Quantitative evaluation of patch test reactions: a comparison between visual grading and erythema index image analysis

Background/aims: The interpretation of patch test reactions may vary between examiners. As test results are graded, an issue also arises when differing degrees of erythema are placed in the same grade. The purpose of this study was to quantitatively evaluate the degree of erythema in patch tests using image analysis and to study the usefulness of this method by comparing it with visual grading. Methods: A total of 121 Japanese patients were patch tested with various materials. At 48 h, digital photographs of the patch test areas were taken, in addition to a visual evaluation by dermatologists. Digital images of the areas were converted to erythema index (EI) images using image processing and both EI and ΔEI (the difference between the patch test site and adjacent normal skin) values of the patch test sites were compared with the corresponding visual grades. Results: An excellent linear correlation (r=0.95) was found between ΔEI and visual grades, although EI also significantly correlated with visual grades. There were significant differences (P<0.0001–0.05) between the mean ΔEI values of any two adjacent visual grades. Conclusion: ΔEI values derived from image processing appear to be suitable for the quantitative evaluation of erythema in patch tests. This method may be helpful in overcoming the subjectiveness of visual evaluation and for training non‐experts in patch testing.     

Quantitation of contact allergy in guinea pigs by measuring changes in skin blood flow and skin fold thickness

Skin blood flow determined by laser Doppler flowmetry (LDF) and skin fold thickness (SFT) have been used to quantitate allergic contact dermatitis in the guinea pig maximization test (GPMT) using chlorocresol as the allergen. The closed patch test procedure itself influenced both LDF and SFT measurements when determined in 12 sham-treated guinea pigs. The LDF was maximal at 24 hours and the SFT at 48 hours. Before correlating the quantitative measurements with the conventional visual scoring in test and control animals the value from a nearby control site was subtracted from the test site values. The correlations were highly significant (p less than 0.001-0.05) indicating that the quantitative methods were useful supplements to the visual scoring as a measure of interobserver and interlaboratory differences.     

Comparative quantitative analysis of ultraviolet B-induced skin blood flow change using laser Doppler perfusion imaging technique

Laser Doppler perfusion imaging (LDI), recently developed, can generate a color-coded image of tissue perfusion, making it possible to assess the spatial distribution of skin perfusion without touching the surface. Using this apparatus, we investigated ultraviolet B (UVB)-induced perfusion profile of the skin and compared the results with those obtained from laser Doppler flowmetry (LDF). Fifteen adult male Korean volunteers were irradiated with several doses of UVB ranging from 25 mJ/cm2 to 186 mJ/cm2. Twenty-four hours later, the erythema reaction was evaluated with LDF and LDI systems. There was a significant correlation between the logarithmic dose of UVB and erythema values. The curves consisted of two parts, an initial, flat phase and then a linear, steep one. Also, there was a good correlation between LDF and LDI. The LDI is as sensitive as conventional LDF, but has the many advantages of measuring blood flow over large areas without contact with the skin surface. This instrument will be useful in the measurement of skin blood flow in many areas of dermatological application.     

Evaluation of contact allergy to chemicals using laser Doppler flowmetry (LDF) technique

Skin blood flow in allergic contact reactions and cross-sensitivity were evaluated using laser Doppler flowmetry (LDF) to study the dose-response relationships in phases of induction and challenge in guinea pigs. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with different doses of DNCB and 2,4-dinitrobenzene sulfonic sodium salt (DNBS). The skin reactions were evaluated by LDF and visual reading score. The results indicated that there were dose-response relationships between the doses of DNCB and LDF measurements in both phases of induction and challenge, that there was a cross-reaction between DNCB and DNBS, and that the reactions at 24 h were greater than that at 48 h after removal of the patches. LDF may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. This is because vascular dilatation and increase of flow already reaches a maximum in weak reactions. The more advanced phases are dominated by oedema formation. This is simply the nature of the inflammatory reaction, rather than a methodological error. The important point is that LDF can separate positive reactions from negative/uncertain reactions. The results indicated that LDF, as a non-invasive technique, may objectively and quantitatively evaluate the dose-response relationships of contact sensitivity of sensitizers.     

