Objective methods for analyzing outcomes in research studies on cosmetic dermatology

Manela-Azulay, Mônica; Cuzzi, Tullia; Pinheiro, Joana Cunha Araújo; Azulay, David Rubem; Rangel, Giuliana Bottino

doi:10.1590/S0365-05962010000100009

Abstracts

Cosmetic dermatology is a field of medicine that is in constant development; therefore, the use of objective methods for validating the findings of scientific studies is crucial. The most commonly used techniques in the majority of these studies include histopathology, immunohistochemistry, morphometry, stereology, digital photography, biometry, optical profilometry and confocal microscopy. The objective of this review was to provide an update on the principal methods used as tools for analyzing outcomes and also to provide the dermatologist with means of sharpening his/her critical judgement with respect to the publications and presentations that use subjective evaluation methods.

Dermatology; Efficacy; Methods

A Dermatologia Cosmética é uma área em constante crescimento, motivo pelo qual se faz necessária a utilização de métodos objetivos para validar os resultados dos estudos científicos. As técnicas mais empregadas na maioria desses trabalhos são: histopatologia, imunoistoquímica, morfometria, estereologia, fotografia digital, biometria, profilometria óptica e microscopia confocal. Esta revisão tem como objetivo trazer informações sobre os principais métodos utilizados como ferramenta de análise dos resultados obtidos, além de auxiliar o dermatologista a aguçar seu senso crítico em relação às publicações e apresentações que se utilizam de métodos subjetivos de avaliação.

Dermatologia; Eficácia; Métodos

REVIEW

^IAdjunct Professor of Dermatology, Dermatology Department, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil

^IIAdjunct Professor of Pathology, Pathology Department, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil

^IIIInternal Medicine and Dermatology Specialist, Cidade (sigla estado), Brazil

^IVHead of the Professor Azulay Institute of Dermatology (IDPA). Assistant Professor, Federal University of Rio de Janeiro. Department Head, Pontifical Catholic University (PUC), Rio de Janeiro, RJ, Brazil

^VInternal Medicine and Dermatology Specialist. Preceptor of the cosmetic dermatology outpatient clinic, Professor Azulay Institute of Dermatology (IDPA). Currently enrolled in a Masters Degree program at the Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil

Mailing Address

ABSTRACT

Cosmetic dermatology is a field of medicine that is in constant development; therefore, the use of objective methods for validating the findings of scientific studies is crucial. The most commonly used techniques in the majority of these studies include histopathology, immunohistochemistry, morphometry, stereology, digital photography, biometry, optical profilometry and confocal microscopy. The objective of this review was to provide an update on the principal methods used as tools for analyzing outcomes and also to provide the dermatologist with means of sharpening his/her critical judgement with respect to the publications and presentations that use subjective evaluation methods.

Keywords: Dermatology; Efficacy; Methods

INTRODUCTION

Cosmetic dermatology is a segment of general dermatology that has developed at an extraordinary pace in recent years. The advent of novel procedures, cosmetic products, cosmeceutics and laser devices has resulted in a need for scientific methodology to appropriately validate the actual efficacy, indications and adverse effects of these new treatments. Many studies present only subjective clinical observations and photographs as a means of evaluating their findings, with no objective methodology.

The objective of the present study was to provide information on methods that may be applied to evaluate the results of cosmetic therapies and also to sharpen the judgement of professionals involved in this area of medical science.

HISTOPATHOLOGY AND IMMUNOHISTOCHEMISTRY

Histopathology is a method traditionally used for observing and analyzing morphological changes in organs. Microscopic analysis procedures are a well-established component of routine diagnosis, particularly when the skin is concerned. The staining techniques used vary from hematoxylin-eosin (routine staining) to other specialized staining techniques used to identify specific structures such as elastic fibers (Orcein, Verhoeff-Van Gieson and Weigert's resorcin-fuscin), collagen (picrosirius red, Gomori's trichrome and Masson's trichrome), mucopolysaccharides (Alcian blue and colloidal iron), melanin (Fontana-Masson), lipids (scarlet-red, oil red O and Sudan), hemosiderin (Prussian blue) and others.^1,2

