 hours after blood sampling . isotype - control antibodies were 
 routinely used in each experiment to detect nonspecific staining ; however the 
 calculation of cd64 and cd163 mfi was done without substracting isotype - control 
 mfi   in order to accurately compare the 
 ratio ( index ) of linearized mfi to mfi alone . comparison between 
 groups was made using the unpaired mann - whitney test and analysis of variance 
 ( anova ) . receiver - operating characteristic ( roc ) curves were drawn to define the optimal 
 sensitivity , specificity , cutoff value , and diagnostic accuracy , determined by 
 the area under the roc curve ( auc ) of the studied surface antigens [ 27 , 28 ] . the cutoff values at which the greatest sum of sensitivity and specificity was 
 obtained were determined by the statistical program . hanely and mcneil 's 
 comparison of diagnostic accuracies was performed . the cd64 
 score point ( combination of two variables : cd64 mfi on neutrophils and 
 monocytes or index cd64 for neutrophils and monocytes ) was calculated for cd64 
 mfi and cd64 index , separately as described elsewhere . when cd64 fluorescence intensity or cd64 index on both neutrophils and 
 monocytes was under the cutoff level , the score point of 0 was assigned . the 
 score point of 1 was assigned when the marker on one type of cells was under 
 and marker on the other type of cells was over the cutoff level and the score 
 point of 2 was assigned when markers on both types of cells were over the 
 cutoff levels . the differences were considered to be statistically 
 significant at the level of p < .05 . the statistical analysis was 
 performed using medcalc for windows , version 5.0 ( medcalc software , mariakerke , 
 belgium ) and statistical package for the social sciences for windows , version 
 12.0 ( spss inc . cd64 mfis , cd163 mfis , and cd64 
 indexes for both neutrophils and monocytes were significantly higher in 
 patients with sirs with sepsis compared with patients with noninfectious sirs , 
 while cd163 indexes showed statistical differences only on neutrophils on day 0 
 ( table 2 ) . statistically significant increase ( p < .05 ) from day 0 to 
 day 1 was found only for cd163 mfi for neutrophils in children with sirs with 
 sepsis and for cd163 mfi and index for monocytes in children with noninfectious 
 sirs . medians of cd64 index for neutrophils were higher in children with sepsis 
 than in neonates with sepsis , although differences were not significant ( p > .05 ) at the time of suspected sepsis and 24 hours later ( figure 2 ) . we did 
 not find any statistically significant differences for cd64 and cd163 indexes , 
 and cd64 and cd163 mfis for neutrophils or monocytes , between gram - positive and 
 gram - negative sepsis ( p > .05 for all comparisons , data not shown ) . optimum diagnostic cutoff levels , aucs , sensitivity and specificity of cd64 and 
 cd163 indexes , cd64 and cd163 mfis for neutrophils and monocytes , and cd64 
 score points , for sirs with sepsis at the time of suspected sepsis and 24 hours 
 later are presented in table 3 . setting sensitivity at more than 95% in roc 
 analysis of cd64 index for neutrophils displayed cutoff point 1.15 on day 0 
 ( sensitivity 96.6% and specificity 40.7% ) and 1.71 ( sensitivity 100% and 
 specificity 75.0% ) on day 1 . there were no significant differences between 
 diagnostic accuracies of mfi and the corresponding index for either parameter , cd64 and cd163 , on neutrophils and monocytes at the time of 
 suspected sepsis and 24 hours later ( p > .05 for all comparisons ) , 
 except for diagnostic accuracy of cd163 mfi for monocytes , which was 
 significantly higher at 24 hours than diagnostic accuracy of the cd163 index 
 for monocytes ( p = .003 ) . the roc curves of different indexes at the 
 time of suspected sepsis and 24 hours later are presented in figure 3 . diagnostic accuracies of cd64 indexes for neutrophils and monocytes at the time 
 of suspected sepsis were both significantly higher compared with diagnostic 
