Extremity wounds are the most common type of modern wartime injury1-8. Modern body armor, improved trauma care systems, and advanced orthopaedic care have contributed to an unprecedented survival rate among recently wounded American service members9-13.
High-energy penetrating blast wounds are especially devastating because the zone of injury is extensive, combining bone, muscle, arterial, and nerve loss with gross bacterial contamination. Up to 75% of these injuries are colonized and/or infected with fastidious environmental bacteria when the patient arrives at the tertiary care military medical facility14,15. Differentiating among wound contamination, colonization, and infection becomes increasingly difficult as the duration of treatment increases, making the use of standard culture techniques for guiding the timing of wound closure unreliable16-23.
The timing of wound closure or flap coverage then becomes a subjective clinical decision, depending mostly on the gross appearance of the bone and soft tissues and less on routine laboratory evaluations or the patient's general condition. Yet, despite seemingly appropriate tension-free wound closure following meticulous serial débridements and antibiotic therapy, some wounds dehisce. Conversely, some wounds with a questionable appearance may possess the ability to heal and undergo unnecessary surgical débridements, adding treatment costs as well as exposing patients to additional anesthetic and surgical risks.
Serum markers have been used to diagnose infection and malnutrition or to predict wound-healing potential in elderly or chronically debilitated patients (Table I)24-37. Patients who have sustained wartime trauma, however, are typically young and healthy, and it has been hypothesized that wound-healing may depend less on chronic comorbidities and more on the degree of bacterial contamination, the adequacy of local wound arterial blood flow, and the acute nutritional status. Few serum markers have been found to accurately predict outcomes in a healthy population with acute traumatic wounds38.
Wound effluent levels of cytokines and chemokines may be less influenced by systemic stresses and more representative of the local wound environment than traditional systemic serum markers of inflammation and infection. With the recognition that the cytokine profiles differ between acute and chronic wounds, it may be possible to differentiate between wounds that will heal uneventfully and those that will become chronic and fail to heal39. Analyses of cytokines and chemokines in fluid from chronic wounds have been performed to understand the pathophysiology of wound-healing and explain the clinical successes associated with negative pressure therapy40-42. Despite a need to understand the pathophysiology of wound-healing, we are not aware of any reported analyses of fluid from acute wounds resulting from high-energy penetrating injuries. Recently, procalcitonin, an amino-acid peptide precursor to calcitonin, has emerged as an important serum indicator of infection severity and the risk of patient mortality43-46. Postoperatively, serum procalcitonin levels return to baseline sooner than do the levels of other acute-phase reactants, making it a more useful marker of infection and inflammation in the perioperative period than traditional serum markers47. Although procalcitonin levels in ascites and salivary secretions have been measured in a few isolated studies, no one has attempted to isolate and quantify procalcitonin levels in the exudate of individual wounds, to our knowledge.
We hypothesized that levels of procalcitonin and other cytokines correlate with wound-healing in severely traumatized extremities. In particular, we theorized that wound exudate levels of procalcitonin may serve as a reliable and objective predictor of individual wound-healing.
Study Methodology
The institutional review board of the National Naval Medical Center, the Naval Medical Research Center, and the Veterans Affairs Medical Center, Washington, DC, approved this study. Wounded United States service members evacuated to the National Naval Medical Center from Iraq and Afghanistan were recruited to be study participants. All servicemen and servicewomen who had sustained high-energy penetrating injuries to one or more extremities, including blast injuries and high-velocity gunshot wounds, were candidates for inclusion in this study. Exclusion criteria were diabetes, atherosclerosis, or other peripheral vascular disease; prior extremity surgery complicated by infection or arterial compromise; a recent prisoner-of-war status or a malnourished state due to another cause; an immunocompromised state; seronegative or seropositive arthropathy; active osteomyelitis; or periodontal disease. After informed consent was obtained, serum and effluent samples were collected prospectively prior to definitive wound closure or flap coverage. Variables recorded included age; sex; date, location, and mechanism of injury; nicotine use; concomitant head, thoracic, or abdominal injury; arterial injury of the affected limb, whether ligated or repaired; antibiotic treatment; and the presence and method of operative fixation. Twenty age and sex-matched healthy active-duty male military volunteers were enrolled to provide serum controls.
Effluent Sample Collection
Effluent samples were collected in the operating room, after removal of the wound vacuum-assisted-closure device (V.A.C.; KCI, San Antonio, Texas) but prior to the final wound irrigation, delayed primary closure, or flap coverage of the wound. Wound effluent was collected directly from the wound with use of a sterile 3-mL syringe. Different sites were sampled from the same wound, depending on effluent location, until the volume collected reached approximately 3 mL. All wounds had sufficient pooling of effluent to accomplish this task.