Topical non-steroidal anti-inflammatory drugs inhibit non-immunologic immediate contact reactions

The effects of 3 topical applications of non-steroidal anti-inflammatory drugs (NSAIDs), diclofenac 1%, naproxene 10% and salicylic acid 5% in gel vehicles, on immediate contact reactions to 500 mM benzoic acid, 500 mM cinnamic aldehyde, 50 mM methyl nicotinate, all in pet., and 14.1 M (100%) dimethyl sulfoxide, were studied in 16 medical students. Erythema and edema reactions were observed visually, and the changes in the skin blood flow were monitored using laser-Doppler flowmetry (LDF). NSAIDs had significant inhibitory effects on erythema induced by all 4 substances tested and on edema induced by methyl nicotinate. The inhibition was probably due in part to percutaneous absorption and systemic effects of NSAIDs. There were differences between visual and LDF assessments, which stresses the importance of monitoring erythematous reactions of the skin both visually and with LDF.     

Loss of contact sensitization evaluated by laser Doppler blood flowmetry and transepidermal water loss measurement

Allergic contact dermatitis, which is mediated by activated T cells through a Type IV reaction, is usually believed to persist throughout life. There have been several reports of spontaneous loss of sensitization, e.g., loss of previous patch test reaction or loss of allergic contact dermatitis withal continuous exposure to the allergens. However, these have not been well quantified. The aim of this study was to confirm the development of desensitization or hyposensitization with the aid of objective measurements. A total of 18 patients with alopecia areata, who had received 4 months diphenylcyclopropenone (DPCP) contact immunotherapy, were patch tested and the responses were measured by laser Doppler blood flowmetry (LDF) and transepidermal water loss (TEWL). Patch tests were performed 2X, before and after 4 months of immunotherapy. To determine whether local desensitization developed, we compared change of DPCP concentrations producing mild contact dermatitis on the scalp. Our results showed that systemic and local hyposensitization occurred in human subjects by weekly applications of low concentrations of DPCP for 4 months. LDF seems to be more correlated with visual scores than TEWL measurement.     

Comparison between visual score and erythema index (DermaSpectrometer) in evaluation of allergic patch tests

Background/aims: During the last decade several new bioengineering methods have been proposed for evaluation of patch test reactions in a more objective manner. The aim of the present study was to investigate the usefulness of erythema index (DermaSpectrometer) in a clinical setting, i.e., reading of allergic patch tests. Methods: Twenty patients with known allergy to formaldehyde participated in the study. Each patient had patch tests for 2 days with formaldehyde solutions from 0 to 10,000 p.p.m. applied. Clinical reading of the test sites and measurement of the erythema index by the DermaSpectrometer were done 24 h after removal. A control group of 20 volunteers with no allergy to formaldehyde were tested in a similar way. Results: Erythema indices were significantly higher for visually rated positive patch tests than for negative tests (P<0.05). The single categories of visually positive reactions (+?, +, ++, +++) could not be unambiguously separated by the DermaSpectrometer. The correlation between clinical readings and the formaldehyde concentration (Spearman's rank correlation coefficient, r=0.60) was higher than between DermaSpectrometer readings and the formaldehyde concentration (r=0.35). Conclusion: In a dilution series of formaldehyde patch testing, readings from a DermaSpectrometer were not found to give better information than visual readings.     