 accuracies of cd163 index for neutrophils ( p = .000 and p = .023 , 
 resp . ) and of cd163 index for monocytes ( p = .028 and p = .050 , resp . ) . diagnostic accuracy of cd64 index for neutrophils at 24 
 hours was significantly higher compared with diagnostic accuracies of cd64 
 index for monocytes ( p = .009 ) , cd163 index for neutrophils ( p = .000 ) , and cd163 index for monocytes ( p = .000 ) . the combinations of two 
 variables : cd64 mfi for neutrophils with cd64 mfi for monocytes or cd64 index 
 for neutrophils with cd64 index for monocytes ( cd64 score point ) insignificantly 
 increased diagnostic accuracy of cd64 mfi ( p = .559 ) or cd64 index ( p = .534 ) for neutrophils on day 0 , while there was an insignificant fall in 
 diagnostic accuracy ( p = .494 and p = .128 , resp . ) on day 1 
 ( table 3 ) . to our knowledge , this is the first study which 
 has evaluated expression of leukocyte antigens cd64 and cd163 in critically ill 
 neonates and children with sirs with sepsis . in this study , we have shown that 
 expression of cd64 and cd163 on neutrophils and monocytes is elevated in 
 patients with sepsis compared with patients with noninfectious sirs . we have 
 also shown that diagnostic accuracy of computer - calculated indexes for cd64 on 
 neutrophils and monocytes and cd163 on neutrophils based on the ratio ( index ) 
 of linearized mfi to the fluorescence signal of standardised beads , is equal to the diagnostic 
 accuracy of manually determined mfi , 
 if proceeded by the same flow cytometer . our results therefore suggest that the 
 cost - effectiveness of tests , based on multiple antibodies ( cd64 and cd163 ) and 
 standardized beads should be reevaluated . cd64 surface upregulation is induced 
 by granulocyte colony - stimulating factor ( g - csf ) and interferon- ( inf- ) 
 within 46 hours of stimulation . our study confirmed the results from 
 studies of the adult population that the level of cd64 expression is 
 significantly higher in patients with sirs with sepsis compared with patients 
 with noninfectious sirs . in our study , the expression of cd64 on 
 neutrophils was significantly higher in critically ill neonates and children 
 with sirs with sepsis compared with those with noninfectious sirs . so far , cd64 
 expression on leukocytes has been studied mostly in the neonatal population . these studies showed that cd64 expression on neutrophils is upregulated in 
 early - neonatal infection [ 9 , 10 ] , late - neonatal infection [ 11 , 12 ] , and in 
 preterm neonates with infection [ 11 , 13 ] . some authors have even suggested that , 
 because of its high sensitivity , measurement of neutrophil cd64 expression may 
 allow clinicians to discontinue antimicrobial treatment if negative within 24 
 hours of suspected infection , without waiting for the definitive 
 microbiological results . the results of our research are in accordance with 
 the results of this other study   only to some extent . the aucs in both 
 studies were similar , but in the other study , higher sensitivity was 
 achieved , probably because the cutoff values were determined in advance to 
 enable better sensitivity with the aim to identify all infected cases . due to 
 the different method of flow cytometry used for the measurement of cd64 
 expression in the other study , the cutoff values can not be compared . in our study the cutoff values were determined 
 by the statistical program to obtain the greatest sum of sensitivity and 
 specificity for comparisons of different markers . in order to increase the 
 sensitivity we determined the cutoff values at which the sensitivity was 96.6% 
 and 100% , respectively , on day 0 and 1 . the specificity was then slightly lower 
 on day 1 than that in the previously mentioned study . three cases of sepsis 
 caused by coagulase - negative staphylococci could have lowered the sensitivity 
 of cd64 expression on neutrophils in our study , whereas in the other study 
 there were no such cases . 