Serum Sample Collection
Peripheral venous blood was drawn from each study participant concurrently with effluent collection, just prior to wound closure or flap coverage. Serum samples were obtained from age and gender-matched controls; all study participants were male. All blood samples were collected in a Red-Top Serum BD Vacutainer (BD, Franklin Lakes, New Jersey).
Sample Processing
All samples were immediately separated with use of a centrifuge (Thermo-Electron, Waltham, Massachusetts) at 3000 rpm for ten minutes. Following centrifuge separation of the cellular debris, the supernatant was removed and was placed into individually labeled 2.0-mL microcentrifuge tubes (Lake Charles Manufacturing, Lake Charles, Louisiana). All samples were stored at -60°F (-16°C) until analysis.
Cytokine/Chemokine Analysis
Cytokine samples were analyzed with use of a Luminex 100 IS xMAP bead array platform (Millipore, Billerica, Massachusetts), in which monoclonal antibodies, with cytokine specificities, are covalently linked to uniquely fluorescent beads. After incubation with the serum or effluent sample, biotinylated monoclonal antibodies that recognize the bead-linked antibody:ligand complex are introduced, and this binding is detected by subsequent streptavidin-phycoerythrin binding. Twenty-two cytokines and chemokines including interleukins (IL)-1 through 8, 10, 12, 13, and 15; interferon (IFN)-? inducible protein-10; eotaxin; IFN-?; granulocyte macrophage colony stimulating factor; monocyte chemotactic protein-1; macrophage inflammatory protein-1a; the protein regulated on activation, normal T expressed and secreted (RANTES); and tumor necrosis factor (TNF)-a were quantitated with a Beadlyte Human 22-Plex Multi-Cytokine Detection System (Upstate/Millipore; catalog number 48-011) according to manufacturer's instructions.
Procalcitonin Analysis
Serum samples were analyzed for procalcitonin (molecular weight, 12741 Da) with use of KRYPTOR-TRACE technology (B.R.A.H.M.S., Hennigsdorf, Germany), which has a functional sensitivity of 60 pg/mL. The more viscous and proteinaceous effluent required a purification process prior to analysis and detection of amino-procalcitonin (molecular weight, 6222 Da) with use of a more sensitive enzyme-linked immunosorbent assay with an analytical sensitivity of 5 pg/mL.
Wound Closure and Follow-up
All wounds were closed at the discretion of the treating surgeon. Although wound-edge tension was not quantified, it is our opinion that a tension-free closure (when possible) is needed in this patient population in order to ensure adequate blood flow to allow healing of already traumatized wound edges. Wounds were examined once daily following wound closure or coverage until the sutures were removed. All wounds were followed clinically for six weeks. The primary clinical outcome measure was wound-healing. For the purposes of this study, failure of wound-healing was defined as a return to the operating room for treatment of dehiscence (spontaneous opening of a previously closed wound), impending dehiscence (a wound with persistent and increasing drainage following surgical closure or flap coverage or a wound with progressive marginal skin necrosis), and/or a clinically suspected infection (turbid or cloudy exudate from the incision or progressive induration, fluctuance, erythema, and calor of a soft-tissue area adjacent to the wound site). In our practice, any high-energy blast wound exhibiting dehiscence, impending dehiscence, and/or infection requires a return to the operating room for formal surgical débridement. Wounds that progressed to healing at six weeks and did not necessitate a return to the operating room were considered to be healed.
Statistical Analysis
The dependent variables of interest were (1) differences in cytokine and chemokine concentrations in serum and the wound effluent relative to serum levels in age and gender-matched controls and (2) differences in effluent analyte concentrations between wounds that healed and those that dehisced. Continuous variable means are reported with standard deviations. Statistical differences between mean continuous variables were evaluated with use of a Student t test. Associations between categorical variables were studied with the Fisher exact test or chi-square test as appropriate. Because only one wound failure was observed in each of four patients with wound dehiscence, wounds were considered independently, and correlations between wound outcome and potentially important covariates were analyzed on a per-patient basis. Differences in mean serum and effluent cytokine and chemokine levels were evaluated with use of analysis of variance to adjust for potentially important clinical factors. The limited number of wounds that dehisced (four) precluded a multivariate analysis to determine independent predictors of wound outcome. A p value of <0.05 was considered to be significant.
Fifty wounds were analyzed in twenty adult male patients. Twenty age and sex-matched healthy adult volunteers were enrolled to provide serum controls. There was no significant difference between the ages of the study subjects and the controls (23 ± 1.8 compared with 28 ± 2.29 years; p = 0.45). Follow-up was complete for all patients. Four of the fifty wounds failed to heal following closure.
