The constant improvement of diagnostic and therapeutic procedures, together with the increase of life-lasting, results in a higher frequency of patients suffering from cutaneous chronic wounds such as vascular, diabetic, and pressure ulcers. These diseases most of the time require a long-term treatment, which is often associated with poor outcomes in terms of quality of life and with a decrease in patients’ hospitalization. Moreover, since the healing process is remarkably slow, the clinical perception of the phases that lead a chronic wound to complete restoration is often penalized: this effect is dramatically amplified in those cases in which the patient is followed by more than one operator. Therefore, the study of wound healing pathophysiology and the development of new tools for the monitoring of the healing process may represent a possible optimization of the treatment efficacy for those lesions .
Monitoring of acute and chronic wounds can be performed by measuring in an objective, precise, and reproducible way and by simply adapting the existing and proven technologies. Wound measurement techniques have received consistent attention in clinical practice and research through the three main areas of interest in wound care: vascular ulcers, diabetic foot ulcers, and pressure ulcers . Recently, the concept of wound bed preparation has been introduced and classified according to the clinical parameters of chronic wounds . This new aspect of wound healing has to be considered as a more comprehensive approach, involving several individual components, which allow the opportunity to investigate clinical, microbiological, and cellular wound aspects from one individual viewpoint. In order to monitor the different aspects of wound bed preparation, various instrumental techniques are now under investigation in order to obtain a better and more objective characterization of the tissue repair process. The advantage of this new scientific discipline is the ability to study living skin in real time. The main physical wound parameters that have received the most attention over the past few years in terms of measurement techniques are area and volume, colour, pH, temperature, wound fluid analysis, odour, pain, and tissue perfusion [4▪▪].
This study reviews current and future diagnostic technologies and the relevance of such methods to clinical practice in wound healing.
THE ROLE OF HISTOPATHOLOGY IN WOUND HEALING
Histopathology of wounds is a very helpful diagnostic tool to make the diagnosis, exclude malignancy and infectious cause, monitor healing progress in the course of treatment, and understand better the pathophysiology of nonhealing wounds. Punch biopsies and incisional biopsies are advocated because they allow more tissue to be examined. Usually, skin biopsies are performed in chronic, poorly responding to treatment ulcers or ulcers increasing in wound size, that is, larger area and/or depth, despite appropriate standard treatment for at least 3 months [5▪▪]. The best site for biopsy is the edge of the ulcer because it enables the comparison between the ulcerated area and the surrounding skin. Furthermore, the base usually shows nonspecific features of dermal necrosis and dense inflammation with secondary vascular damage to the underlying vessels which simulates vasculitis or vasculopathy and is referred to as pseudovasculitis or pseudovasculopathy. All wounds with abnormal granulation should be biopsied to exclude malignant transformation because as per recent study the overall skin cancer frequency in chronic leg ulcers is increasing and around 10.4%. In cases where malignancy is suspected a biopsy from the ulcer bed is necessary too to exclude sarcomatous malformation as this is the most common site where these tumors arise. In conclusion, we currently recommend two punch or incisional biopsies from the edge and one from the wound bed in any chronic wound unresponsive to treatment within 3 months.
Haematoxylin and eosin (H&E) is the most widely used stain in wound pathology as in general dermatopathology. As changes may be subtle, at least 10–12 H&E-stained step-sections are advised per case. Special stains (non-H&E) and immunohistochemical markers are applied to highlight tissue components, microorganisms, or foreign materials nonvisible or less visible on the H&E sections. The conventional set of special stains form infections organisms includes: Periodic acid-Schiff (PAS) for fungal organisms, Gomori-methenamine silver (GMS) for fungal organisms and particularly for the dematiaceous fungi causing phaeohyphomycosis, Alternaria sp., chromoblastomycosis which can present as chronic nonhealing skin ulcers. Acid fast bacillus (AFB) or Ziehl-Neelsen as well as Faraco-Fite stains are used to identify the capsule of acid-fast microorganisms such as mycobacteria. Other less commonly applies special stains for infectious organisms include: Brown and Brenn for bacteria, Giemsa stain for Leishmania, and Histoplasma.
SPECIAL STAINS HIGHLIGHTING TISSUE COMPONENTS IN WOUND PATHOLOGY
Periodic acid-Schiff (BM) highlights the vessel wall which is particularly helpful in cases of hyalinized thick walls due to deposition of plasma proteins, lipids, and basal membrane material in hypertensive and diabetic ulcers as well as in vasculopathies such as atrophie blanche and cryoglobulinaemia.
Phosphotungstic acid–haematoxylin (PTAH) – apart from striated muscle fibres, it highlights fibrin in blue-purple; this is particularly useful to identify fibrin thrombi in vasculopathy.
