Lymphocytes and Langerhans cells in patch tests

The distribution of immunocompetent cells was analysed in allergic (nickel) and irritant (dithranol) patch tests using conventional transmission electron microscopy and labelling with monoclonal antibodies in an avidin-biotin immunoperoxidase study. The biopsies were taken 24 or 48 h after the allergen/irritant application. In allergic and irritant reactions, most inflammatory cells were OKT11 positive (pan T lymphocytes). The majority of these cells were also OKT4 positive (helper/inducer T lymphocytes), while the minority were OKT8 positive (suppressor/cytotoxic T lymphocytes). NK9 positive cells (natural killer cells) were observed in small numbers. The number of dendritic OKT6 and OKIal positive cells (Langerhans cells) in the epidermis was unaffected in allergic reactions. In irritant reactions, a normal number of OKT6 positive Langerhans cells was observed, while the number of OKIal positive cells had increased in the epidermis. Dithranol caused prominent fine structural changes in the mitochondria of the Langerhans cells, while the keratinocytes appeared largely unaffected. The present study indicates that allergic and irritant patch tests cannot be differentiated reliably using current immunohistopathological or electron microscopic techniques, in spite of the small differences observed.     

健康成人皮肤对十二烷基硫酸钠刺激反应的反射式共聚焦显微镜特征分析

目的 观察十二烷基硫酸钠（SLS）刺激后18 ~ 60岁健康人皮肤反射式共聚焦扫描显微镜（RCM）特征的差异,分析年龄和性别对皮肤反应的影响,同时初步探讨RCM在客观评价皮肤反应中的价值。方法 采用封闭式斑贴试验,分别将0.1%和0.5% SLS贴敷于120例健康受试者背部48 h,并于去除后不同时间点进行临床评估和RCM检测。结果 0.1%和0.5% SLS组皮肤刺激反应的RCM特征主要有角化不全、角质层结构不清、棘层海绵水肿、表皮炎症细胞浸润和真皮乳突毛细血管扩张等。去除0.1%和0.5%SLS 刺激后24 h,RCM特征发生率达到高峰,其中真皮毛细血管扩张的发生率分别高达66.7%和95.0%。0.5% SLS去除后24 h,男性海绵水肿的发生率为68.9%（42/61）,显著低于女性［84.7%（50/59）,χ2 = 4.24,P < 0.05］; 0.1%SLS去除后24 h,18 ~ 40岁年龄组人群棘层海绵水肿的发生率为53.3%（32/60）,显著高于41 ~ 60岁［35.0%（21/60）,χ2 = 4.09,P < 0.05］;其余RCM参数,在0.1%和0.5% SLS刺激去除后,不同性别或不同年龄组之间差异均无统计学意义（P > 0.05）。临床评估显示去除0.1%和0.5% SLS后24 h,男女皮肤刺激反应发生率差异均无统计学意义（均P > 0.05）,18 ~ 40岁和41 ~ 60岁两个年龄组之间差异也无统计学意义（均P > 0.05）。Spearman相关性分析显示,临床评估结果与RCM特征之间具有良好的相关性,其中,去除0.1% SLS后24 h,海绵水肿和真皮毛细血管扩张与临床评估结果的相关系数均高达0.77（P < 0.001）。但去除SLS 0.5 h后,0.1%和0.5% SLS组表现出2项以上RCM特征的受试者比例分别为17.5%（21/120）和51.7%（62/120）,比同一时刻临床评估的阳性率［2.5%（3/120）和12.5%（15/120）］更接近去除SLS 24 h后的临床评估结果34.2%（41/120）和85.0%（102/120）。结论 性别和年龄对0.1%和0.5% SLS诱导的皮肤刺激反应无明显影响;相对临床评估,RCM在刺激反应早期能更加客观准确地评估皮肤反应。     

Correlation between laser Doppler perfusion imaging and visual scoring of patch test sites in subjects with experimentally induced allergic and irritant contact reactions

