Cellular Characterization of Wound Exudate as a Predictor of Wound Healing Phases

This study was designed to characterize the cell populations present in wound exudate, to examine the validity of these populations in identifying the wound phase to find possible predictors of healing, and to anticipate events in favor of a more rapid recovery. Materials and Methods. Fourteen patients who presented with postsurgical wounds with negative pressure therapy were included in the study. The authors collected samples from 51 wounds in the inflammatory phase and 29 in the proliferative phase. Results. The number of neutrophils and platelets in the exudate collected from wounds in the inflammatory phase was significantly higher (P < 0.05), while the number of lymphocytes, eosinophils, and basophils was significantly higher in exudate from wounds in the proliferative phase. The receiver operating characteristic analysis gives an area under curve of 0.815 for neutrophils (P < 0.001, CI: 0.698-0.933) and 0.760 for platelets (P < 0.001, CI: 0.647-0.873). The results showed 45.8% of neutrophils have a sensitivity of 84.4% and a specificity of 66.7% for detecting the inflammatory phase. For platelets, a level of 135.000 has a sensitivity of 71.1% and a specificity of 61.1%. The percentage of neutrophils and the platelet levels remained significant predictors of the inflammatory phase. Conclusion. The study results suggest that platelets and neutrophils in wound exudate might be useful prognostic indicators of the inflammatory or proliferative phase of wound healing.

  In everyday pathology, wounds remain a challenging clinical problem, with early and late complications presenting a frequent cause of morbidity and mortality.^1-2 Therefore, wound healing remains a challenging clinical problem, and correct efficient wound management is essential.

  Wound healing is an interactive process that involves soluble mediators, extracellular matrix components, resident cells (eg, keratinocytes, fibroblasts, endothelial cells, and nerve cells), and infiltrating leukocyte subtypes, which participate differentially in the classically defined 3 phases of wound healing: inflammation, tissue formation, and tissue remodeling.^3-7

  The inflammatory response is regarded as the first of a number of overlapping processes that constitute wound healing. In skin repair, the infiltrating leukocytes are the principal cellular components of the inflammatory response.^7-9 As such, an excessive or reduced influx or activation of infiltrating leukocytes into the damaged tissue may have profound effects on downstream cell migration, proliferation, differentiation and, ultimately, the quality of the healing response. Immediately after injury, extravasated blood constituents form a hemostatic plug. Platelets and polymorphonuclear leukocytes entrapped and aggregated in the blood clot release a wide variety of factors that amplify the aggregation response, initiate a coagulation cascade, and/or act as chemoattractants for cells involved in the inflammatory phase.^7,10,11

  The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction.¹² In angiogenesis, new blood vessels are formed by vascular endothelial cells.¹³ In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix by excreting collagen and fibronectin.¹² Concurrently, reepithelialization of the epidermis occurs, in which epithelial cells proliferate and “crawl” atop the wound bed, providing cover for the new tissue. Observing for clinical signs is the most practical way to assess the wound phase, but the extent to which these signs are present in wounds with high bioburden is unclear. The classic signs of pain, erythema, edema, heat, and purulence have traditionally been used to identify a wound in the inflammatory phase.

  The proliferative phase of wound healing can be recognized by the presence of 3 lines (white, red, pink) in the wound bed (Figure 1). The red line represents the angiogenic phase, indicating the end of the dermal layer of the skin. The white line indicates collagen production in the skin. Collagen is the main protein in human connective tissue that serves as the supporting structure of the epidermal layer. The pink line indicates the beginning of epidermal layer. These 3 signs are representatives of the proliferative stage. The problem of using these clinical signs is that it is a subjective method that can vary from one individual to another.

  In an attempt to reduce the wound burden, much effort has focused on understanding the physiology of healing and wound care with an emphasis on new therapeutic approaches and the continuing development of technologies for acute and long-term wound management.^2,14,15 Correct clinical management may positively influence the wound healing course and reduce potential complications.^16-18 This study was designed to characterize the cell populations (leukocytes and platelets) present in wound exudate throughout the healing process and to examine the validity of these populations in identifying the wound phase. In this way, the authors’ objective was to find possible predictors of healing and to anticipate events in favor of a more rapid wound recovery.

  Fourteen patients were included in the study (7 males and 7 females, median age 66.6 years, range 26-88 years). The patients presented with postsurgical wounds that were healing by secondary intention, and pressure ulcers in stage III and stage IV. Six patients presented with abdominal wounds while the remaining patients presented with pressure ulcer wounds. The patients were receiving negative pressure wound therapy (NPWT).19 Patients with metabolic, endocrine, hepatic, or renal disease, or those receiving medication known to interfere with wound healing, were excluded from the study.

  At each visit, the wound was assessed through the traditional method of direct observation by the same person, in this case, Salvador Cervero, MD, to prevent observational errors. A series of macroscopic parameters were taken from the wound to ascertain which phase of healing the wound was in: 1) the wound was classified in the inflammatory or proliferative phase according to clinical signs such as pain, erythema, edema, heat, purulent exudate, and the presence of the 3 white, red, or pink lines; 2) the quantity of exudate was categorized as being small, moderate, or abundant; and 3) the color of the exudate was categorized as serum, hematic, green, or purulent.

  The accumulated wound fluid was collected once the NPWT was ended (90 mm Hg). Wound fluid was withdrawn from the wound drain with a sterile syringe. The specimens were collected in EDTA tubes and the cellular content was analyzed on the hematology analyzer (Advia 2120i Hematology System with Autoslide, Siemens, Erlangen, Germany).