Erythema-index of clinical patch test reactions

Background: Methods for the precise non-invasive and continuous grading of disease intensity are potentially of great use in a clinical setting. The erythema index offers a good method for assessing UVR induced erythema. Due to the development of a convenient hand-held spectrometer, it is possible that the method could be used for the grading of eczematous reactions in a clinical setting as well. Objective: To assess the usefulness of the erythema index for the quantification of eczematous reactions using the Derma-Spectrometer (Cortex technology, Hadsund, Denmark) in a clinical setting. Method: The erythema index of 56 patch test reactions ranging from +? to +++, was compared to regional controls and negative patch tests (189). The effects of intrumental application pressure was studied in 5 volunteers. Statistical analysis was carried out using Mann-Whitney and Jonckheere-Terpstra tests. Results: The erythema-index was significantly higher in all degrees of patch test reactions than in uninvolved regional skin or negative patch tests. It also showed a significant positive trend for higher values in +, ++ and +++ reactions (P<0.0001). In direct comparison, the erythema index was unable to distinguish between?+ and +, or between ++ and +++ reactions. Only the distinction between + and ++ appeared significant (P<0.04). It is suggested that application pressure of the instrument is only of importance in high grades of erythema. Conclusion: Measurement of erythema index in eczematous reactions appear to be related to the intensity of the reaction, although the method does not reflect the many other changes which occur with increasingly strong reactions (oedema, etc.). The method therefore offers an objective quantitative supplement to clinical grading, especially in weak reactions. It is suggested that the method is better suited for experimental studies with a strict control of confounding factors.     

Ultraviolet B dose-dependent inflammation in humans: a reflectance spectroscopic and laser Doppler flowmetric study using topical pharmacologic antagonists on irradiated skin.

Normal skin responds acutely to ultraviolet (UV) light exposure with complex inflammatory mechanisms. In the present study UVB irradiation ranging from subclinical erythema doses to twice the minimal erythema dose (24 mJ/cm2 to 96 mJ/cm2) was delivered to the skin of 8 volunteers. Pre-irradiated sites were immediately afterwards exposed to a 24-h occlusive patch containing 1 of 4 anti-inflammatory agents or vehicle control. The resultant change in erythema (vascular reaction) was measured objectively using laser Doppler flowmetry (LDF) and reflectance spectroscopy (RS). The 4 anti-inflammatory compounds reduced the UVB-induced vascular reactions in different and dose-dependent ways. Betamethasone-17-valerate and diphenhydramine were most effective at the 24 mJ/cm2 dose site and indomethacin and acetylsalicylic acid were more effective at sites > or = 48 mJ/cm2. Ranking the reduction in oxygenized hemoglobin (OH) content was as follows: betamethasone-17-valerate (OH reduction = 37.4%) > indomethacin (OH reduction = 21.5%) > acetylsalicylic acid (ASA) (OH decrease = 21.0%) > diphenhydramine (OH reduction = 18.4%). Using LDF, the total ranking of the cutaneous blood flow (BF) reduction was: indomethacin (BF reduction = 39.7%) > betamethasone-17-valerate (BF reduction = 32.7%) > acetylsalicylic acid (BF decrease = 17.5%) > diphenhydramine (BF reduction = 12.3%). Diphenhydramine significantly reduced erythema only at the lowest irradiation dose (24 mJ/cm2) and the decrease in OH was associated with an increased amount of deoxygenized hemoglobin (DOH). A similar slight venous dilatation was present at acetylsalicylic acid-exposed sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term repetitive sodium lauryl sulfate-induced irritation of the skin: an in vivo study

Skin may adapt to topical irritants through accommodation. This study focuses on long-term exposure to irritants and attempts to demonstrate accommodation. Sodium lauryl sulfate (SLS) induced irritant contact dermatitis at 3 concentrations (0.025% to 0.075%). Distilled water, acetone and an empty chamber served as controls. Experimental compounds were applied to forearms of 7 healthy volunteers for 24 hr before replacing by a fresh chamber for 6 non-consecutive weeks over 103 days. Possible accommodation was quantified by visual scoring (erythema and dryness) and by bioengineering parameters: transepidermal water loss (TEWL), capacitance, chromametry and laser Doppler flowmetry (LDF). Significant erythema, dryness, elevated TEWL, skin colour reflectance and LDF values occurred during the exposure periods. Upon repeat exposure, an immediate and augmented response in erythema, TEWL, skin colour reflectance and LDF developed. However, irritant skin changes were not sustained. Irritation parameters return to baseline after cessation of exposure. There was no evidence of sustained irritation or accommodation after the last exposure. Study findings do not document sustained accommodation or adaptive hyposensitivity after long-term repetitive irritant exposure under these test conditions. Alternative models should be developed to prove or disprove the accommodation hypothesis.     

Simultaneous assessment of blood flow in UVB-inflamed human skin by laser Doppler flowmetry and the 133-xenon washout technique.