Purpose: To compare laser Doppler perfusion imaging (LDPI) measurements in experimentally induced allergic contact reactions and irritant contact reactions. The degree of correlation between visual scores and LDPI measurements was also studied.
Methods: Fifteen patients with known contact allergy to nickel or fragrance were patch tested with nickel sulfate 5% pet., fragrance mix 8% pet., sodium lauryl sulfate (SLS) 0.5% pet., SLS 1.0% pet. and two empty control chambers. Visual readings and LDPI measurements were taken at 0, 48 and 96 h.
Results: There was a positive correlation between visual scores and LDPI measurement in the nickel sulfate and fragrance mix patch test sites. However, no correlation between visual scores and LDPI measurements was seen in the SLS 0.5% and SLS 1.0% patch test sites. There was no significant difference ( P =0.125) in LDPI measurements between contact-allergic reactions and contact-irritant reactions.
Conclusion: LDPI correlates with visual scoring in contact-allergic reactions, but not in irritant reactions. LDPI is not useful in distinguishing between allergic and irritant reactions.     

Reproducibility of irritant patch test reactions to sodium lauryl sulfate in a double-blind placebo-controlled randomized study using clinical scoring

Reading of doubtful patch test reactions can be improved by comparing them to dened negative, allergic and irritant controls. For the latter, an irritant patch test is needed that gives sufficiently reproducible results. In our double-blind, placebo-controlled, randomized study, we have analysed the synchronous reproducibility of patch tests with 0%, 0.0625%, 0.125%, 0.25%, 0.5% and 1.0% sodium lauryl sulfate (SLS). Tests and readings were done according to ESCD guidelines. 139 patients (75 women, 64 men) 18 to 77 years old were tested. The % of positive patients as well as the % of reproducible positive reactions increased with rising concentrations of SLS to a maximum of approximately 90% and 85%, respectively, with 1 % SLS. Time courses of reactions were also related to the concentration of SLS: with 1 % SLS, plateau-type time courses were most common (65%), followed by decrescendo- (23%) and crescendo-type reactions (12%). With 1% SLS, very similar mild to moderate reactions were obtained on days 2 and 3. The reaction threshold and reproducibility of tests were not related to age and sex of patients or to their own assessment of skin irritability. We conclude that 1% SLS is appropriate for an irritant patch test that gives sufficiently reproducible results.     

Allergic contact dermatitis from ophthalmic products: can pre‐treatment with sodium lauryl sulfate increase patch test sensitivity?

In patients suspected of allergic contact dermatitis because of topical ophthalmic medicaments, patch tests performed with patients' own products are often negative. The irritant anionic surfactant sodium lauryl sulfate (SLS) may alter the stratum corneum and increase antigen penetration. Pre-treatment of the skin with SLS 0.5% for 24 h was performed in the sites of patch tests with patients' own products in 15 selected patients. In patients previously negative to their own products tested with conventional patch tests, SLS pre-treatment showed 6 new relevant positive reactions and induced a stronger positive reaction in 1 patient. SLS pre-treatment could be proposed as an alternative promising method, which may increase sensitivity of patch tests with patients' own products.     

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.     

Irritant patch testing with sodium lauryl sulphate: interrelation between concentration and exposure time

BACKGROUND: It is well known that the degree of skin reaction to an irritant depends on its concentration and exposure time. OBJECTIVES: To determine the interrelationship between the concentration of sodium lauryl sulphate (SLS) and exposure time in both weak (subclinical) and severe reactions. METHODS: Patch testing with SLS was performed at different concentrations (0.125%, 0.25%, 0.5%, 1.0% and 2.0%) and with different exposure times (3, 6, 12, 24 and 48 h). Evaluation was conducted by measurement of transepidermal water loss and by laser-Doppler flowmetry both 30 min and 24 h after patch removal. RESULTS: We found more reliable and constant skin reactions 24 h after patch removal, and a higher correlation between SLS concentration and skin reaction. CONCLUSIONS: We conclude that the concentration of SLS influences the test outcome to a larger degree than the exposure time. We present formulae by which the outcome of SLS patch testing at various SLS concentrations ranging from 0.125% to 2% and any exposure time between 3 and 24 h can be estimated.     