Verhoeff-van Gieson (VVG) and orcein colour the elastin and therefore are useful to distinguish between venules and arterioles as well as to highlight the internal elastic lamina reduplication and hyaline arteriosclerosis and intimal thickening in hypertensive ischemic leg ulcers.
Von Kossa stain for calcium salts is applied to highlight calcification of the media in the affected vessels in calciphylaxis and Mockenberg's medial calcific stenosis.
IMMUNOHISTOCHEMICAL DIAGNOSTIC STAINS
CD31 is the most sensitive and specific endothelial marker just as D2–40 is the most sensitive marker for lymphatic endothelia. D2–40 recognizes podoplanin, a transmembrane glycoprotein that is constitutively expressed in lymphatic endothelial cells, allowing distinguishing dermal blood vessels from lymphatic vessels. By using D2–40, a recent study showed lymphatic abnormalities in chronic venous insufficiency venous ulcers [6▪▪]. Factor VIII is a common endothelial marker that stains also mast cells and platelet thrombi.
PROTEASES AS PREDICTIVE MARKERS
Several biomarkers have already been identified in nonhealing wounds that could potentially be used as the basis for diagnostic tests . At the present time proteases appear to represent the best available biomarker to provide useful information concerning wound status and whether a particular treatment would be appropriate or likely to improve clinical outcomes. Research has shown that wounds which are progressing towards healing have reduced levels of serine and matrix metalloproteinases, whereas nonhealing wounds often contain excessive and high levels of these substances . Therefore, a test which measures protease activities could be useful as a predictive biomarker and determine if a patient would likely respond to a protease-modulating therapy.
Researchers have hypothesized that measuring the combined effects of multiple inflammatory proteases offers greater potential as a predictive marker than any single protease. The fact that all previous testing and data have been based on wound fluid analysis is also advantageous; noninvasive and pain-free sample collection could facilitate ease of use and foster repeat testing when necessary. Maximum benefits and routine usage of this predicative marker could be advanced by a ‘point-of-care’ test carried out at the patient's bedside or treatment chair where results could be obtained within the normal timeframe of a patient visit; clinicians could quickly determine an appropriate dressing selection for that wound in real time.
DIAGNOSTIC WOUND MEASUREMENT
Noninvasive wound diagnostic includes the measurement of bacterial burden, tissue blood flow, and determination of tissue viability. These parameters are clues to the definition of the cause, pathophysiology, and status of the wound, but we believe that a complete and detailed history and physical examination are also fundamental.
The technique of ultrasonography involves the detection of reflected sound waves through tissues that have inherently different acoustic properties. Images of the skin can be displayed in one-dimensional A mode (adequate for skin thickness measurements), two-dimensional B mode (which produces a vertical cross-section of the tissue being scanned), or C mode (which produce images horizontal to the skin surface). Generally a higher ultrasound frequency provides a better resolution of the image . Units used in dermatology need to achieve much greater resolution to be able to visualize the skin. Generally, with 20 MHz ultrasound, the dermis and hypodermis can be distinguished easily, but the epidermis over most of the body, except on the palm and sole, cannot be visualized as a separate structure because it is too thin to be resolved. High-frequency ultrasound is used to analyse the ultrastructure in chronic wounds, hypertrophic scars, keloids, and normal surrounding skin . The parameters investigated are the depth between skin surface and the inner limit of the dermis and the tissue density. The depth measurement, expressed in millimetres, gives an estimate of wound volume and scar thickness. Ultrasonography is characterized by the high echogenicity of the dermis, which is sharp compared to a hypoechogenicity of subcutaneous fat. This technique allows an accurate determination of granulation, sloughy/necrotic tissue, and the physical dimensions of ulcers, while also providing an index to the structural components of the ulcer.
Laser Doppler systems
Skin microcirculation is known to consist of two functionally different networks: the superficial, nutritive and the deeper, mainly thermoregulatory vascular bed. Laser Doppler flowmetry is commonly used because it is a noninvasive, simple, objective measurement, which evaluates cutaneous blood flow 1–2 mm under the skin surface and gives a continuous or near-continuous record. This technique is useful in the evaluation of wound healing and it is used in burn scarring for the constant evaluation of local skin microcirculation . Laser Doppler perfusion imaging (LDPI) is a technique that has been finding increasing utility in skin research. It measures cutaneous perfusion by scanning a low-power laser beam over a region of skin. The images obtained by LDPI are displayed on a computer monitor using various colours to depict the variations in perfusion that occur in different regions of the skin. The measurements obtained are objective and reproducible. The instrument has been used to evaluate the effects of postural changes on blood flow, as well as postural vasoregulation and mediators of reperfusion injury in venous ulceration .