Some studies have shown that, as skin ages, histopathological changes may occur in comparison with younger skin.^3-6 Findings in the chronologically aging skin may include thinning of the epidermis, flattening of the dermal-epidermal junction and a decrease in the cellularity of the dermis.⁴ The papillary dermis forms ridges that maintain contact with the epidermis. A progressive flattening of these ridges often occurs over an individual's lifetime, resulting in decreased surface contact between the dermis and epidermis.³

In skin that has been chronically exposed to the sun, the epidermis is thicker, and atrophy may occur in the final stage of photoaging; acanthosis may be accompanied by cell atypia, loss of nuclear polarity and irregularities in cell size and in tinctorial properties.^5,6 The melanocytes increase in number and size, whereas a reduction occurs in the Langerhans cells and their function is compromised. Furthermore, in photoaged skin, unlike skin that is only chronologically aged, there are more fibroblasts, mastocytes and histiocytes in the dermis, characterizing the inflammatory process referred to as dermatoheliosis.⁵ The most marked histological characteristic of photoaging is solar elastosis, in which the mature collagen fibers are substituted by collagen with a basophilic appearance (basophilic degeneration of collagen) in the papillary and reticular dermis.^5,6

Fragmentation of the elastic fibers and changes in the structure and composition of the anchoring fibrils, proteoglycans and glycosaminoglycans, have also been described in photoaging skin.

It is interesting to note that the stains mentioned in the section on histopathology, particularly picrosirius red for collagen and resorcin-fuscin for elastic fibers, may contribute greatly in objectively analyzing whether or not an improvement has occurred in the cosmetic therapies applied. In the case of resorcin-fuscin, the elastic fibers are marked in black, collagen in pinkish red and the others in yellow.¹ It is also possible to quantify collagen and elastic fiber using specific stains in morphometry.^7-9

The principle of immunohistochemistry involves an antibody-antigen reaction; therefore the method is considered highly specific. Various antibodies are used to identify the different antigens, the most common of which are: collagen types I and III¹⁰; elastic fibers using anti-elastin antibody; muscle tissue using vimentin and alpha-actin; and melanocytes using Melan-A and HMB-45, among others. CD68, among others, is one of the markers used for macrophages.¹

In the case of idiopathic cutaneous hyperchromia of the periorbital region (dark shadows), one study found that the pigment involved in this condition is composed of melanin within the macrophages, visible using the CD68 marker rather than the accumulation of melanocytes, which would have been identified using Melan-A.¹¹

MORPHOMETRY AND STEREOLOGY

Morphometry and stereology are similar techniques for measuring anatomical structures; however, morphometry is simpler to apply in image analysis. It is used to facilitate the collection, presentation and analysis of data obtained in research studies and in routine laboratory work, and even permits different anatomical structures to be related with their functions.⁷

Stereology interprets the three-dimensional internal structural arrangement based on an analysis of structure sections that provide only two-dimensional data; it works with density. Morphometry is a two-dimensional, quantitative method that determines lengths, areas and perimeters, and offers the benefits provided by image analysis software packages. According to Aherme and Dunnill, despite discussions regarding the nomenclature, morphometry and stereology should be considered to constitute a single method.¹²

Application of this methodology improves the pathologist's ability to establish a diagnosis and often even to predict prognosis in some pathological processes.¹³ To do so, equipment and accessories for macroscopic and microscopic analysis are used.

In macroscopy, sophisticated accessories may be used in combination with computer graphics or even simple tools such as a ruler and measuring tape.^3,7

For microscopic analysis, the resources most commonly used include the integrating eyepiece, which identifies the points or lines of the eyepiece that intersect with certain structures and the clear chamber, which, when attached to the light microscope or magnifier, permits the field under observation to be drawn on a sheet of paper or on a digital table. A micrometer drum may also be used to measure the diameter of structures and a microscope with a grid eyepiece to evaluate areas.⁷

Although these tools remain in use today, their use was more common prior to the advent of computer morphometry, which may be interactive or automatic. The interactive form requires the investigator to make the measurements directly, whereas the automatic form, which has been available since the eighties, allows measurements to be made automatically, a faster procedure that involves less interaction from the investigator.^7,14

The results are expressed numerically; therefore, there is no subjectivity and they may be reproduced and verified at any other time by any other specialized laboratory.