The effect of grenz rays on irritant skin reactions in man

To investigate the effect of grenz rays on irritant contact reactions, eleven healthy volunteers were studied. They were given 3 Gy of grenz rays, once a week for 3 weeks, to a defined area of the back. Twenty-four hours after the last treatment, serial dilution sodium lauryl sulphate patch tests were applied both on the grenz ray treated area and on the untreated control skin. Biopsy specimens were taken from the irritant reactions both from the grenz ray treated area and from the control area and different cell populations in dermis and epidermis were identified by monoclonal antibodies (Leu 2, 3, 4, 7, Leu M1, B1, OKT6). In the grenz ray treated epidermis there was a pronounced reduction of OKT6-positive cells but the composition of the dermal cellular infiltrate did not differ between control and grenz ray treated skin. The assessment of the patch test reactions did reveal a tendency towards weaker reactions in the grenz ray pre-treated skin but this difference was not statistically significant. It is concluded that grenz rays do not have a marked effect on the elicitation of irritant reactions.     

Probable irritant contact dermatitis from phenylephrine in eyedrops with irritant (false-positive) phenylephrine patch test reactions

A 61-year-old white woman developed an acute eczema of the eyelids and periorbital region after application of eyedrops containing 10% phenylephrine hydrochloride (HCI) for an ophthalmologic examination. The patient showed false-positive (irritant) reactions both to eyedrops as is and phenylephrine HCI 10% aqueous after 48 hours, quickly fading at 72 hours, and unremarkable at 96 hours. There were false-positive results in 2 of 19 controls. Patch testing with phenylephrine HCI 1% aqueous, which is the correct testing concentration, was negative in both case patients and controls. Open tests showed a soaplike irritant effect only to 10% aqueous phenylephrine after 48 hours sharply marginated to the test site area. Repeated open application test further confirmed the above findings. Intradermal testing with phenylephrine showed no delayed reactions either in the affected patient or 19 controls. Irritant patch testing reactions do not necessarily indicate a diagnosis of irritant contact dermatitis; however, it is likely that in our patient, phenylephrine at its higher concentration (10%) was responsible for the clinical picture of irritant dermatitis.     

Clinical morphology of sodium lauryl sulfate (SLS) and nonanoic acid (NAA) irritant patch test reactions at 48 h and 96 h in 152 subjects

In this study of 152 women, comparison of patch test responses between 2 irritants over 96 h at 2 symmetrical anatomical sites is studied. 2 irritants, each at 4 different concentrations (nonanoic acid (NAA) 80%, 40%, 20%, 10%; sodium lauryl sulfate (SLS) 3%, 2%, 1% and 0.5%) and using propan-lol and'water for injection'as the respective controls, were placed as 15 μl aliquots, soaked onto filter paper discs in Finn Chambers, on the volunteer's left and right lower back. The patches were removed at 47, and read at 48 and 96 h. Irritant reactions were evaluated for erythema and surface changes by degree and area affected. Statistical analysis of the results showed that erythema decreased with time for all concentrations of NAA, and at higher concentrations for SLS. Surface changes increased with time for SLS and at higher concentrations of NAA. There was no statistically significant difference comparing left and right sides. Traditionally in patch testing, reactions which fade after 48 h have been regarded as irritant rather than allergic. This study refutes that assumption. Data from our left to right comparisons, made in the same individuals at the same time, show that irritant reactions may be more reproducible than previously appreciated.     

