Histologic Comparison of Pressure and Autoimmune Wounds

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1 Histologic Comparison of Pressure and Autoimmune Wounds Item Type Thesis Authors Nanda, Alisha Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the College of Medicine - Phoenix, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 13/05/ :57:32 Link to Item

2 Histologic Comparison of Pressure and Autoimmune Wounds A thesis submitted to the University of Arizona College of Medicine Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine Alisha Nanda Class of 2017 Mentor: Marc Gottlieb, MD

3 Abstract Introduction: The cell make up and architecture of a wound is generally not explored before treatment is started. This pilot study will potentially be able to differentiate the histologic makeup of different wound etiologies and therefore start to elucidate a more targeted therapy to treat a wound with the hypothesis that etiology of wound is associated with a set of histologic characteristics. Methods: 12 samples of pressure wounds and 13 samples of autoimmune connective tissue wounds were examined and characterized under microscopy. Types of cells, necrosis, granulation, and inflammation, among other characteristics were studied. Results: The autoimmune wounds displayed a statistically significant increase in lymphocytes, chronic inflammation, and fibrosis than in the pressure wounds. Discussion: There are apparent differences in histology and morphology of wounds of different etiologies, as hypothesized. This suggests the possibility of requiring specific treatments for the varying wound types. 2

4 Table of Contents Introduction... 1 Materials and Methods... 3 Results... 5 Discussion Conclusions References

5 List of Figures and Tables Table 1: Characteristics of pressure and autoimmune wounds Table 2: Statistical significance of findings using Fisher s Exact test. Figure 1: Example images of (A) pressure, (B) autoimmune connective tissue, and (C) normal wounds 4

6 Introduction Background for the Research Question The cell make up and architecture of a wound is generally not explored before treatment is started. All wounds are generally treated similarly. This pilot study will potentially be able to differentiate the histologic makeup of three different wound etiologies and therefore start to elucidate a more targeted therapy to treat a wound. Wounds need a diagnosis specific treatment, but it is not always easy to make a diagnosis. Histology can help with a diagnosis and therefore guide treatment. Some differences are already seen between these wound types, but not quantified or described well. Wounds can be differentiated based on if they are caused by trauma, burns, chronic and pathologic (CAP) by extrinsic causes or chronic and pathologic by intrinsic disease, or acute [1]. Treatment options vary from topical care, supportive and natural closure, surgical modalities, and non operative pharmacophysiologic modalities. Wound assessment is done by clinical, visual examination: checking for dead, gray connective tissue, necrotic tissue, yellow slough, red granulation tissue, exudate, colonization [1]. The cause of the wound is usually attributed to clinical diagnoses, but choice of treatment is based on limited tools for wound treatment or history and demographics of the patient but not a specific diagnosis of causation (nosology). The history and physical is used to arbitrarily determine the appropriate treatment. This method, however, is not easily transferrable or reproducible between observers. The three wound types chosen for this study: autoimmune, trauma, and pressure ulcers are very dissimilar in etiology, thus making histological differences more apparent. The anatomy of the wound is not well understood and this study would aid in understanding the nuances and improving treatment in future studies. Trauma wounds do not display the same pathophysiology as autoimmune connective tissue disorders, thus it is hypothesized that on histology there would be clear differences between 1

7 the two. Trauma wounds are healthy wounds and would be the benchmark of normality, or the control in this study. Connective tissue diseases often have common pathogenetic mechanisms and many times affect the skin [2,3]. Many people suffer from autoimmune connective tissue disorders, including lupus, rheumatoid arthritis, scleroderma, Sjogren s syndrome, dermatomyositis, and polymyositis. Abnormalities that result from these conditions can affect the epidermis and upper dermis, often chronically, defined as longer than 3 months [2,3]. Chronic wounds are characterized by a disruption of normal healing and present as a state of cutaneous inflammation [4]. Neutrophils and macrophages collect in the wound and lead to excessive numbers of proinflammatory cytokines (IL 1, IL 6, TNF alpha). Sites of chronic wounds are also susceptible to bacterial colonization and tissue hypoxia. In a study of pressure ulcers in 20 patients, there were four general types of histopathology. These included a dense fibrin matrix, inflammatory cells, cells ranging from normal to necrotic, and occluded blood vessels. Pressure wounds are due to trauma, but have become chronic. Acute and chronic wounds have a different histologic profile and we thus expect the chronicity to alter the histology of the pressure wound, compared to the acute, trauma wound. Pressure ulcers differ from autoimmune wounds in that pressure ulcers do not result from disease of the wound healing mechanism [5]. There has not been a definitive study explicitly showing the differences in histology between the types of wounds. This project aims to find histologic patterns amongst wound types. Hypothesis/research question Primary objective: The goal of the study is to determine the normal histopathology of two types of wounds: pressure and autoimmune connective tissue disorder wounds. 2

