9.2.4 Non-invasive Methods in Dermatology: Functional Tests

A great part of dermato-venereological diseases can be diagnosed by the naked eye, the palpating finger and the appropriate long-term experience and expertise in the clinical setting. However, several situations in diagnosing diseases and monitoring treatment efficiently need invasive techniques such as the gold standard of dermatopathology ( see chapter 9.2.2 ) and / or non-invasive techniques such as imaging technology ( see chapter 9.2.1.). In addition to these, functional tests have been developed over decades to give measurable objective data of the skin and its appendages in health and disease. Some tests are easy to perform, others require more advanced technology, material, time and energy.

Tests for epidermal function include in particular those measuring desquamation, hydration and transepidermal water loss, pH and colour including pigmentation which together with some other more specific ones are a common test set. Several stand alone or computer assisted devices are on the market. For other methods see chapters mentioned above.

The epidermal barrier function is composed of a multifunctional physical and biochemical structure and different set of functional tests of the skin exist (see also chapter 9.1.1. to 9.1.4 ). Its measurement includes methods such as those written below. Several methods used together deliver a good overview on the current status of function of the skin.

Str.corneum cell desquamation is a permanent and essential physiologic part of normal skin. Different factors may influence via outer and inner environmental signals the degree of desquamation (hyperproliferation of basal layer, dryness, repair signals, wounding, cytokine signals, genetic factors ,clothes, profession). There are for example dermatoses characterized  by high degree of turnover of cells, such as psoriasis, and others such as some ichthyoses with low degree of turnover by retention hyperkeratosis. Aging of the skin mostly has a low turnover.

Instruments to measure the desquamation are chamber techniques with a device to include the loose scales of the upper skin layer. One can measure serial tape strippings to evaluate the recovery function of the epidermis. Densitometric and light reflectance analysis of the adherent scales together with a specialized software calculates the desquamation index.

Epidermal hydration is essential for skin integrity. If skin becomes dehydrated by increased loss or by decreased supply, function is disturbed and the barrier breaks off.

Hydration of the skin can be measured by electrical conductance, impedance or capacitance. Corneometers (Evaporimeter /Tewameter) are used to measure for example the loss of hydration due to mechanical stress of the hands in occupational diseases such as hand eczema or the success of external rehydration via a moisturizer. Trans-Epidermal Water Loss (TEWL ) is measured in g/hcm2 . The system measures the water vapour flow density: JV= g/(m x h) (mass of water vapour (in g) per area (in cm2) and time (in h)).

The pH value is a biomarker for the skin’s natural acid mantle (around pH 5). Not only the hydration from the pressure of water from the inside via capillaries and take up by the epidermal cells but the amount of sweat from eccrine and apocrine glands, lipids from the epidermal and the sebaceous cells are important. A neutral pH value can lead to lower resistance to infections, barrier function disorders and the spread of skin diseases.

Oxygen tension can be measured as a function of capillary oxygen supply to the epidermis which is reduced during aging, loss of capillary network in diabetes ,chronic renal failure, sclero- and pseudoscleroderma but may increased to vasodilatation in inflammation or drug related.

Skin pigmentation is roughly related to the skin types I to VI. However, those are not helpful in measuring the individual persons skin pigmentation and changes due to UV light exposure or chemical or other physical stimuli acting on melanocytes . For testing the efficacy of light protection factors in sun screens for example the use of Colorimetry (reflectance methods via chromameter) is ideal. The method helps to identify not only the degree of melanin but also the other physical colour components which contributes to skin colouring ( CIE colour system / L*a*b* system). For example inflammation redness or colour of skin deposition materials can be measured.

Tape stripping can be used to measure the recovery time of epidermal basal cells or for allergen testing (see below). A simple technique to evaluate if Malassezia spores or hyphae are on the skin surface is to make a tape strip ( 5-10 x) and to glue it on a glass slide. With a droplet of methylene blue, the microbe can be visualized under the microscope.

Mechanical properties of the skin are in particular influenced by the function of the dermis. The function of elasticity in skin aging, in scleroderma or the adherence of the epidermal-dermal connection at the basal membrane are targets for twistometry, identometry, ballistometry or suction blister techniques.