 in children , there are only two reports of 14 infants and children , and 8 
 children , respectively , hospitalized because of different bacterial infections 
 ( mostly pneumonias and pyelonephritides ) in whom cd64 expression on neutrophils 
 was significantly higher compared with noninfected controls [ 13 , 14 ] . so far , 
 cd64 expression has not been studied in septic critically ill children . in 
 adult patients , higher expression of cd64 on neutrophils was found in gram - negative 
 sepsis compared with gram - positive sepsis . in the recent study in neonates 
 , as in our study , this difference was not confirmed . the cause for this 
 difference between adults and neonates could be a less expressed neutrophil 
 response to infection with gram - negative bacteria in neonates . indeed , we found 
 lower cd64 index in neonates with sepsis than in children , although the 
 difference was not statistically significant . a novel marker of infection , the 
 cd64 score point , which incorporates the quantitative analysis of cd64 
 expression on both neutrophils and monocytes has 
 recently been 
 introduced as a marker in adults which could distinguish between infection and 
 healthy states . in our study , the cd64 score point was able to discriminate 
 between sirs with sepsis and noninfectious sirs as well , although its 
 diagnostic accuracy was not higher than that of cd64 expression on neutrophils 
 themselves . to our knowledge , cd64 expression on 
 monocytes in neonates and children has not previously been studied . there are 
 some reports of elevated cd64 expression on monocytes in adult patients with 
 sepsis [ 5 , 15 , 16 ] . in our study , the results of cd64 on monocytes and 
 neutrophils were similar at the time of suspected sepsis , but 24 hours later 
 the diagnostic accuracy of cd64 on monocytes was significantly lower than on 
 neutrophils . this data could indicate that activation in monocytes is faster or 
 more rapidly completed than in neutrophils . the increased expression of cd163 is 
 a part of the maturation of a monocyte to a phagocytic macrophage . the 
 expression of cd163 is upregulated by interleukin-6 and glucocorticoids 
 together with interleukin-10 , and downregulated by lps and interferon- . although the absolute value of median expression of cd163 was much lower on 
 neutrophils than on monocytes in our patients with sepsis , the expression of 
 cd163 on both types of cells could differentiate between patients with sirs 
 with sepsis and noninfectious sirs in our study . as dynamics of fluorescence intensity for cd64 and cd163 on neutrophils was diagnostically very significant , 
 it is highly unlikely that a nonspecific staining would have been the cause . also , 
 the representative facs diagrams made possible quite a sharp distinction 
 between cell populations without causing serious problems in cell gating . our 
 results therefore indicate that , like monocytes , neutrophils are able to some 
 extent to express cd163 . only one author found increased bone marrow infiltration 
 with cd163-positive macrophages in postmortem analysis of samples from patients 
 who died from severe sepsis or septic shock compared with controls . in 
 contrast , high concentrations of soluble cd163 ( scd163 ) have been described in 
 adult septic patients in several studies [ 3 , 21 ] . it has been shown 
 experimentally with cultured monocytes that cd163 can be shed from the cell 
 membrane after lps stimulus , cross - linking of the fc receptor for 
 immunoglobulin g   or oxidative stress . the shedding of cd163 from 
 monocyte membrane correlates with decreased expression of membrane cd163 on 
 cultured monocytes [ 20 , 34 ] . the decreased expression of membrane cd163 is 
 probably partly due to shedding of cd163   and partly due to decreased cd163 
 mrna synthesis [ 34 , 36 ] . in healthy adult volunteers , cd163 surface expression the precise time point 
 of increased expression is not yet exactly known , but it was estimated to be 
 024 hours after lps stimulation in adult volunteers   or even later in 
 cultured monocytes . it was shown that stimulation with lps induced 
 short - term suppression of cd163 mrna expression in cultured monocytes , while 
 long - term cultures of monocytes treated with lps showed intereleukin-10 
 dependent recovery of surface cd163 expression . in our clinical 
 study , we did not observe the transitional fall of membrane cd163 expression on 
 monocytes . the expression of cd163 on both monocytes and neutrophils was higher 
 in septic patients than in patients with noninfectious sirs both at the time of 
 suspected sepsis and 24 hours later . the reason may be that clinical signs are 
 observed with significant delay after endotoxinemia . most clinical studies in the past 