8 Methods and Materials This is a retrospective, qualitative research study. The goal is to understand and compare the histology of two wounds and potentially develop further studies that tailor treatments to the tissue characteristics of each type of wound. Wound samples, which are put on slides and assessed by the pathologist, are standardly taken during treatment. The more than 20 samples were chosen arbitrarily, after meeting the inclusion and exclusion criteria below. Gender, race, employment, language, or geography were not be used to exclude or include tissue samples. There was no intervention or interaction with live human subjects. The slides used for this study are those that have already been collected and are stored at Banner University Medical Center Phoenix. These samples are stored in the hospital and are taken from the patient routinely. Additional materials from patients did not need to be solicited to implement this study. More than ten samples each of pressure and autoimmune wounds were collected from unique patients. The surgery log will be checked and the corresponding tissue slides will be found in the lab. This number of slides of each wound type is a reasonable to determine some statistically significant parameters. No extra measures are needed to collect these samples from the patients as they are already collected as part of the treatment regime anyway. The samples were stained with H & E stain and/or Trichrome Blue stain and examined under light microscopy in Dr. Gottlieb s office. The samples will be chosen based upon the inclusion criteria outlined below. A research coordinator in the wound healing department will do the blinding numbering the slides in a way that will randomize the wounds and conceal the identity of the patient and the etiology of the wound. There will be a numerical code for each slide and a corresponding document listing the wound type. The analysis will be done blindly, without knowing the etiology of the wounds. An exhaustive list of the wound characteristics examined cannot be given at this point, because this is an exploratory study, but some of the characteristics that will be studied may include: lymphocyte cluster, and eosinophil counts, 3

9 neutrophil induction, thickness of stratum, wound layer comparison, lymphoid infiltration, lymphoid clustering and palisading, lymphoid aggregates, germinal centers, location of cells, lymphocytes in lymphatics, architecture compared to normal, connective tissue distribution, edema, signs of tissue lysis, alteration of blood vessels, acute vs. chronic inflammation, and active ulceration. After characteristics have been noted and compiled, the cause of the wound will be revealed to complete the analysis. Inclusion Criteria More than 10 pressure wound samples will be collected from otherwise healthy patients who do not have a diagnosis of Sjogren s syndrome, dermatomyositis, systemic lupus erythematous, rheumatoid arthritis, scleroderma, or a similar connective tissue disorder. Age range More than 10 autoimmune wounds will be collected from patients with a diagnosis of Sjogren s syndrome, dermatomyositis, systemic lupus erythematous, rheumatoid arthritis, scleroderma, or a similar connective tissue disorder and the wound is likely a result of their autoimmune condition. Age range A pressure wound sample will be excluded from this study if the patient has an underlying autoimmune connective tissue disorder. 4

10 Results Characteristic Pressure Wounds Autoimmune Wounds No. present Percentage No. present Percentage out of 12 (%) out of 13 (%) samples samples Granulation Tissue Neutrophils Lymphocytes Gangrenous Necrosis in Epidermis Eosinophils Fibrinoid Necrosis Hyperkeratosis Hemorrhage Fibroblasts Osteomyelitis Multinucleated Giant Cell Foreign Body Inflammation Calcification Coagulative Necrosis Chronic Inflammation Fibrosis Table 1: Characteristics of pressure and autoimmune wounds 5

