Patch testing 101, part 1: performing the test.

Understanding the basics of patch testing is essential to caring for patients with contact dermatitis. Several screening or standard series are available, and additional allergens or series may be necessary based on the patient's history. A delayed reading should be performed 72 to 144 hours after patch placement. Certain oral medications, phototherapy, or topical products may interfere with patch test results.

Dietary supplement product composed of natural ingredients as a suspected cause of erythema multiforme: A case report and identification for the confident false positivity of lymphocyte transformation test.

Growing and sustainable consumption of health-care products raises a controversial issue underlying the reliability of an in vitro diagnostic approach for adverse skin reaction. This report aimed to: (i) discuss the causative nature of a commercial dietary supplement composed of natural ingredients, particularly an Euglena-containing product, suspicious for erythema multiforme in our exemplified case; and (ii) to address the assay suitability of the lymphocyte transformation test (LTT) for identifying allergic reaction to any ingredient(s) of the product. A Japanese elderly man developed erythema multiforme after intake of a commercially available natural dietary product, whose LTT was positive. His clinical course and positive LTT suggested a provisional diagnosis of natural dietary product-induced eruption. We conducted an inquiry survey for the standard LTT with any commercial products containing Euglena in three major Japanese laboratory services and identified 22 subjects, almost all of whom (21/22, 95.6%) showed a positive LTT for any Euglena-containing products as a suspected causative. Seven normal healthy volunteers who had no intake history of Euglena-containing products showed an equivalent LTT positivity rate with the same product taken by our case; culprit components of the product included Euglena, Angelica keiskei, Barley grass and Chlorella. A cell-free culture system and enzyme-linked immunoassay suggest that the high LTT positivity relies on the non-specific lymphoproliferative activity, and not contamination of uncharacterized microorganisms and endotoxins. Because of the constitutive false positivity of LTT, this assay is unreliable for in vitro supportive diagnosis of adverse skin events caused by dietary products containing particular natural ingredients, as well as herbal materials.

Safety profile of silver sulfadiazine-bFGF-loaded hydrogel for partial thickness burn wounds.

In the present investigation, the safety of novel combinational silver sulfadiazine-bFGF-loaded hydrogel was assured by performing acute skin irritation, sensitization, acute dermal toxicity, and eye irritation in compliance with the Organization for Economic Co-operation and Development guidelines. In the skin irritation study, placebo, test, and positive control (0.8% w/v aqueous solution of formaldehyde) were applied on New Zealand rabbits and monitored for abnormal skin responses including erythema and edema. The placebo and test formulation did not induce any adverse reactions and were classified as nonirritating materials. In the skin sensitization test, guinea pigs were sensitized by positive control (0.1% w/v 1-chloro-2,4-dinitrobenzene in 10% of propylene glycol as a standard skin sensitizing agent), placebo, and test formulations. Weak sensitization was observed in the placebo and test formulation treated groups. Additionally, acute dermal toxicity test was performed in Wistar rats, where no signs of toxicity were observed in biochemical, hematological, and histopathological studies. Moreover, the acute eye irritation test was carried out in rabbits and no abnormal clinical signs were evident in the cornea or iris. As a whole, these findings suggest that the hydrogel formulation does not cause any skin irritation, skin sensitizationand dermal toxic effects, and eye irritation following dermal and ocular applications, respectively. Therefore, all the findings obtained from this preclinical study indicated that this hydrogel formulation is nontoxic and safe for use in animal models.

Are allergen batch differences and the use of double skin prick test important?

BACKGROUND: Skin prick tests (SPT) are widely used both in clinical diagnostics and in research. The standardization of allergen extracts is well documented to be crucial for the validity of SPT, whereas less emphasis has been placed on reproducibility and the SPT procedure itself. The objectives of this study are to clarify how the double skin prick test procedure influence the sensitivity and specificity of the test and to analyse the differences in weal size in skin prick tests between two batches of allergen extracts from the same vendor. METHODS: The association between rhinitis and SPT was assessed among 1135 persons from a general population sample. SPT was performed twice with 10 common aeroallergens. In a subsample of 90 persons SPT was performed simultaneously with five of the allergens using different batches. RESULTS: Thirty percent had at least one positive SPT. Among asthmatics this number was 62%. Only minor differences were seen between the sizes of two weals from the same batch. A second SPT with the same batch did not change the association between rhinitis and sensitization. When performing SPT with two different batches disagreement was observed in 2% (Birch) to 11% (Cat) of the subjects. CONCLUSIONS: Performing SPT twice with the same allergen batch does not enhance the validity of the test, and value of double testing can be questioned. Considerable differences in SPT response with different batches from the same manufacturer were observed. Thus inter batch differences in allergen extracts might be a source of variability.

Allergen skin prick test should be adjusted by the histamine reactivity.

BACKGROUND: Skin prick test results are mostly reported as mean wheal diameter obtained with one concentration of allergen. Differences in technique between personnel causes variation in wheal size. The research question was whether the influence of differences in skin prick test technique among assistants and centers can be reduced by relating the allergen wheal response to that of histamine. METHODS: Two methods for estimating skin reactivity, the method of Nordic Guidelines using histamine as a reference and the method of Brighton et al. [Clin Allergy 1979;9:591-596] not using histamine as a reference, were applied to data from two biological standardization trials, using the same batch of freeze-dried timothy pollen preparation. RESULTS: The concentration defining the Nordic biological unit, defined as a concentration of allergen eliciting a wheal of the same size as that of histamine dihydrochloride 10 mg/ml, did not differ between the centers. When not using histamine as a reference, applying the method of Brighton et al., there was a 15-fold difference in the estimate of the biological activity between the trials that was eliminated by adjusting the allergen response to that of the histamine reference. CONCLUSIONS: To reduce the influence of differences in test technique among assistants and centers responses to allergen-induced skin prick tests should be compared to that of histamine.

