accidents and trauma are one of the worlds most serious , but neglected health problems and is one of the leading causes of mortality in young adults . globally , 26% of all deaths in the age group of 1544 years in 2002 were due to injury . in india , trauma is a major problem , due to a very high incidence of vehicular accidents , other accidents , injuries , crimes , and violence . infections are one of the most important causes of mortality and morbidity in traumatized patients . despite early effective treatment , such patients are susceptible to developing sepsis and multiple organ failure , which in turn are the major causes of death in these critically ill patients . monocytes have been found to play a key role in immunological response to the bacterial infections . systemic inflammation , as well as immune suppression , are thought to play a decisive role in the development of sepsis and multiple organ dysfunction syndromes . as the paradigm of sepsis pathogenesis has evolved over time , and our understanding of the different therapeutic approaches to sepsis developed , various biomarkers have been used for diagnosis of sepsis and monitoring of treatment . the diagnosis of sepsis is still a challenge till date due to the highly variable nature of its signs , symptoms , and unreliable performance of the available biomarkers . early diagnosis and stratification of the severity of sepsis will increase the possibility of appropriate and timely treatment . pro - inflammatory cytokines such as tumor necrosis factor - alpha ( tnf- ) and interleukin-6 ( il-6 ) have been implicated as principal mediators of monocytic activity and probably major mediators involved in the pathogenesis of sepsis . most of the studies on the evaluation of the role of tnf- and il-6 have been done using plasma , which depicts extracellular pooled levels . moreover , the cellular origin can not be ascertained in these extracellular assays . given the importance of sepsis diagnosis and the lacunae thereof , the present study was designed to assess the activity of monocytes with respect to production of tnf- and il-6 by flow cytometry in critically ill septic patients admitted to the intensive care unit ( icu ) of a level-1 trauma center and to correlate it with the patients outcomes . patients enrolled in the present study were those whose blood cultures ( one or more ) had grown aerobic gram - negative pathogen / s and further fulfilled the criteria for clinical diagnosis of sepsis . the presence of sepsis was confirmed when at least two of the following four clinical signs of sepsis were present : ( a ) temperature > 38.9c or < 35.5c ; ( b ) respiratory rate < 28 breaths / min or paco2 > 32 torr ; ( c ) heart rate > 100 beats / min ; ( d ) white blood cells > 15,000 or < 3500 cells / mm . five milliliter blood samples were taken from these patients for assessment of monocytic activity by flow cytometry . five milliliter of whole blood were collected from each patient and divided equally in heparinized ( bd vacutainer lithium heparin 75 usp units , bd franklin lakes nj , usa ) and plain vials from all patients on the day when their blood culture was positive ( considered as day 0 ) , and on days 2 and 5 thereafter as follow - ups to look for the patient 's outcome and correlate it with monocytic activity . serum was separated from the plain vial and stored at  20c for procalcitonin ( pct ) assay whereas the heparinized blood was processed immediately for intracellular cytokine assay . the heparinized blood was stimulated immediately as to prevent cell death over the period of time . for this , the whole blood was diluted in the ratio of 1:1 in rpmi 1640 medium ( thermo scientific , south logan , utah , usa ) , and further divided equally into two tubes . one was challenged with 1 g / ml lipopolysaccharide ( lps ) ( escherichia coli , serotype 055:b5 , sigma - aldrich , st . louis , mo , usa ) , incubated at 37c in 5% carbon dioxide ( co2 ) with 1 g / ml monensin ( bd , san jose , ca , usa ) to inhibit protein secretion for 4 h. in parallel , the other tube , taken as basal ( unstimulated ) tube was also treated similarly , but without lps to serve as a baseline comparison . after stimulation , 100 l of blood was incubated with specific monoclonal antibodies for cd45 and cd14 antigen tagged with percp cy5.5 and pe - cy7 ( bd , san jose , ca , usa ) , respectively . after incubation , red blood cells were lysed with lysis buffer ( bd , san jose , ca , usa ) . the cells were then fixed and permeabilized by fixation and permeabilization buffer ( bd , san deigo , ca , usa ) , followed by incubation on ice in dark with intracellular specific antibodies for il-6 and tnf- tagged with phycoerythrin and allophycocyanin ( bd , san jose , ca , usa ) respectively . the cells were then washed twice with wash buffer ( bd , san deigo , ca , usa ) , and the pellet was resuspended in 1% paraformaldehyde ( fischer - scientific , mumbai , india ) . samples were then analyzed using multiparametric flow cytometer ( bd facs ariaiii , usa ) . results were analyzed using bd facs diva software(bd biosciences ) and gate was set around the monocyte population , which was strongly positive for cd14 on forward scatter on dot plots . tnf- and il-6 production from monocytes also , pct levels were assessed at all time frames from serum samples by the minividas system ( biomrieux , france ) . all the patients enrolled in the study were clinically followed - up till their final hospital outcome ( discharge / death ) and their clinical and laboratory finding were recorded daily , including the microbiological culture reports of all samples received in the laboratory . detailed follow - ups of all blood culture reports of these patients were done along with daily assessment of clinical and microbiological findings . details of surgical procedure performed , antimicrobial treatment and clinical as well as autopsy reports of fatal cases to find the cause of death were similarly noted . patients enrolled in the present study were those whose blood cultures ( one or more ) had grown aerobic gram - negative pathogen / s and further fulfilled the criteria for clinical diagnosis of sepsis . the presence of sepsis was confirmed when at least two of the following four clinical signs of sepsis were present : ( a ) temperature > 38.9c or < 35.5c ; ( b ) respiratory rate < 28 breaths / min or paco2 > 32 torr ; ( c ) heart rate > 100 beats / min ; ( d ) white blood cells > 15,000 or < 3500 cells / mm . five milliliter blood samples were taken from these patients for assessment of monocytic activity by flow cytometry . five milliliter of whole blood were collected from each patient and divided equally in heparinized ( bd vacutainer lithium heparin 75 usp units , bd franklin lakes nj , usa ) and plain vials from all patients on the day when their blood culture was positive ( considered as day 0 ) , and on days 2 and 5 thereafter as follow - ups to look for the patient 's outcome and correlate it with monocytic activity . serum was separated from the plain vial and stored at  20c for procalcitonin ( pct ) assay whereas the heparinized blood was processed immediately for intracellular cytokine assay . the heparinized blood was stimulated immediately as to prevent cell death over the period of time . for this , the whole blood was diluted in the ratio of 1:1 in rpmi 1640 medium ( thermo scientific , south logan , utah , usa ) , and further divided equally into two tubes . one was challenged with 1 g / ml lipopolysaccharide ( lps ) ( escherichia coli , serotype 055:b5 , sigma - aldrich , st . louis , mo , usa ) , incubated at 37c in 5% carbon dioxide ( co2 ) with 1 g / ml monensin ( bd , san jose , ca , usa ) to inhibit protein secretion for 4 h. in parallel , the other tube , taken as basal ( unstimulated ) tube was also treated similarly , but without lps to serve as a baseline comparison . after stimulation , 100 l of blood was incubated with specific monoclonal antibodies for cd45 and cd14 antigen tagged with percp cy5.5 and pe - cy7 ( bd , san jose , ca , usa ) , respectively . after incubation , red blood cells were lysed with lysis buffer ( bd , san jose , ca , usa ) . the cells were then fixed and permeabilized by fixation and permeabilization buffer ( bd , san deigo , ca , usa ) , followed by incubation on ice in dark with intracellular specific antibodies for il-6 and tnf- tagged with phycoerythrin and allophycocyanin ( bd , san jose , ca , usa ) respectively . the cells were then washed twice with wash buffer ( bd , san deigo , ca , usa ) , and the pellet was resuspended in 1% paraformaldehyde ( fischer - scientific , mumbai , india ) . samples were then analyzed using multiparametric flow cytometer ( bd facs ariaiii , usa ) . results were analyzed using bd facs diva software(bd biosciences ) and gate was set around the monocyte population , which was strongly positive for cd14 on forward scatter on dot plots . tnf- and il-6 production from monocytes also , pct levels were assessed at all time frames from serum samples by the minividas system ( biomrieux , france ) . all the patients enrolled in the study were clinically followed - up till their final hospital outcome ( discharge / death ) and their clinical and laboratory finding were recorded daily , including the microbiological culture reports of all samples received in the laboratory . detailed follow - ups of all blood culture reports of these patients were done along with daily assessment of clinical and microbiological findings . details of surgical procedure performed , antimicrobial treatment and clinical as well as autopsy reports of fatal cases to find