Demographic Data and Risk Factors for Wound Dehiscence (Table II)
With the numbers studied, only two of the clinical factors differed between the patients in whom the wounds healed and those in whom a wound dehisced. An increased rate of wound dehiscence was observed in the patients with a concomitant closed head injury as well as those with an associated arterial injury, whether ligated or repaired, of the affected limb.
Comparison of Serum Cytokine and Chemokine Concentrations Between the Study Population and Controls (Table III)
Concentrations of serum procalcitonin, TNF-a, and IL-7 in the study group were significantly higher than those in the control group (p < 0.05). Concentrations of serum IL-4, IL-13, granulocyte macrophage colony stimulating factor, and IL-12p40 in the study group were significantly lower than those concentrations in the control group (p < 0.05).
Cytokine and Chemokine Concentrations in the Wound Effluent and Serum of the Study Population (Table IV)
The effluent concentrations of several cytokines were found to be markedly elevated compared with the serum levels in the study group. These cytokines included monocyte chemotactic protein-1, IL-3, IL-5, IL-6, IL-8, IL-13, IL-15, TNF-a, macrophage inflammatory protein-1a, IL-1a, IFN-?, and IL-12p40 (p < 0.05). Effluent procalcitonin levels were lower than serum procalcitonin levels (p < 0.001).
Markers Associated with Wound Dehiscence (Table V)
The most significant associations between wound dehiscence and the chemokine and cytokine levels were derived from the effluent analysis. Procalcitonin (p = 0.01), RANTES protein (p = 0.002), and IL-13 (p = 0.03) levels differed significantly between the wounds with dehiscence and those that healed uneventfully. No wound with an effluent procalcitonin concentration of <220 pg/mL, an IL-13 concentration of >12 pg/mL, or a RANTES protein concentration of >1000 pg/mL failed to heal.
The use of traditional serum markers to assist surgical decision-making regarding individual extremity wounds in patients who have sustained traumatic injuries is limited by several coexisting factors. First, the insult associated with trauma raises metabolic demand and primes the inflammatory cascade, elevating systemic levels of white blood cells and proinflammatory cytokines48,49. Second, trauma patients often undergo several operations within a relatively short period of time. Combined with the systemic effects of the initial trauma, these multiple treatment interventions universally increase the levels of the most commonly referenced acute-phase reactants such as IL-6, C-reactive protein, and the erythrocyte sedimentation rate. Third, the presence of local wound or systemic infection increases systemic acute-phase reactants and further upregulates proinflammatory cytokine production. As a result, nonspecific serum markers do not possess sufficient positive and negative predictive value to be clinically relevant in terms of the appropriate timing of extremity wound closure.
Because cytokines and chemokines are produced locally, wound effluent levels are most representative of the local wound environment and are less susceptible to the influences of systemic factors. Animal models have been used to correlate white blood cell counts and total serum protein, lactate, and albumin levels with the degree of wound and/or fracture-healing50-52. Wound effluent analysis has been used to characterize the nature of chronic, but not acute, wounds in humans53-57. Unfortunately, studies involving such analyses have provided little insight into the dynamic conditions of infected traumatic wounds. To our knowledge, the literature contains no reports on wound effluent sampling in humans for assessment of acute extremity injuries.
In the current study, arterial injury of the affected limb and closed head injury were associated with wound dehiscence. It is possible that these injuries are associated with higher-energy trauma that results in a larger, more devastating zone of injury. Also, an arterial injury, whether ligated or repaired, disrupts normal blood flow to the limb and may increase the amount of dysvascular tissue within the zone of injury. However, the limited number of wound failures in the current study precludes definitive statistical comparisons of the levels of procalcitonin and the other markers in patients with and without vascular or cerebral trauma. Defining the clinical importance of these two factors awaits larger studies with greater numbers of wound failures in order to assess the independent effect of these injuries on wound outcome.
The procalcitonin concentration was elevated in both the serum and the wound effluent of patients in whom the wound dehisced (p < 0.05). Normally, only neuroendocrine cells (e.g., thyroid and lung cells) produce calcitonin; however, in the presence of infection, procalcitonin is produced by several tissues, including skin, thyroid, fat, liver, lung, kidney, brain, and heart, and by neutrophils. The presence of extracirculatory procalcitonin in ascitic and salivary fluid has been mentioned in a few reports58,59. Interestingly, preliminary data suggest that extracirculatory (effluent) procalcitonin could reflect local wound conditions in response to an isolated infection60, presumably as a result of the ubiquitous cellular mRNA expression, which has been demonstrated in experimental models of sepsis60,61. Alternatively, since procalcitonin is considered a "late" marker (and possibly a mediator) of severe systemic infections with a slow rate of degradation62,63, the concentration gradient of procalcitonin in the serum (mean, 114.2 pg/mL) relative to that in the effluent (mean, 18.0 pg/mL) in wounds that healed uneventfully may reflect a small amount of excess procalcitonin not trapped by protein binding64. As the serum levels of procalcitonin observed in this study are considered to be below levels associated with severe systemic infection (~2000 pg/mL), the narrowed gradient of the serum level compared with the effluent level in patients with wound dehiscence (mean, 560.8 pg/mL compared with 169.3 pg/mL) suggests that the elevated procalcitonin in the effluent stems at least in part from a local source.