  The results were analyzed using the statistical software program (Statistical Package for Social Sciences 18.0, SPSS Inc, Chicago, IL). Quantitative data were expressed as means ± standard deviation. Qualitative parameters were expressed in percentages. For continuous variables the means of the groups were compared using the Student t test. Categorical variables were compared using the chi-squared test. Correlation studies were done using Pearson’s regression test. Receiver operating characteristic (ROC) curve and the area under curve (AUC) were calculated for each biomarker measured. Receiver operating characteristic analysis was used to determine whether the parameters had any predictive value with respect to wound healing. Results are expressed in terms of sensitivity, specificity, positive predictive value (PPV%), and negative predictive value (NPV%) according to a confidence index (CI) of 95%. Odds ratios (OR) and their 95% CI were computed by logistic regression model analysis to clarify the impact of several potentially independent prognostic factors. A P value of < 0.05 was considered statistically significant.

  The study was approved by the ethical committee of the General Hospital University of Elche (Elche, Spain), and each patient gave written informed consent.

 There were no clinical complications during the course of the study. Fluid samples from 80 wounds were collected from a total of 14 patients. The characteristics of the wound fluid collected from each patient are described in Table 1 and continued. Fifty-one sample wounds were in the inflammatory phase and 29 in the proliferative phase. The results of cell counts are presented in Table 2. The percentage of neutrophils and the number of platelets in exudate collected from wounds in the inflammatory phase were significantly higher (P < 0.05) than in exudate from wounds in the proliferative phase. Moreover, the percentage of lymphocytes, eosinophils, and basophils were significantly higher in exudate from wounds in the proliferative phase. No significant difference was observed for the percentage of monocytes.

  Univariate analyses were primarily used for the selection of variables, based on a P value < 0.05 (Table 2). The selected variables including neutrophils, lymphocytes, eosinophils, basophils, and platelets were further analyzed by multiple logistic regression analysis. Receiver operating characteristic analysis was used to determine whether the variables had any predictive value with respect to the phase of the wound. To this aim, those patients with wounds in the inflammatory stage were considered to be sick patients, whereas those with wounds in the proliferative stage were considered healthy patients. The results of ROC analysis and multiple logistic regression analysis are presented in Table 3. Only variables which present a significant curve are shown.

  The ROC analysis gives an AUC of 0.815 for neutrophils (P < 0.001, CI: 0.698-0.933) and 0.760 for platelets (P < 0.001, CI: 0.647-0.873). A percentage of neutrophils of 45.8% has a sensitivity of 84.4% and a specificity of 66.7% for detecting the inflammatory phase. For platelets, a level of 135.000 has a sensitivity of 71.1% and a specificity of 61.1%. The ROC curves for neutrophils and platelets are presented in Figure 2.

  The percentage of neutrophils (OD: 7.6 ; P < 0.001; 95% CI: 2.6-21.9) and the platelets levels (OD: 9.3; P = 0.002; 95% CI: 1.8-47.7) remained significant predictors of the inflammatory phase (Table 3).

  The assessment and choice of treatment for chronic wounds could be improved if a simple biomarker were available to predict healing. Since fluid collection from wounds is a noninvasive procedure, this method was chosen for the purposes of this study; if a component of the fluid could be identified to distinguish the phases of wounds, it could have predictive value. In this study, the authors examined the leukocytes and platelets present in the wound exudate throughout the healing process. Preliminary results show that neutrophils and platelet levels were increased in wounds in the inflammatory phase, suggesting that these cell components play a crucial role during skin repair in the adult cell organism.

  The early stage of the repair response is dominated by the inflammatory phase, and neutrophils are the predominant cell type.^8,9 The current study’s results were in agreement, and show neutrophils can help ascertain which phase of healing a wound is in. These findings demonstrate that high levels of neutrophils ( > 45.8%) indicate a wound is in the inflammatory phase and, if progress is favorable, this percentage decreases and the wound will progress on to the proliferative stage.

  Platelets also play a fundamental role in the healing of wounds.^8,10 Upon injury to the skin, a set of complex biochemical events take place in a closely orchestrated cascade to repair the damage. Within minutes post-injury, platelets aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding, which helps the process of coagulation and the closing of the wound. This has a positive effect in the scarring process.¹¹ This is why we see high levels of platelets (> 135.500) in the first inflammatory phase. When the wound is undergoing a scarring process, the number of platelets falls since the wound has closed and passed on to the proliferative phase.

  Platelets and neutrophils in wound exudate might be useful prognostic indicators of the inflammatory or proliferative phase. Elevated levels of neutrophils and platelets in wound exudates may serve as a predictor of progression to the inflammatory phase. It must be noted, however, that this investigation lacks from clinical follow-up of the full wound healing and does not provide information about which wounds are reinfected. The authors are working to fill in this gap with a longitudinal study on the overall wound healing process.

  The authors would like to thank the Foundation for Biomedical Research of General Hospital University of Elche for its financial support of this study.

The authors are from the General University Hospital of Elche, Elche, Spain.

Address correspondence to:
Natividad López, PhD
Clinical Laboratory Department
General University Hospital of Elche
Camí de l´Amazara 11
03203 Elche, Spain
sagona@terra.com

Disclosure: The authors disclose no financial or other conflicts of interest.