In her doctoral thesis, Manela-Azulay objectively measured the increase in collagen following treatment with 5% topical vitamin C in photoaged skin using morphometric evaluation techniques. The results were highly significant.⁸

DIGITAL PHOTOGRAPHY

Digital photography represents an extremely valuable tool in dermatological practice and research. Scientific studies in cosmetic dermatology have often used this resource to demonstrate the results of treatment. It is often the only means of verifying results, thereby implying an extremely subjective nature in the interpretation of data. If this were not enough, it should also be taken into consideration that parameters such as luminosity, distance and the angle of the photographs are often far from adequate. Contracting professional photographers or taking photographs with adequate equipment are ways in which to compensate for this lack of a more appropriate methodology.

It should be emphasized that the scientific journals of greatest credibility require an audit of the authenticity of a photograph prior to accepting it for publication in order to analyze whether the photograph in question has undergone prior editing.¹⁵

A digital image is composed of pixels, which are points of light in the image, captured by an electronic sensor. Each pixel registered is codified according to its topographical location on the photographic image and has a certain intensity of color. This allows investigators to calculate distances, measure areas, evaluate the intensity of color and recognize patterns in the digital images.^15,16

In one study, morphometry of a digital image allowed melanin to be quantified in each area prior to and following treatment for idiopathic cutaneous hyperchromia of the periorbital region with intense pulsed light. In this case, the combination of these two methods improved analysis of the findings.¹⁷

WOOD'S LAMP AND ULTRAVIOLET PHOTOGRAPHY

The Wood's lamp was invented in 1903 by Robert Wood. ¹⁸ The emission of long-wave ultraviolet radiation from the Wood's lamp is generated by a high-pressure mercury arc through a filter made of barium silicate with 9% nickel oxide referred to as the Wood's filter.¹⁹ This filter is opaque to all forms of light except those within the 320-400 nm range, with a peak at 365 nm. Tissue fluorescence occurs when the shortest wavelength, in this case between 340 and 400 nm, initially emitted by the Wood's lamp, is absorbed and only the longer wavelength radiations, generally those within the visible light spectrum, are emitted.¹⁹

Whereas melanin from the epidermis and dermis absorbs light within this wavelength range, when the collagen in the dermis absorbs this light, it becomes fluorescent, thanks to the longer waves such as those within the visible light range, principally those within the visible blue light spectrum. Therefore, collagen is highlighted with the use of the Wood's lamp.

It is important to emphasize that the fluorescence of skin is generally poor. The spectrum of this fluorescence changes with chronic exposure to the sun, probably as the result of an alteration in elastin in the dermis.^20,21 The fluorescence of the tissue results primarily from the constituents of elastin, collagen, aromatic amino acids, nicotinamide adenine dinucleotide (NAD) and perhaps from the precursors or products of melanin. The patient should be examined in a dark room, preferably without windows.^22-24

Within the field of cosmetic dermatology, the Wood's lamp may be used in cases of pigmentation disorders. In hypopigmented or achromic lesions resulting from too little melanin in the epidermis, the Wood's lamp can be used to visualize the autofluorescence of the collagen in the dermis, the lesion appearing as bright blue. One example of its use is in the early follicular repigmentation found in the treatment of vitiligo with the use of photochemotherapy.²⁵ In the case of cutaneous hyperpigmentation, when light reflects on the skin, photons of shorter UVB (290 - 320 nm) and UVA wavelengths (320 - 400 nm) are more easily distributed in the stratum corneum and in the epidermis. The opposite occurs with photons of longer wavelengths such as those within the visible light range (400 - 800 nm), which penetrate more deeply into the dermis.¹⁹

Melanin absorbs light intensely, both in the ultraviolet range and in the visible light range. When the Wood's lamp illuminates an epidermis with a large quantity of melanin, most is absorbed, while the adjacent, less pigmented skin reflects light as usual, resulting in contrasts at the borders between the areas with different melanization gradients. The variations in the pigmentation of the epidermis thus become more apparent under the Wood's lamp compared to normal light.

In dermal pigmentation, this contrast is less apparent under the Wood's lamp, since some of the collagen autofluorescence is distributed above and below the dermal melanin; therefore, less fluorescence is visible.²⁶ According to these findings, the Wood's lamp may contribute towards diagnosing melasma as epidermal, dermal or mixed, thus defining treatment-related prognosis.