11 Table 2: Statistical significance of findings using Fisher s Exact test. 6

12 When comparing wounds from autoimmune connective tissue disorders to pressure ulcers. There was increased granulation tissue, fibrinoid necrosis, hyperkeratosis, and fibroblasts in the pressure wounds. The autoimmune wounds displayed increased inflammation, calcification, chronic inflammation, and fibrosis than in the pressure wounds. However, statistically significant characteristics were lymphocyte presence, chronic inflammation, and fibrosis. significance was calculated using Fisher s Exact test. Statistical 7

13 Figure 1: Example images of (A) pressure, (B) autoimmune connective tissue, and (C) normal wounds (A) Pressure wound at 10X magnification, showing granulation tissue, neutrophils, fibrinoid necrosis, and hyperkeratosis. 8

14 (B) Autoimmune connective tissue disorder wound displaying lymphocytes, calcification, chronic inflammation, and fibrosis. 9

15 (C) Normal wound healing. Taken from Biology of Wound Healing chapter of Dermatology [6] 10

16 Discussion Wound healing generally consists of three phases. These phases include inflammation, tissue formation, and then tissue remodeling [6]. As described previously, cell cell interactions, cytokines, and extracellular matrix proteins play a key role in the wound healing response [6]. However, wounds incurred, for example, from local stressors (i.e. pressure) or from systemic disease (i.e. autoimmune connective tissue disorders) can lead to chronic, non healing lesions. Limitations to the study included the lack of controls. Healthy wounds, or wounds incurred in trauma, would have been helpful controls. The comparison of autoimmune and pressure wounds with these controls would have further elucidated patterns in wound healing depending on type. Due to lack of these trauma wound samples, the comparison was completed with just the two wound types. We appreciated previously reported data on normal wound healing to aid in our comparisons. 11

17 Future Directions In the future, expanding the IRB to include harvesting samples from trauma wounds would aid in providing a control in the study. That would help in providing clearer differentiations between the wound etiologies. Once differences in histology have been identified, it is now possible to delve into treatments that would address the varied wound etiologies based on cell types present and architecture. Other factors leading to poor wound healing could also be investigated in a similar manner. 12

18 Conclusions It is seen that there are variances in histology based on the etiology of the wound. This pilot study identified signs of chronic inflammation, fibrosis, and calcification to be more apparent in the autoimmune connective tissue wounds than in the pressure wounds. This study delved into pressure wounds and autoimmune wounds, however other etiologies that lead to poor healing include vasculitis, age, diabetes associated microangiopathy, poor nutrition, ischemia, bacteria, and growth factor deficiency amongst others [6]. Treatments for these chronic wounds are commonly dressings, and can even be gene therapy, stem cells, and molecular modeling of growth factors. These therapies success depends on understanding the mechanism of the wound healing. Better understanding of the variations in wound healing on etiology will improve treatment [6]. 13

19 References [1] Clinical Protocol for Wound Management and Wound Management Standards. Wirral Community ntandwoundmanagementstandardsjune2013.pdf [2] Marinovic, B., Jukic, I. L., & Lipozencic, J. (2012). Wounds in autoimmune bullous dermatoses and systemic connective tissue diseases. [Rane kod autoimunih buloznih dermatoza i sistemskih bolesti vezivnog tkiva] Acta Medica Croatica : Casopis Hravatske Akademije Medicinskih Znanosti, 66 Suppl 1, [3] Sontheimer, R. D. (2004). Skin manifestations of systemic autoimmune connective tissue disease: Diagnostics and therapeutics. Best Practice & Research.Clinical Rheumatology, 18(3), [4] Dantzer, E. (2011). Role of skin substitutes in surgical repair of the sequelae of burn injuries. [Place des dermes equivalents en chirurgie reparatrice des sequelles de brulures] Annales De Chirurgie Plastique Et Esthetique, 56(5), [5] Vande Berg, JS, Rudolph, R. Pressure (decubitus) ulcer: Variation in histopathology A light and electron microscope study. Human Pathology [6] Eming, Sabine. Biology of Wound Healing. Surgery section. Dermatology,

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