The purpose of the study was to compare skin blood flow by laser Doppler flowmetry (LDF) and the 133-Xenon washout technique in UVB-inflamed human skin. Six healthy subjects participated in the study. Forearm skin blood flow was measured prior to irradiation and then 8, 24, 48 and 72 h after the exposure to twice the minimal erythema dose of UVB. Baseline blood flow as measured by the 133-Xenon washout method was 5.2 (range 3.0 to 10.4) ml/100g/min and LDF flux was 3.1 (range 2.7 to 5.7) arbitrary units. Following irradiation, maximum blood flow increase as evaluated by the 133-Xenon washout was 10.8 (95% confidence interval 3.7 to 11.3) times baseline blood flow, corresponding to an absolute blood flow of 47.5 (range 33.8 to 56.3) ml/100g/min. Maximum increase after induction of inflammation as assessed by LDF was 34.6 (95% confidence interval 24.6 to 56.5) times baseline flux. The two methods showed comparable time courses in all subjects. A significant correlation between the two methods was found, Spearman's rho = 0.54, p = 0.006. The relative LDF blood flow increase was 4.2-fold (95% confidence interval 2.7 to 5.0) greater than the increase measured by the 133-Xenon washout method. These results are at variance with previous comparative studies of the two methods. Some explanations are discussed.     

Irritation and staining by dithranol (anthralin) and related compounds. IV. Visual estimation of erythema compared with contact thermometry and laser Doppler flowmetry

Irritation and staining caused by equi-irritant doses of dithranol (anthralin) and 10-butyryl dithranol (butantrone) were observed for one week after a single 24-hour exposure under occlusion. The test chambers were applied on the uninvolved dorsal skin of 11 psoriasis patients. The estimates of erythema were made with a refined reading scale, and to test the visual discriminatory power they were compared with objective measurements of contact temperature and blood flow of the skin. A sensitive thermistor and a novel laser Doppler flowmeter were used. On the whole, erythema, contact temperature and superficial blood flow, as interdependent parameters of cutaneous inflammation, all showed a time-dependent statistically significant correlation to the dose of dithranol and 10-butyryl dithranol. The intercorrelations between erythema and blood flow and between erythema and temperature were statistically significant, too. With the doses used, staining of the skin did not hamper the measurement of superficial blood flow, but it did exaggerate visual estimates of erythema at the 4th and 7th day readings.     

Local cold exposure test as therapy control in patients with Raynaud's phenomenon: comparison between laser Doppler fluxmetry and simultaneous red blood cell velocity measurements in nailfold capillaries

Summary Synchronous measurement of laser Doppler flux (LDF) and capillary red blood cell velocity (CBV) was performed in adjacent areas of the same nailfold during a local cold stress test in 12 healthy controls (eight women and four men) and in 22 patients (17 women and seven men) with secondary Raynaud's phenomenon before and after treatment. Two questions were addressed: Are there any differences in the signal pattern between LDF and RBV? Is it possible to detect early on in therapy, before clinical benefit becomes obvious, whether a treatment is successful or not? Despite the fact that the resulting signal patterns recorded by these two techniques are widely compatible, certain differences could be observed. In healthy controls, decrease of values during cooling time and increase after cooling were more distinct in RBV than in LDF. Compared with control values, CBV and LDF in patients with Raynaud's phenomenon were lower. After cooling CBV took an average of 3 min to reach initial value again as compared with 40s in healthy controls. During 4 min observation time, pretest values of LDF were not achieved again in patients, whereas it took 50s in healthy controls. If, after a few days of vasospasmolytic therapy, test results improved or normalized, clinical symptoms subsided gradually during the next weeks. Clinical improvement was not observed in those patients in whom cutaneous blood flow remained decreased despite therapy. CBV indicated this more clearly than LDF. Duration of flow stop at the end of cooling showed a marked improvement in patients treated successfully. Discrepancies between CBV and LDF are interpreted as being due to LDF detecting other vessels in addition to the superficial, nutritional capillaries. LDF seemed to be a poor tool for evaluating the effect of treatment. Determination of CBV and flow stop duration during local cold exposure may help in early selection of the best treatment for a patient with Raynaud's phenomenon by predicting later possible clinical benefit.     