Technological advances have led to a new way of evaluating patients' skin prior to and following dermatological therapies by a combination of digital photography and ultraviolet light. This is referred to as ultraviolet or UV photography. The filter applied in UV photography uses the same wavelength as the Wood's lamp. This combination of technologies allows images to be recorded with the inherent peculiarities described above, thus permitting posterior analysis and comparison. Therefore, despite the limitations of the technique, this tool permits physicians to monitor the natural progression of treatments offered to their patients.

BIOMETRY

Biometry is a science that works by translating biological phenomena into numerical data, thereby establishing relationships between the data obtained in order to determine the laws that govern them. It includes a set of methods applied for various measurements. Non-invasive instruments are used to evaluate the physicochemical properties of the skin such as its oiliness, hydration, tonus, pH, transepidermal water loss and the frictional properties of the skin, among others. These evaluations are performed using different devices.^27-29

Transepidermal water loss measures the water vapor lost through the surface of the skin by passive diffusion. This measurement is important in evaluating the competency of the skin barrier, which may be reduced in sun-exposed skin, for example. Subcutaneous hydration is determined by measuring electrical capacity. The capacity of intracellular fluid to conduct electrons in the subcutaneous tissue is measured using a device called a Corneometer.^27,30,31 Indexes of desquamation and skin dryness can also be evaluated using, respectively, the Corneofix^® and Visionscan^® devices. The corneocytes can be detached by applying a small adhesive over the skin. The thickness of the corneocytes indicates the degree of dryness of the skin (desquamation index). Skin pH is measured using a pH meter. pH is measured three times at the same site and statistical calculations are made.²⁸ Lipid index is calculated using the Sebumeter^®. The quantity of sebum in the skin is expressed in ±g /cm2. Lipid measurement is based on photometry of the fat in the area in question.²⁹

The humidity of the surface of the skin may also be measured using biometry. The measurement of the angle of contact formed between the skin surface and the water is an indicator of its hydrophobic or hydrophilic tendency. An angle of contact between 0º and 9º indicates that the quality of the skin is hydrophilic, whereas a greater angle of contact is more indicative of hydrophobia (lipophilic) on the skin surface. The device consists of a surgical microscope with a mirror directed at 45º to the surface under evaluation. A video camera permits visualization and storage of the image.^27,28

The elastic and viscoelastic properties of skin may also be measured using suction methods (Cutometer^®) or acoustic propagation waves (Reviscometer^®).³² Skin properties can thus be measured prior to and following the use of any given product to calculate its efficacy.

OPTICAL PROFILOMETRY

Optical profilometry is a digital imaging process used as a quantitative method to evaluate micro-topographical characteristics. Imprints in silicone rubber are used to make a mold of the skin that copies the topography of the surface, obtaining negative replicas of the skin surface. A digital imaging process system consisting of a video camera with high-resolution black and white images interfaces with a computer that contains the specifically designated image. A fiber-optic illumination device placed at a fixed angle is used to obtain details of the skin surface. The image is digitalized onto a 256 x 512 pixel matrix with different levels of lighting.³³

Gary L. Grove et al. showed that digital imaging processing of replicas of the skin surface provides a convenient method for quantifying wrinkles.³³ Various studies have used this method to evaluate results in the field of cosmetic dermatology.^34,35

CONFOCAL MICROSCOPY

Confocal microscopy is a non-invasive imaging method that permits evaluation of the physical characteristics of the skin and its annexes, in vivo and three-dimensionally. Images of the section of skin under evaluation may be obtained without physically dissecting the tissue or fixing it. Correlation with histology is good when skin is examined up to a depth of 350 ±m, i.e. up to the superficial reticular dermis, this constituting a limiting factor.³⁶

Laser-scanning confocal fluorescence microscopy, referred to as confocal microscopy, uses fluorescence to acquire images. Fluorescence is a type of luminescence (light emission) in which the body absorbs light and after a short interval of time re-emits it. Fluorophores are chemical compounds used to produce fluorescence in the material under evaluation.^36,37

Confocal microscopy uses a laser source to promote excitation of the fluorophores. Using a set of lenses, the microscope is able to focus a cone of laser light to a predetermined depth in the sample under evaluation. By altering the focal point, it is possible to illuminate the entire plane under study, point by point. Only the light of the points in focus is registered and will be processed by the computer; therefore, extremely precise two-dimensional images may be constructed.³⁶

Obtaining successive images of different planes from the same sample permits three-dimensional moving images to be constructed. This method has led to great advances in research with living organisms. The combination of the principles of optics and physiochemistry have finally made it possible to "take a close look at" various types of living cells and measure biological phenomena in real time and space.