Artificial disruption of skin barrier prior to irritant patch testing does not improve test design

BACKGROUND: Irritant patch testing is often performed as a 24- or 48-h occlusive patch test with low concentrations of sodium lauryl sulphate (SLS). OBJECTIVES: The aim of this study was to investigate potential ways to shorten this test procedure and obtain precise test results. PATIENTS AND METHODS: Thirty-six healthy volunteers underwent irritant patch testing with different pretreatments (PT) of the test fields. Occlusive test chambers were applied on the upper back with SLS 0.5%, 1%, 2% and 5% in large Finn Chambers(R). The patches were removed after 4 and 24 h, respectively, depending on the concentration used. Test fields were pretreated as follows: PT 0, field without any PT (control); PT 1, prick with lancet; PT 2, prick with test stamp; PT 3, scratch with lancet; PT 4, incision with standardized incision instrument (0.1-0.2 mm depth). Skin reactions were evaluated by transepidermal water loss (TEWL), skin erythema and skin hydration and as well by a visual score (VS) at 4, 24 and 72 h. RESULTS: Our data show an obvious distinction between PT 0-2 and PT 3-4 at all measurement methods. The average TEWL values with PT 3-4 were higher than those with PT 0-2, especially on the 4-h course. This distinction may derive from the shape and size of the skin impairment achieved by PT 3-4, leading to a mechanical barrier disruption. However, SLS may infiltrate directly into deeper skin layers supported by capillarity. Consequently, no or little penetration through the epidermis and interaction with its structures occurs, which is responsible for irritant skin reactions. The SLS dose in the upper skin layers is therefore lower at these PTs. The lower remaining dose of SLS also explains this distinction, especially for the VS. Additionally, there are presumed reactions in deeper layers of the epidermis and dermis at PT 3-4. CONCLUSIONS: In summary, all data suggest a different reaction pattern from the classical irritant response. Therefore, application without any PT seems to be best suited for irritancy skin testing, especially for visual assessment. PTs prior to irritant patch testing have been shown to be unjustifiable.     

Simultaneous sodium lauryl sulphate testing improves the diagnostic validity of allergic patch tests. Results from a prospective multicentre study of the German Contact Dermatitis Research Group (Deutsche Kontaktallergie-Gruppe, DKG)

Background There is evidence that a higher skin susceptibility may induce nonspecific erythematous or weak positive reactions to contact allergens in patch testing. Objectives To evaluate whether simultaneous application of sodium lauryl sulphate (SLS) along with diagnostic patch tests with contact allergens can provide information regarding skin irritability which may help to discriminate allergic from nonspecific irritant reactions to contact allergens. Methods Between July 2001 and June 2003, this prospective study collected patch test data of 5971 patients from 19 centres in Germany and Austria in the Information Network of Departments of Dermatology (IVDK). In addition to contact allergens (standard series and eight known ‘problematic’ allergens with a low reaction index and a high positivity ratio: 1,3‐diphenylguanidine, amerchol L‐101, benzalkonium chloride, benzoyl peroxide, cocamidopropyl betaine, octyl gallate, phenyl mercuric acetate and propylene glycol), patches with SLS 0·5% and 0·25% aq. were applied. Reactions to the allergens and to SLS were analysed at the IVDK data centre. The association between an erythematous or positive reaction to a certain allergen and an irritant reaction to SLS was assessed with logistic regression analysis, at the same time controlling for the influence of age and sex. Results Of the 29 allergens of the standard series, 23 and 21 gave a higher percentage of nonspecific erythematous reactions in patients with an irritant reaction to 0·25% and 0·5% SLS, respectively, in comparison with SLS‐negative patients. All eight ‘problematic’ allergens gave an increased percentage of nonspecific erythematous reactions. Similarly, 22 and 21 allergens of the standard series gave a higher percentage of positive allergic reactions in patients with an irritant reaction to 0·25% and 0·5% SLS, respectively, and seven of the eight ‘problematic’ allergens gave a higher percentage of positive allergic rections (exception: octyl gallate). For most allergens, the markers of skin reaction (reaction index and positivity ratio) were worse in SLS‐positive patients. Differences were more pronounced when testing with SLS 0·25% than with SLS 0·5%. Conclusions Because there is a convincing association between skin irritability (evaluated by SLS test) and the degree of skin reaction to contact allergens, the SLS test may help in deciding whether a doubtful erythematous or weakly ‘positive’ skin reaction should be interpreted as allergic or irritant.     