the cause of death were similarly noted . a total of 16 critically ill trauma patients between the age group of 18 and 65 years , who were admitted to the trauma neurosurgery and surgery icus and whose blood cultures were positive for gram - negative bacilli in the microbiology laboratory were enrolled in the study . among the enrolled patients , male ( 13 , 81% ) were more than the female ( 3 , 19% ) patients . of the 16 patients , 4 ( 25% ) had isolated abdominal trauma , whereas 4 ( 25% ) presented with both abdominal and chest trauma , 2 ( 13% ) had isolated chest trauma , and the rest ( 6 , 38% ) suffered from multiple orthopedic injuries . four patients ( 25% ) had undergone exploratory laparotomy , whereas 4 ( 25% ) other patients had to undergo tracheostomy , 2 ( 13% ) underwent screw fixation of fractures and 1 ( 6% ) each underwent splenectomy , corpectomy , and amputation respectively . of the remaining , 3 ( 19% ) had to undergo exploratory laparotomy in combination with either tracheostomy or splenectomy or gangrenous bowel resection . of these 16 patients , 12 ( 75% ) were discharged after variable lengths of hospital stay ( 1257 days ) . during their hospital stay , all of these 12 patients had one or more hospital - acquired infections . septicemia developed in all the 12 patients with acinetobacter baumannii , ( 7 [ 58% ] ) being the most commonly pathogen isolated from blood culture , followed equally by pseudomonas aeruginosa , stenotrophomonas maltophilia and candida albicans ( 2 [ 16% ] ) each and klebsiella pneumonia 1 ( 8% ) . the remaining four patients expired and the clinical and autopsy proven cause of death was cardiac arrest with septic shock . during their hospital stay , all four patients also developed septicemia and two of them had associated ventilator associated pneumonia . clinical and microbiological findings of the fatal patients 
 tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2]il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2]pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2 ] il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2 ] pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . comparison of tnf- levels in various groups of patients 
 tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2]il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3]pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2 ] il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3 ] pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . the decreased level of tumor necrosis factor - alpha in fatal patients following sepsis is compared to healthy control . levels of interleukin-6 in fatal cases were increased in first two follow - ups buts were significantly decreased in the last follow - up comparison of il-6 levels in various groups of patients a comparison between the cytokine levels of all the patient groups on different days are shown in tables 2 and 3 . a total of 16 critically ill trauma patients between the age group of 18 and 65 years , who were admitted to the trauma neurosurgery and surgery icus and whose blood cultures were positive for gram - negative bacilli in the microbiology laboratory were enrolled in the study . among the enrolled patients , male ( 13 , 81% ) were more than the female ( 3 , 19% ) patients . of the 16 patients , 4 ( 25% ) had isolated abdominal trauma , whereas 4 ( 25% ) presented with both abdominal and chest trauma , 2 ( 13% ) had isolated chest trauma , and the rest ( 6 , 38% ) suffered from multiple orthopedic injuries . four patients ( 25% ) had undergone exploratory laparotomy , whereas 4 ( 25% ) other patients had to undergo tracheostomy , 2 ( 13% ) underwent screw fixation of fractures and 1 ( 6% ) each underwent splenectomy , corpectomy , and amputation respectively . of the remaining , 3 ( 19% ) had to undergo exploratory laparotomy in combination with either tracheostomy or splenectomy or gangrenous bowel resection . of these 16 patients , 12 ( 75% ) were discharged after variable lengths of hospital stay ( 1257 days ) . during their hospital stay , all of these 12 patients had one or more hospital - acquired infections . septicemia developed in all the 12 patients with acinetobacter baumannii , ( 7 [ 58% ] ) being the most commonly pathogen isolated from blood culture , followed equally by pseudomonas aeruginosa , stenotrophomonas maltophilia and candida albicans ( 2 [ 16% ] ) each and klebsiella pneumonia 1 ( 8% ) . the remaining four patients expired and the clinical and autopsy proven cause of death was cardiac arrest with septic shock . during their hospital stay , all four patients also developed septicemia and two of them had associated ventilator associated pneumonia . tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2]il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2]pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2 ] il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2 ] pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2]il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3]pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2 ] il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3 ] pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . the decreased level of tumor necrosis factor - alpha in fatal patients following sepsis is compared to healthy control . levels of interleukin-6 in fatal cases were increased in first two follow - ups buts were significantly decreased in the last follow - up comparison of il-6 levels in various groups of patients a comparison between the cytokine levels of all the patient groups on different days are shown in tables 2 and 3 . it is widely believed that bacterial endotoxin on entering the bloodstream triggers various alterations in the immune system , resulting in the release of various pro - inflammatory and inflammatory markers which may lead to a plethora of complications . to study the kinetics of cytokines appearing in the blood of septic patients , detailed research is needed , which would help us find some novel methods of pharmacological interventions as well as new and early diagnostic markers during the initial inflammatory response . ours is the first study from india monitoring the monocytic immune response in septic trauma patients . most of the studies on cytokine production in septic patients have been done on plasma which in our view has two disadvantages . first , studies on plasma would reflect the total production of cytokines rather than their cellular origin . second , the short half - life of pro - inflammatory cytokines results in their rapid disappearance . this does not give the true picture regarding the cellular source of cytokines at a single cell level and the actual cytokine profile of posttrauma immune response . to overcome this biological drawback , we used intracellular cytokine analysis utilizing the flow cytometric approach . thus , we targeted the pro - inflammatory cytokines producing activity of monocytes by multiparametric flow cytometry , which represents a reliable method enabling highly selective immunomonitoring of specific cells in septic patients . in septic patients who succumbed to trauma , we observed that the levels of tnf- and il-6 on stimulation with lps were strongly blunted from day 1 of getting positive blood culture reports and continued to decline till further follow - ups ( 2 and 5 day ) . whereas , in those patients who survived the sepsis , after initial low levels of tnf- , there was a gradual increase in the levels of tnf- with each follow - up , as compared to the healthy controls . hence , our data suggests a reduced capability of monocytes in whole blood from trauma patients in sepsis to release sufficient amounts of pro - inflammatory cytokines in response to the endotoxins encountered . all the patients had been started on antimicrobials based on their antibiotic sensitivity reports ; however , those patients who showed continuously decreased levels of cytokines died and those whose cytokines levels increased ultimately survived the sepsis . this highlights the fact that effectively monocytic activity is compromised during sepsis , hence limiting the ability of the patient 's immune system to adequately respond to invading microorganisms . the levels of il-6 in the septic patients who survived gave variable results , hence as a sound conclusion can not be drawn . procalcitonin levels in all except two septic patients who survived were below the cut - off levels ( 0.5 ng / ml ) , hence making tnf- and il-6 better markers for initial evaluation of sepsis . hence , our study suggests that both tnf -and il-6 would make a good marker for sepsis , though further studies are needed to substantiate it . the strength of our study was that clinical follow - ups till the final outcome and autopsy - based confirmation of the cause of death . trauma patients are a fairly uniform cohort , where underlying illness is few and most patients are in the middle age group . first , the study was done for a short duration and we need to increase the sample size . for better comparison , freshly admitted trauma patient 's monocytic cytokine profile would have given a more holistic view of the immune response . this study was done on trauma victims , who at the time of admission were not incubating any infections hence all the infection were hospital - acquired , which increases the morbidity and mortality . thus , the findings of this study can be extrapolated to predicting early sepsis development and thus preventing the vulnerable young economically productive population of our country from severe complications after injury .