In dermatology, it has been used in patients with psoriasis, prior to Mohs micrographic surgery to visualize surgical borders, in actinic keratoses, basocellular and squamous cell carcinomas, melanomas, and fungal, viral and bacterial skin infections. More recently, it has been used in the area of cosmetic dermatology.^37-40

This modality is useful not only for diagnosis but also to monitor therapies and evaluate the efficacy of treatments, principally due to the non-invasive character of the technique. Some examples are the comparative studies that use confocal microscopy to define histopathological response to the treatment of actinic keratoses with photodynamic therapy and imiquimod. This tool also permits evaluation of the dynamic processes occurring in response to treatment. A study using confocal microscopy showed that after some minutes of treatment of a hemangioma with pulse dye laser, the blood flow inside the lesions ceased and was substituted by amorphous material.⁴¹

DISCUSSION

Cosmetic dermatology is a field that is overexposed to novelties. The appearance of new technologies and new creams remains in most cases unsubstantiated by scientific studies and, when studies do exist, they are generally associated with the manufacturers of the product. It is extremely important for the dermatologist to be aware of the studies that are available, to be critical of them and to have information on the evaluation methods that were used in these studies in order to analyze the actual validity of findings.

Histopathology and immunohistochemistry are important evaluation techniques in a research study; however, in the case of an in vivo study, the patient has to be submitted to skin biopsy. In this case, choosing the appropriate site is fundamental to ensure that no methodological failures occur and, without doubt, to guarantee optimal final esthetic results with respect to scarring.

Morphometry is a more objective method than histopathology for the quantification of the histological changes found in any given study. It is common to find histopathology images showing an improvement following treatment. It is important to emphasize that single histopathology findings may not reflect the actual results. For this reason, techniques must be used that objectively quantify various randomly selected areas prior to and following treatment, thus avoiding possible errors in reporting results. Morphometry may be performed not only on a histopathological specimen but also using digital photography.^17,18,26 The inconvenience is that this is a method that requires specialized training in this area and specific equipment.

Digital photography is without doubt an extremely important tool for use in research studies; however, it generally has to be used in association with other methods. Accurate analysis will be successfully achieved when the technique of photography is used appropriately. For comparative before-and-after photographs, lighting must be identical and the patient must be photographed in the same position.²⁴

Principally used to evaluate the mechanical properties of the skin, biometry is often applied in studies in the area of pharmacology to evaluate the efficacy of cosmetics and cosmeceutics. With this technique, it is possible to measure the state of oiliness and hydration of the skin, as well as other properties; however, each modality of evaluation requires a specific device.^27,29,30

Optical profilometry is a method that has already been widely used in scientific studies. It must be performed by a very well-trained professional, since the silicone replicas of the skin surface should be meticulously made so as not to yield false results.³³ In view of this limitation, the technique is seldom used nowadays.

Confocal microscopy offers advantages compared to conventional histopathology, since it involves no incisions and provides a good quality image. Nevertheless, it is a method that has limitations because of issues such as the size of the equipment, the optical technique that is prone to visual artefacts, the technical/physical challenge imposed by the limited depth of optical penetration of currently available lasers and, finally, the educational challenge related to the visual interpretation of the images by physicians.³⁷

CONCLUSIONS

There are various methods for the analysis of intervention outcomes within research protocols in the field of cosmetic dermatology. It is of fundamental importance to identify the objective of the study in question and then to opt for an evaluation method or even a combination of methods. With this approach, the subjectivity so often found in the conclusions presented in studies in this field of medicine would be eliminated.

To consolidate the advances in cosmetic dermatology even further, the use of reliable techniques are necessary to guarantee the validity of data resulting from scientific studies.

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