A stronger patch test elicitation reaction to the allergen hydroxycitronellal plus the irritant sodium lauryl sulfate

Household and cleaning products often contain both allergens and irritants. The aim of this double-blinded, randomized, paired study was to determine whether patch testing with an allergen (hydroxycitronellal) combined with an irritant [sodium lauryl sulfate (SLS)] cause a stronger patch test elicitation reaction than patch testing with the allergen (hydroxycitronellal) alone, in patients previously patch tested positive to hydroxycitronellal. A stronger patch test elicitation reaction was defined as at least 1 day of patch test reading showing more positive patch tests (+, ++ or +++) on the forearm patch tested with 6 concentrations of SLS plus hydroxycitronellal than on the forearm tested with 6 concentrations of hydroxycitronellal alone and no day of patch test readings showing more positive tests on the hydroxycitronellal forearm. 15/20 (75%) had at least 1 day of patch test reading with more positive patch tests on the forearm patch tested with SLS plus hydroxycitronellal and no day of patch test readings with more positive patch tests on the forearm tested with hydroxycitronellal (P = 0.0253). Estimation of the blood flow with laser Doppler supported these findings.     

Sodium lauryl sulfate irritant patch tests: Degree of inflammation at various times

Irritant reactions were induced on the forearms of 10 normal subjects with 10% aqueous sodium lauryl sulfate under patch test occlusion for 24 h. Test sites were observed at 24, 26, 28, 30, 48, 72 and 96 h and the degree of inflammation recorded. Inflammation was most prominent at 28 h and decreased in intensity over the time course of the study. Inflammation at 48 and 72 h was similar to when patches were removed. This suggests that inflammatory responses in skin for at least certain irritants like sodium lauryl sulfate do slowly decrease in intensity after 48 h. However, the inflammatory response may initially accelerate after patch test removal and remain intense for at least 48 h. Fading of irritant reactions by 48 or 72 h may not reliably distinguish irritant from allergic patch test reactions. This does not refute the usefulness of a delayed (96 h) reading since inflammation from sodium lauryl sulfate had decreased significantly by this time.     

Epidermal Langerhans cell apoptosis is induced in vivo by nonanoic acid but not by sodium lauryl sulphate

Exposure to irritants may cause chronic irritant contact dermatitis (ICD), characterized by irregular epidermal thickening and a predominantly dermal mononuclear cell infiltrate. The mechanisms involved, and why only certain individuals are affected, are not clearly understood. Different irritants may trigger different cellular and molecular interactions between resident skin cells and recruited inflammatory cells. In some individuals these interactions may become self-perpetuating resulting in persistent inflammation in the absence of continued exposure. This study examined Langerhans cell (LC) density in clinically normal skin of 46 patients with chronic ICD and 10 healthy individuals, and compared the action of the two irritants nonanoic acid (NA) and sodium lauryl sulphate (SLS) on the LCs and keratinocytes of clinically normal skin in patients with chronic ICD. There was a higher number of LCs/mm basement membrane in patients compared with controls, although there was no difference in the number of dendrites/LC nor in dendrite length. SLS induced keratinocyte proliferation after 48 h exposure, had no effect on LC number or distribution, and induced keratinocyte apoptosis after 24 and 48 h exposure. In contrast, NA decreased keratinocyte proliferation after 24 h exposure but this returned to basal levels after 48 h, and induced epidermal cell apoptosis after only 6 h exposure. NA dramatically decreased LC number after 24 and 48 h exposure, which was accompanied by basal redistribution and decreased dendrite length. Most significantly, NA induced apoptosis in over half of the LCs present after 24 and 48 h exposure.     