The role of pH in wound healing has proven to be of fundamental importance, and prolonged chemical acidification of the wound bed has been shown to increase the healing rate in chronic venous leg ulcers . The mechanism of interaction between acidic pH and the wound healing process is related to the potential to increase tissue oxygen availability through oxygen dissociation and to reduce the histotoxicity of bacterial end products, thus stimulating the wound's healing process. The change of value is in accordance with the stage of the ulcer, moving to an acidic state during the healing process [14▪]. Two significant methods are widely used for measuring cutaneous pH: the colourimetric technique and the glass electrode potentiometric measurement. The most common pH instrument is a flat glass electrode connected to a metre and applied to the skin, with one or two drops of bi-distilled water interposed between the electrode and the skin. The use of a flat electrode is important in order to provide appropriate contact with the skin surface. The electrode is applied onto the skin at intervals of 10 s until stabilization of the reading. Measurements are performed at a room temperature that is below 23°C and a relative humidity of less than 65%, because sweat can influence the results. Readings should be taken 12 h after the application of detergents or creams to the skin. Measurement of pH is a noninvasive technique, simple, easy to use, and provides important information about changes in wounds.
Confocal microscopy has recently become quite commonly used in the dermatological field. The basic principle uses a light source and a lens to focus on a specific plane within the sample of tissues. The returning light from this focal point is detected by the instrument and used to create an image that is a composite of a large number of imaged points. The final image acquired is clear, because the system is trained to detect mainly the light that is directly backscattered from the focal point and to exclude any scattered and reflected light from out-of-focus planes, thus minimizing image blur. The light source used can be either intense visible light or near-infrared light, as used with the video-rate laser-scanning confocal microscope. The main advantage of confocal microscopy is that it can allow the skin to be evaluated in its native state either in vivo, or when freshly biopsied (ex vivo) without the fixing, sectioning, and staining that is necessary for routine histology. Confocal microscope imaging of normal skin in vivo gives clear images of the cellular layers of the epidermis and upper dermal region. The quality of images obtained by confocal microscopy can depend on the type of confocal microscope used and the ability of the operator. Traditional confocal microscopes require the use of fluorescent dyes in order to achieve adequate tissue contrast in creating a clear image. For in-vivo skin examination, some commercially available confocal microscopes can achieve image contrast entirely through the detection of reflected light from within the skin, whereas others require an intradermal injection of a fluorescent contrast agent before different skin-cell layers can be clearly visualized. The completely noninvasive nature and high-resolution capability of a confocal microscope has made it a useful instrument in wound healing research . Confocal microscopy has been shown to characterize the pattern of neovascularization and reinnervation in a model of human skin equivalent grafted in a pig, confirming that angiogenesis occurs first and act as an influencing and guiding factor on innervation in experimental wound healing . Currently, confocal microscopes are costly instruments and, although available commercially, their use is mainly confined to research. However, as the technology of confocal microscopy improves, and smaller, more affordable instruments become available, the technique could have immense potential as a diagnostic tool in wound healing, enabling clinicians to characterize wound parameters, image skin lesions, and diagnose them without the need for biopsy, and to define the margins of skin lesions prior to any intended excision.
MRI is a valuable diagnostic technique that can be used to image a variety of body tissues. MRI procedures may accelerate the decision for surgery or closed drainage in patients with signs of severe infected lesions and significantly improve survival. In infected wounds, MRI provided excellent anatomo-radiologic correlations by precisely defining the extent of infection . One important aspect of the wound healing phases is represented by angiogenesis, which is a process characterized by new vessel growth toward and into the tissue . Without new vessels, which ensure an adequate supply of blood, oxygen, and nutrients to the wound area, wound healing cannot proceed. MRI provides a noninvasive quantitative assay for the wound-healing process, but its use in clinical settings requires further development.
Wound healing assessment is becoming a more and more sophisticated section in wound management due to the introduction of different types of equipment, which are able to monitor noninvasively the various phases of tissue repair. With the availability of biomedical engineering technologies, wound measurement instruments are rapidly evolving, as evidenced by the considerable amount of data produced in recent literature. The wide range of clinical and biochemical parameters to be assessed represents the main challenge for the years to come, while caregivers will hopefully be provided with user-friendly tools to be used routinely and safely. A better reproducibility on the part of the various devices and a reduction in costs is to be expected in the near future, allowing widespread diffusion of the techniques in question among end users. In clinical use, applications of these technologies will be differentiated from basic research and particular emphasis will be placed on therapeutic control and the prevention of recurrences. The objective assessment of chronic wounds during tissue repair will become a specific aspect within wound management, which will not replace the clinical assessment of expert caregivers but may bring numerous advantages in terms of understanding and awareness of the problem.