Evaluation of skin susceptibility to irritancy by routine patch testing with sodium lauryl sulfate

Irritant patch testing with sodium lauryl sulfate (SLS) will become more and more a routine test determining skin susceptibility in men. Recently, it has been shown that for practical reasons, irritant SLS patch testing can take place on the back simultaneously with a routine allergic patch test to other contact allergens. However, SLS patch testing has mostly been performed on the forearm in studying experimental skin irritation so far. The aim of this study was to determine whether there is a relationship in skin response to aqueous SLS (0.125%; 0.25%; 0.5% and 1.0%) between the forearm and the back assessed by visual scoring and measurement of transepidermal water loss (TEWL). We found a pronounced reaction of the forearm compared to the back. TEWL values as well as visual scores correlated well with SLS concentration. There was also a high correlation in visual scoring between the forearm and the back. Based on test sensitivity and specificity we suggest a 48 hrs patch test for routine screening with 0.5% SLS on the forearm evaluated by TEWL measurement or visual scoring 24 hrs after patch removal. A mild erythema (scored as < or =1) is considered to be normal. If for practical reasons, the SLS patch is placed on the back simultaneously with the allergic patch test, 0.5% SLS may be sufficient, too. TEWL measurement so far provides a reliable method and will certainly be necessary for experimental studies on irritant skin reactions, particularly when different SLS concentrations are used. After a 48 hrs patch test with SLS 0.5% TEWL measurement should be performed at 72 hrs. A value of < or =31.6 g/m(2)hr seems to follow the normal distribution.     

Objective assessment of nickel sulfate patch test reactions with laser Doppler perfusion imaging

The laser Doppler perfusion scanning technique was used to assess the superficial blood flow of nickel sulfate hexahydrate patch test reactions. There was good agreement between laser Doppler and visual assessments when the highest assessment values of reactions were studied. Earlier detection of reactions was possible with the laser technique. There was great inter-individual variance in perfusion between identically tested patients. 4 patients were visually negative when a TRUE Test^TM patch test dose of 0.20 mg/cm² was applied for 48 h and the test area read 4× up to 168 h. These 4 showed a dose-related increase in perfusion and visually positive reactions using longer application times. The instrument allowed a dose reduction not possible for visual assessments. Reading transparent patches in contact with the skin through transparent semi-occlusive plastic foil or through windows in the tape strip over the patches, allowed us to detect perfusion at 48 h, where a longer application would have been needed using tape and visual assessments.     

The irritant effect of different metalworking fluids

The irritant effect of different water-based cutting fluids (CFs) on the skin was investigated in healthy subjects ( n = 10) using 2 non-invasive bioengineering methods. Transepidermal water loss (TEWL) was measured by an evaporimeter EP1 (Servo Med), skin blood flow (SBF) was recorded with a laser Doppler flowmeter (MBF 3D, Moor Instruments England). Additionally all test sites were evaluated by visual scoring before measurement. 3 cutting fluids A, B and C from different manufacturers were tested at use concentrations of 4% or 5%. Single 24-h patch tests and repeated patch tests were performed on the volar side of the right forearm. Measurements were carried out before and after exposure to assess basal values and the skin response to CF. Tests with CFs A and C resulted in significant increase in TEWL after 24 h and after cumulative treatment, compared with controls ( p < 0.01). The TEWL values for CF B did not differ from the water probe. An increase in SBF was found only for CFs A and C after cumulative patch testing ( p < 0.01). In spite of their similar alkalinity, the 3 CFs showed different irritant skin reactions, due to their chemical components. This was verified particularly by measurement of TEWL.     