 have used manually determined fluorescence intensity to describe cd64 
 expression on neutrophils . this method , however , is subject to day - to - day 
 operational and instrumental fluctuations and can not be used for comparison 
 between different laboratories . the newly developed method for 
 computer - calculated indexes uses calibration beads which serve as an internal 
 fluorescence intensity standard and , together with fully automated software for 
 index calculation , which removes the subjectivity in data analysis by the end 
 users , provide a novel approach in minimizing lot - to - lot variation , and in 
 removing the subjectivity due to different range of experience and skills of 
 operators . in our study , comparison of manually determined fluorescence 
 intensity and computer - calculated index revealed that diagnostic accuracy of 
 both parameters was equal for cd64 on neutrophils and monocytes and cd163 on 
 neutrophils , but not for cd163 on monocytes . our explanation is that automated 
 index calculation was originally developed for evaluation of cd64 index on 
 neutrophils in different clinical conditions   and the method is probably 
 not universally transmittable to evaluate all other antigens on different cell 
 types for diagnostic purposes . the calculation of cd64 index for neutrophils , 
 which achieved the highest diagnostic accuracy in our study , was optimal and we 
 suggest that this parameter should be used in future evaluations . we found no 
 studies , except for a poster presentation at sepsis 2007 in paris 
  , which have used index calculation 
 for evaluation of cd64 expression on neutrophils . firstly , the definition of clinical sepsis with culture - negative 
 patients , particularly critically ill neonates , is still a matter of 
 discussion . . a greater 
 number of neonates and children is needed for a more precise evaluation of diagnostic 
 accuracies of selected infection markers ( cd64 and cd163 index for monocytes 
 and neutrophils ) and for comparison with routinely used infection markers in 
 the future . the blood samples were processed within 36 hours in our study . for 
 clinical use , the method should be available every day , particularly because 
 some authors suggest processing of blood specimens for analysis of cell surface 
 markers immediately after obtaining them to avoid cell apoptosis . however , 
 other authors claim that measurements for neutrophil cd64 assay are stable for 
 up to 30 hours at room temperature and up to 72 hours refrigerated . as we 
 already mentioned , no significant differences in levels of cd64 and cd163 
 expressions were obtained in the first 36 hours after blood sampling in our 
 study . the data from the present study show 
 that cd64 and cd163 expressions on neutrophils and monocytes are elevated in 
 critically ill neonates and children with sirs with sepsis . diagnostic 
 accuracies of computer - calculated indexes for 
 cd64 on neutrophils and monocytes and cd163 on neutrophils based on the ratio ( index ) 
 of linearised mfi to the fluorescence signal of standardized beads are equal to 
 diagnostic accuracy of manually determined mfi , if processed by the same flow 
 cytometer . therefore , cd64 mfi , cd163 mfi , cd64 
 indexes for neutrophils and monocytes and cd163 index for neutrophils can all be 
 used for discrimination of sirs and sepsis in critically ill neonates and 
 children .
objective .   to evaluate the expression of cd64 and cd163   on neutrophils and monocytes in sirs with / without sepsis and to compare the diagnostic accuracy of cd64 and cd163 molecules expression determined as ( 1 ) mean fluorescence intensities ( mfi ) of cd64 and cd163 ; and ( 2 ) the ratio ( index ) of linearized mfi to the fluorescence signal of standardized beads . 
 patients and methods . 
 fifty - six critically ill neonates and children with systemic inflammatory response syndrome ( sirs ) and suspected sepsis , classified into two groups : sirs with sepsis ( n = 29 ) and sirs without sepsis ( n = 27 ) . results . 
 cd64 and cd163 mfi measured on neutrophils and monocytes were elevated in patients with sirs with sepsis . 
 diagnostic accuracy of indexes was equal to diagnostic accuracy of mfi for cd64 on neutrophils ( 0.833 versus 0.854 for day 0 and 0.975 versus 0.983 for day 1 ) and monocytes ( 0.811 versus 0.865 for day 0 and 0.825 versus 0.858 for day 1 ) , and cd163 on neutrophils ( 0.595 versus 0.655 for day 0 and 0.677 versus 0.750 for day 1 ) , but not for cd163 on monocytes . 
 conclusion . 
 cd64 mfi , cd163 mfi , cd64 indexes for neutrophils and monocytes , and cd163 index for neutrophils can all be used for discrimination of sirs and sepsis in critically ill neonates and children . 
 cd64 
 index for neutrophils , however , is superior to all other markers .