Twistometry is used via a disk glued to the skin which is rotated via a small motor thereby measuring the twisting capacity to a given force. An indirect measurement of strength and vitality of elastic fiber and collagen matrix in healthy and diseased skin is possible. Another technique is Levarometry. 

Identometry Most used instrument is the Cutometer working with a light source and negative air pressure giving results on the elasticity of the dermis. The light intensity depends on the penetration depth into the skin. The resistance of the skin to the negative pressure (firmness) and its ability to return into its original position (elasticity) display the functional capacity.

Frictiometry is a method using a constant pressure on the skin by the weight of a rotating disk which is incorporated in handhold device. It gives results on the elasticity and friction related return to normal and disturbances (loss of elastic fibers, atrophy, sclerosis).

Skin temperature is related to body temperature and blood flow as well as fever signals (cytokines, HSP neuroregulation and others). Thermoregulation detected by thermal imaging devices gives an exact overview on different areas of the body surface. Skin functions as a thermal interface between body and environment. The temperature or heat flow can be measured by conduction (contact thermometry), convection (Schlieren`s photography) or radiation imaging (thermal infrared cameras).

Follicular biopsies are performed to measure the content of the follicular infundibulum and, in particular, to count the number of microcomedones per area. A droplet of cyanoacrylate is applied on a glass slide, then placed with light pressure for example on the forehead and removed after 30 – 60 seconds. Under the microscope one can count on the surface of the glass slide the number of microcomedones important in acne. It is also used for counting demodex mites in rosacea. The follicular cast can also be used for measuring the number and types of other microbes, content of drugs and lipids produced by the gland.

Sebumetry is the method to measure the temporary composition of surface lipids and amount of lipids coming from the follicular canal of the gland in quantity and quality and for monitoring over time. Different methods are used. Sebutapes are microporous adherent tapes applied on defatted skin. The lipids are absorbed and can be measured by image analysis. The gravimetric technique is of much higher precision. It uses absorbent paper after defatting of the forehead skin, left there over three hours and then under given conditions regarding air pressure and humidity the amount is measured according to mcg of sebum weight per area and time.

Sweat gland activity can be reduced or increased in several local or systemic conditions on the body in eccrine and/ or apocrine gland rich areas. For example, emotional stress, thermoregulation, paraneoplastic related or endocrine driven, drug induced or idiopathic reasons may stimulate its activity. Diseases such as cystic fibrosis, scleroderma, congenital ectodermal dysplasia, sympathectomy or alcohol and diabetes induced neuropathy can go along with reduced sweating or even anhidrosis (primary autonomic disorders with acquired anhidrosis). 

Methods to measure: filter paper collection, the quantitative sudomotor axon reflex test (QSART), silastic mold or iodine-impregnated paper imprint after pilocarpine stimulation. Sweat is visualized by a topical indicator such as iodinated starch or sodium alizarin sulfonate (alizarin Red S).

The iodinated starch powder test is prepared by adding 0.5–1.0 g of iodine crystals to 500 g of soluble starch. For smaller body areas, a Minor’s starch-iodine test for excessive sweating may be performed by applying betadine on Q-tips to the affected area. Once the iodine solution has dried, cornstarch can be lightly sprinkled on the area. Deep purple color change will develop over time in the areas affected with hyperhidrosis.The amount of sweat secretion per minute and cm2 is practicable for definition of hyperhidrosis. Those areas demarcated can be used for injection of botulinum toxin (hands, axillae) or for sweat gland subcutaneous currettage (axillae).

A new customized camera device designed to quantify secretions of individual sweat glands (SGs) after sweating was stimulated on a 2.25 cm2 skin area by iontophoresis of pilocarpine can be used The camera imaged sweat from 50 to 400 sweat ducts. One calculates secretion rate of individual SGs, total sweat volume, and number of secreting SGs of different body sites.

Different methods exist for evaluation of hair loss and hair growth or the strength and elasticity of hair. Several diseases affecting the hair growth need a biopsy for histological evaluation.