Conflicts of interest
The authors disclose no conflict of interest for this study.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 127).
1. Romanelli M, Gaggio G, Coluccia M, et al. Technological advances in wound bed measurements. WOUNDS 2002; 14:58–66.
2. Dargaville TR, Farrugia BL, Broadbent JA, et al
. Sensors and imaging for wound healing
: a review. Biosens Bioelectron 2012.
3. Schultz G, Mozingo D, Romanelli M, Claxton K. Wound healing
and TIME: new concepts and scientific applications. Wound Repair Regen 2005; 13 (4 Suppl):S1–S11.
4▪▪. Matzeu G, Pucci A, Savi S, et al. A temperature sensor based on a MWCNT/SEBS nanocomposite. Sensors and Actuators A 2012; A178:94–99.
The study describes the use of a new sensor for temperature monitoring made of nanocomposites which will give the clinician essential information about prevention and treatment of chronic wounds. Wound temperature is an essential parameter to be taken under control during wound healing in order to prevent the risk of infection particularly in patients with diabetes and at risk for pressure ulcer development.
5▪▪. Senet P, Combemale P, Debure C, Lok C. Angio-Dermatology Group of The French Society of Dermatology. Malignancy and chronic leg ulcers: the value of systematic wound biopsies: a prospective, multicenter, cross-sectional study. Arch Dermatol 2012; 148:704–708.
The authors consider the role of wound biopsy to distinguish vascular ulcers from neoplastic ulcer. The study offers a careful analysis of prevalence of malignant wounds.
6▪▪. Fernandez AP, Miteva M, Roberts B, et al. Histopathologic analysis of dermal lymphatic alterations in chronic venous insufficiency ulcers using D2-40. J Am Acad Dermatol 2011; 64:1123e1–1123e12.
The study describes a new diagnostic biomarker for lymphatic vessels in patients with chronic venous insufficiency (CVI). The findings further implicate lymphatic dysfunction in the pathogenesis of CVI ulcers and allow the formulation of a hypothesis concerning lymphatic changes that may be tested in future studies.
7. Hahm G, Glaser JJ, Elster EA. Biomarkers to predict wound healing
: the future of complex war wound management. Plast Reconstr Surg 2011; 127 (Suppl):21S–26S.
8. Soo C, Shaw W, Zhang X, et al. Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair. Plast Reconstr Surg 2000; 105:638–647.
9. Wendelken M, Alvarez O, Markowitz L, et al
. Key insights on mapping wounds with ultrasound. Podiatry Today 2006; 7:70–74.
10. Dyson M, Moodley S, Verjee L, et al. Wound healing
assessment using 20 MHz ultrasound and photography. Skin Res Technol 2003; 9:116–121.
11. Bray R, Forrester K, Leonard C, et al
. Laser Doppler imaging of burn scars: a comparison of wavelength and scanning methods. Burns 2003; 29:199–206.
12. Widmer LW, Vikatmaa P, Aho P, et al. Reliability and repeatability of toe pressures measured with laser Doppler and portable and stationary photoplethysmography devices. Ann Vasc Surg 2012; 26:404–410.
13. Romanelli M, Dini V, Barbanera S, Bertone MS. Evaluation of the efficacy and tolerability of a solution containing Propyl-betaine and Polihexanide for wound irrigation. Skin Pharmacol Physiol 2010; 23 (Suppl 1):41–44.
14▪. Sharpe JR, Booth S, Jubin K, et al
. Progression of wound pH during the course of healing in burns. J Burn Care Res 2012.
In this study the authors provide a clear correlation between the diagnostic use of wound surface pH assessment and the healing of burns. The acidic ambient to be provided in the wound bed during tissue repair is able to keep bacterial burden under control and to facilitate cells interaction at dermal and epidermal compartment.
15. Terhorst D, Maltusch A, Stockfleth E, et al
. Reflectance confocal microscopy for the evaluation of acute epidermal wound healing
. Wound Repair Regen 2011; 19: 671–679.
16. Ferretti A, Boschi E, Stefani A, et al. Angiogenesis and nerve regeneration in a model of human skin equivalent transplant. Life Sci 2003; 73:1985–1994.
17. Escher KB, Shellock FG. An in vitro assessment of MRI issues at 3-Tesla for antimicrobial silver containing wound dressings. Ostomy Wound Manage 2012; 58:22–27.
18. Helbich TH, Roberts TPL, Rollins MD, et al. Noninvasive assessment of wound healing
angiogenesis with contrast-enhanced MRI. Acad Radiol 2002; 9 (Suppl 1):S145–S147.