Although the role of procalcitonin as a marker for infection has been known for some time, its usefulness was limited to predicting severe infection because commonly available assays were of limited sensitivity, with a detection threshold of 300 to 500 pg/mL65. The assay used to analyze the wound effluent in this study is a novel and highly sensitive method to detect amino-procalcitonin at an analytical threshold of 5 pg/mL. Sample processing methods were adjusted to allow the determination of both effluent and serum procalcitonin concentration. These technique modifications are well suited for determining levels of serum and exudate procalcitonin that correlate with lesser degrees of systemic and local infection and inflammation.
Both IL-13 and RANTES protein levels were suppressed in wounds that dehisced in the current study. IL-13 is normally expressed by T-helper cells that downregulate macrophage activity, induce differentiation of monocytes, and induce proliferation, differentiation, and isotype switching in B-cells. IL-13 inhibits macrophage activity by decreasing the expression of inflammatory cytokines and chemokines, including TNF-a, IL-1a, and certain matrix metalloproteinases, thereby suppressing the production of nitric oxide54,58. Low levels of IL-13 are associated with macrophage activation and with the subsequent production of inducible nitric oxide synthetase by inflammatory cytokines and chemokines. High levels of serum TNF-a in our patient population signified a systemic inflammatory state. High levels of nitric oxide strongly suppress RANTES protein production despite elevated TNF-a concentrations, which normally would serve to upregulate RANTES protein production66. Serum levels of IL-10, the stereotypical anti-inflammatory cytokine, did not significantly differ between the study subjects and the controls.
It has been hypothesized that wounds progress from the acute to the chronic state after a protracted inflammatory phase. Suppression of IL-13 and subsequently RANTES protein levels by means of nitric oxide production in the wound effluent supports the hypothesis that wounds that dehisce demonstrate characteristics consistent with a chronic wound or inflammatory state. It is important to note that the concentrations of most cytokines differed between the serum and the wound exudate, suggesting at least partial local production of these cytokines (Table V). This finding further supports the belief that direct measurement of effluent concentrations is an important adjunct to serum analysis.
This pilot study was limited not only by the small number of wound failures (four), but also by certain characteristics unique to our study population. Many patients in this study had sustained multiple wounds involving multiple organ systems. This may have had a confounding effect on our attempt to correlate serum cytokine and chemokine concentrations with wound dehiscence, although no patient in this series had clinical evidence of ongoing chest or abdominal infection and no more than one wound failure was observed in any patient. Given that a large number of cytokines and chemokines were analyzed, some observations may be statistically, but not clinically, significant. Considering the significant p values observed when procalcitonin (p = 0.01) and RANTES protein (p = 0.002) levels were correlated with wound dehiscence, however, these associations appear important. Unfortunately, the large standard deviations observed in the analyses of IL-13 and RANTES protein levels preclude absolute interpretation of those particular results.
Validation of these findings in future controlled clinical trials could have a profound influence on the treatment of traumatic and infected wounds of the extremity. The orthopaedic community currently lacks an objective means of rapidly determining the degree of bacterial inflammation or bioburden of a given wound in order to determine the appropriate timing of wound closure. In this pilot study, 8% of the wounds failed and required an unplanned return to the operating room. No wound with an effluent procalcitonin concentration of <220 pg/mL, an effluent IL-13 concentration of >12 pg/mL, or an effluent RANTES protein concentration of >1000 pg/mL failed to heal. It is probable that some of the wounds that healed uneventfully would have been closed successfully earlier in the débridement process had the levels of procalcitonin, IL-13, and RANTES protein been below these identified thresholds. Considering the escalating cost of health care, particularly for the treatment of infected and traumatic wounds, the implications of a rapid and reliable marker for predicting wound failure in terms of minimizing patient morbidity and treatment costs are substantial.
To our knowledge, this is the first study to detect the presence and quantify the concentration of procalcitonin and inflammatory cytokines in the effluent of acute traumatic extremity wounds. Effluent cytokine and chemokine analysis may be an objective means of determining the likelihood of wound-healing and the timing of traumatic wound closure. 