Allergic and irritant skin reactions evaluated by laser Doppler flowmetry

Skin blood flow (SBF) in allergic and irritant reactions was determined by laser Doppler flowmetry (LDF) in an attempt to differentiate these reactions in an objective way. 12 subjects with known allergy to nickel were patch tested with nickel sulphate, nickel chloride, and with sodium lauryl sulphate (SLS) 1, 2, 5 and 10%. A positive correlation between the concentration of SLS and SBF was found. Compared with control sites, SBF in weak (+) and strong (++) positive allergic and irritant reactions was increased approximately 5-10 fold 1 and 2 days after application, and decreased towards normal 7 days after application. At day 7, the mean SBF was higher in allergic reactions than in irritant reactions. However, no consistent differences were found. It is concluded that although LDF can be used to quantify the strength of allergic and irritant skin reactions, this method cannot be used solely to separate these 2 types of reaction.     

The calcipotriol dose–irritation relationship: 48 hour occlusive testing in healthy volunteers using Finn Chambers®

Calcipotriol is widely used in the treatment of psoriasis. Adverse lesional and perilesional irritation may occur. Allergy may occasionally be suspected. Allergy patch testing with calcipotriol may be difficult or impossible because calcipotriol is a local irritant. The aim of the present study was to assess the calcipotriol dose–irritation relationship, and establish a non-irritant patch test concentration for calcipotriol allergy patch testing. The study was a prospective, double-blind, randomized, dose titration evaluation in 180 healthy volunteers never previously exposed to calcipotriol. All individuals were patch tested with a calcipotriol dilution series (range 0.016–250 μg/mL). Clinical reading of test sites and measurement of erythema using a Minolta ChromaMeter were performed on days 2 and 3. Laser Doppler perfusion imaging of cutaneous blood flow was performed on day 3.
Doubtful reactions (score ½) and weak reactions (score 1) were frequent and observed even at low dose exposure. Reactions declined in strength between the readings on day 2 and day 3. Only score 2 reactions with moderate erythema and some infiltration showed a threshold of no irritation. This threshold was confirmed by colorimetry and flowmetry. Cases of suspected allergy to calcipotriol may, to avoid irritant reaction and false positive readings, be patch tested with calcipotriol 2 μg/mL citrate-buffered isopropanol solution applied under occlusion for 48 h using small Finn Chambers^®. Score ½ and 1 reactions are likely to reflect irritation. A positive test should be repeated after a minimum period of 3 months to ensure its consistency over time. A repeated open application test may be indicated.     

Individual and instrumental variations in irritant patch-test reactions—clinical evaluation and quantification by bioengineering methods

A major purpose of irritant patch testing is to differentiate between normal, delicate and less sensitive skin. To assess the usefulness of irritant patch testing, knowledge of variation in responses to identical patch tests is essential. In the present study inter- and intra-individual variation in irritant patch test reactions due to sodium lauryl sulphate and nonanoic acid is given when evaluated by traditional visual scoring as well as different non-invasive methods, i.e. measurement of transepidermal water loss, the hydration state of stratum corneum by electrical conductance, cutaneous blood flow by laser Doppler flowmetry, and measurement of skin thickness by 20-MHz ultrasound A-scan. The intra-individual 'site-to-site' variation was considerably less than the inter-individual variation, which is essential to differentiate between persons with delicate and less sensitive skin. Methods used were relevant for this purpose except for the measurement of electrical conductance.     

Atopy patch tests in young adult patients with atopic dermatitis and controls: dose-response relationship,objective reading,reproducibility and clinical interpretation

The clinical interpretation and reproducibility of atopy patch tests was studied in 23 selected young adult patients with atopic dermatitis and 25 healthy controls using standard inhalant allergens. Non-invasive measurements were used for objective assessment of test reactions and the participants were retested after 6 weeks. Ten of 19 (53%) evaluable patients with atopic dermatitis had at least one positive atopy patch test. However, there was no clear clinical relevance of the atopy patch test results when related to patient history and distribution of dermatitis. Reproducible and dose-dependent results were obtained with Dermatophagoides pteronyssinus, grass and cat with a reproducibility rate of 0.69 to 0.81 in patients and 0.60-0.96 in controls. A unique finding was a significant positive correlation between a positive atopy patch test, allergen dose and increase in transepidermal water loss and erythema, while measurement of capacitance did not distinguish between positive and negative reactions. The results of the present study do not support the routine use of atopy patch tests in the evaluation of adult patients with atopic dermatitis.