Delayed systemic dermatitis from amylase cellulase,and protease

A 69-year-old woman was referred to us presenting generalized eczema and mild systemic symptoms after taking digestive tablets (AeroRed Eupeptico; UPSA Medica, San Sebastian, Spain) 7 days before consultation. Patch tests with the com ponents of these tablets (tested as is) were performed. Amylase, cellulase, and protease elicited a papulovesicular reaction at 48 and 96 hours. The other components, even a lipolytic enzyme, were negative. The same patch tests in 20 controls were negative. Several extracts of fungi and products related to the culture medium were tested by prick tests (read at 20 minutes and 48 hours) and patch tests (read at 48 and 96 hours) with negative results. Intradermal skin tests with Aspergillus were also negative at 20 minutes and 48 hours. Enzymes are well-known allergens in immunoglobulin E—mediated reactions, but a few cases of type IV hypersensitivity have been reported. This patient developed systemic contact-type dermatitis caused by cellulase, amylase, and protease taken orally in which a delayed allergy was confirmed by positive patch tests. We stress the efficacy of patch tests to establish the etiologic diagnostic mainly in cases of drug-induced dermatitis.     

免疫磁性技术分离提纯酵母样真菌的研究

用包被有羊抗鼠ＩｇＧ的磁珠结合鼠抗白念单抗后，对白念珠菌，类星形念珠菌，类星形念珠菌，热带念珠菌，新生隐球菌，光滑念珠菌菌悬液及白念珠菌与新生隐球菌两菌混悬液进行了分离纯化研究。结果：免疫磁球和白念珠菌，类星形念珠菌结合尤为明显，与热带念珠菌少量结合而不和新生隐球菌，光滑念珠菌结合。     

Intra-individual variation of irritant threshold and relationship to transepidermal water loss measurement of skin irritation

Irritant susceptibility studies have used either visual assessment or transepidermal water loss (TEWL) to determine subject response. We have developed a visual assessment method which determines subject irritant threshold. We examined the relationship between sodium lauryl sulfate (SLS) irritant threshold and TEWL measurements from normal skin and SLS patch tests. 19 subjects were recruited. The irritant threshold of each subject was measured and TEWL measurements made from the applied SLS patch tests. Individuals with a lower irritant threshold (easily irritated skin) had elevated TEWL levels compared to those with higher thresholds. The irritant threshold test had a low intraindividual variation. This study showed that the 2 methods grouped patients in a similar manner. The variation seen may reflect the different outcomes measured: irritant threshold visually assesses the skin inflammatory response while TEWL measures skin barrier modification.     

Clinical allergy to cocamidopropyl betaine: reactivity to cocamidopropylamine and lack of reactivity to 3-dimethylaminopropylamine

7 patients allergic to cocamidopropyl betaine (CAPB) were detected by positive patch test reactions to Tegobetaine L7TM. These patients were then asked to participate in further testing to its potential impurities, cocamidopropylamine and 3-dimethylaminopropylamine (DMAPA). 4 of the 7 patients were tested to purified CAPB and cocamidopropylamine, with 2 reacting to the purified betaine on allergy patch testing and 3 reacting to cocamidopropylamine 0.1%. At another date, 6 of the 7 were successfully recalled for testing to DMAPA in the presence of sodium lauryl sulfate (SLS) or pure CAPB; 1/6 reacted to DMAPA in water, but only at very high concentrations, at least 3 orders of magnitude higher than that to which skin exposure would occur from use of products containing CAPB. Both SLS and CAPB increased the number of reactions recorded to high levels of DMAPA. However, positive reactions to much lower concentrations of DMAPA (>or=100 ppm) were found in only 1/6 subjects and then only in the presence of the irritant SLS. 0/6 reacted to pure CAPB alone. Taken together, these results suggest that DMAPA is unlikely to be an important contact allergen in CAPB of appropriate quality. They also confirm that CAPB of suitable purity, where levels of both cocamidopropylamine and DMAPA are minimized, is unlikely to trigger reactions in those ostensibly allergic to the material.