Trichogram is a classical method to study the proportion of anagen, catagen and telogen hairs and its proportion. In addition, dysplastic and dystrophic or broken hairs can be counted. For example to study the degree of hair loss in androgenetic or androgenic alopecia one samples a bundle of hair from the frontal or parietal and from the occipital hair line after 5 days non washing the hair. With forceps, the hair is pulled out of its roots and moved to a glass slide, imbedded in Corbit balsam and evaluated under the microscope.

Phototrichogram is a non invasive and reproducible method to quantify hair density and hair growth parameters . The hairs at a selected site are trimmed for the applied diagnostic optical equipment ( 0.8-1.5 cm ). With a syringe with insulin needle or with a tattoo apparatus basically invisible by bare eye tattoo marks are set. The test area is covered with a coverslip glass to allow the positioning of the trichoscope lens. An image is analyzed by special computer program evaluation. The program will calculate the total number of hairs /cm2 as well as the number of terminal and vellus-like, anagen and telogen hairs.

For methods see chapters 5.1.2. and 9.2.1. (ultrasound imaging, laser flow assessment).

Glasspate/glass slide

A simple glasspate/glass slide can be used to evaluate if a bleeding like lesion of the skin is a vessel dilatation or a permanent capillary defect with erythrocytes outside the vessel wall. Small pressure to the lesion followed by diminishing or complete abolishing of the erythema speaks towards functional reasons. If the erythematous lesions (petechia, purpura) stays a capillary leakage of different origin can be supposed.

The glasspate can also be used to test the capillary reflex related to pressure and friction followed by erythema or whealing. Paradoxical whitening of the provocated skin area for example can be seen in atopy constitution. Whealing is a sign of dermographism of acute and persisting type and needs further evaluation of the skin or other organs (mastocytosis, physical urticaria, deaminoxidase deficiency). See also chapter urticaria / physical urticaria.

Test techniques for different test procedures and protocols regarding allergies are highly standardized. Irritant patch testing, standard epicutaneous patch testing and photo-patch testing or prick testing are the most widely used ones.

Epicutaneous contact allergy testing is used to evaluate amount and degree of and to differentiate allergic and irritant factors behind eczemas. Standardized or in some cases individualized substances in appropriate vehicles are applied via for example Finn chambers with occlusive protection to the back of a patient. It is left there for 48 hrs and removed for first reading according to a grading protocol. Additional readings follow after 72 and 96 hrs. Some substances require reading after 120 hrs or one week. A record is to be written and a test protocol handed out to the patient.

Photopatchtesting is performed if suspicious agents/allergens need photoactivation to become immunologic. In addition to the standard test one set of suspicious allergens is additionally irradiated with UVA light and the read out follows the same protocol.

Prick Testing is used for evaluating type I allergy. Standardized solutions of allergens compared to a NaCl and a histamine control field are applied to the skin in a droplet . Each individual droplet is then separately pierced with a lancet,transporting the substance in the epidermis and papillary and mid dermis. Reading of positive wheals according to standardized reading protocols follows continuously and after 20 minutes it is recorded. Sometimes reactions appear later. A record is to be written and a test protocol handed out to the patient.

Other tests not mentioned here in detail are scratch - ,rubber-, prick-to-prick or intradermal test. Systemic provocation tests for drugs, food ,preservatives and colouring of food allergies are mentioned in the chapters on special diseases ( see chapters….).

Sensation tests are done in the peripheral nervous system first in a dermatomal pattern . Tests for light touch, sharp/blunt, temperature and often two-point discrimination are the most common bedside tests (small vs large diameter nerve fibres).

Electromyography and nerve conduction tests are performed by neurologists to evaluate paresthesias and peripheral neuropathy. This concerns in particular dermatologic diseases such at leprosy or scleroderma, chronic graft versus host disease at the skin , late borreliosis or late stage syphilis. Patients with neurotrophic ulcers or diabetes mellitus , deposition disorders such as amyloidosis and traumatic nerve damage are other indications for testing.

Alloknesis is a specific test of peripheral nerve endings for evaluating itch signals. With a small brush one strikes over the skin. It is a primarily physiological and non-pruritogenic stimulus which leads in predisposed patients to different reactions of prickling, burning and / or itching to be measured on a score.