Characterization and standardization of allergen extracts.

This paper summarizes the development of the extraction and characterization of allergens responsible for the induction of immunoglobulin (lg) E-induced allergies from the beginning of the 20th century, including the nomenclature of allergens. The majority of papers characterizing allergens and allergen extracts state that the lack of standardization of allergen extracts is the reason for the paper, and so it has been for more than 100 years. A natural part of that process might be the isolation of an allergen molecule and this starts the speculation of 'what makes that allergen an allergen?' To achieve the perfect standardization is a desirable end that is still awaited. So far none of these problems have been finally solved. I started in allergy shortly after the discovery of IgE in 1967. Since that time the history as I remember it is based on the literature, my interpretation of it, and of course may be a little biased due to personal prejudice! The history of the last 10-15 years has still not matured and it might be a little early to draw conclusions. However, at the end of this chapter I do dare to make a few conclusions after having followed the development in this field for 40 years. As this is history it is not meant to be either comprehensive or technically and scientifically precise in all aspects, but rather draws on some thoughts as to what in my mind have been important developments until now. Specific techniques are only mentioned by name and not intended to be discussed in depth. This activity has, however, pushed me to reflect on my hopes and speculations at the time of my introduction to the field of allergen chemistry. To my surprise I realize that far more than I ever expected at that time has been fulfilled. It has been extremely exciting to be a part of that development.

Inhibition of IgE binding to cross-reactive carbohydrate determinants enhances diagnostic selectivity.

BACKGROUND: Allergy diagnosis by determination of allergen-specific IgE is complicated by clinically irrelevant IgE, of which the most prominent example is IgE against cross-reactive carbohydrate determinants (CCDs) that occur on allergens from plants and insects. Therefore, CCDs cause numerous false-positive results. Inhibition of CCDs has been proposed as a remedy, but has not yet found its way into the routine diagnostic laboratory. We sought to provide a simple and affordable procedure to overcome the CCD problem. METHODS: Serum samples from allergic patients were analysed for allergen-specific IgEs by different commercial tests (from Mediwiss, Phadia and Siemens) with and without a semisynthetic CCD blocker with minimized potential for nonspecific interactions that was prepared from purified bromelain glycopeptides and human serum albumin. RESULTS: Twenty two per cent of about 6000 serum samples reacted with CCD reporter proteins. The incidence of anti-CCD IgE reached 35% in the teenage group. In patients with anti-CCD IgE, application of the CCD blocker led to a clear reduction in read-out values, often below the threshold level. A much better correlation between laboratory results and anamnesis and skin tests was achieved in many cases. The CCD blocker did not affect test results where CCDs were not involved. CONCLUSION: Eliminating the effect of IgEs directed against CCDs by inhibition leads to a significant reduction in false-positive in vitro test results without lowering sensitivity towards relevant sensitizations. Application of the CCD blocker may be worthwhile wherever natural allergen extracts or components are used.

Relevance of the directionality of skin elasticity to aging and sagging of the face.

BACKGROUND: Forces acting in facial skin have been suggested to show directionality. Non-invasive methods of measuring this directionality may thus provide information related to aging processes. The Reviscometer(®) RVM600 device is capable of measuring directionality of forces on the skin. This device has not been used previously in a published study to evaluate changes in directionality of forces on facial skin with aging. AIM: The first objective of this pilot study was to investigate relationships between mechanical directionality using the Reviscometer(®) RVM600, the Cutometer(®) MPA580, and aging of the facial skin in a supine position. In addition, the study investigated relationships between mechanical directionality and 'skin sagging,' which may be caused by gravity. To validate this as a new measurement of mechanical directionality, we also performed double-blinded trials on two groups of subjects, with one group using a product containing an anti-aging substance and the other group using a placebo product without an anti-aging substance. METHODS: We examined 91 healthy Japanese women with a mean age of 48.5 years (range, 20-79 years) at the three sites on the face using the Reviscometer(®) RVM600 and the Cutometer(®) MPA580, and evaluation was performed for skin sagging in September and November 2008, and January 2009. The Reviscometer(®) RVM600 was used to measure resonance-running time (RRT) every 10° from 0° to 350°. Evaluation of skin sagging was undertaken by making marks on the face and using face photographs taken in both sitting and supine positions to calculate the sagging index. Usage testing was conducted on 38 healthy Japanese women in a double-blinded study with one group, using a preparation containing Yomogi AGEs Clearing (YAC) extract and another group using the same preparation without the YAC extract from October 2008 to April 2009. Mean age of these subjects was 44.0 years (range, 30-60 years). Measurements were taken at the three sites on the face using the Reviscometer(®) RVM600 and the Cutometer(®) MPA580 and sagging index. RESULTS: A significant correlation was identified between RRT parameters and subject age at all three measurement sites. Significant correlations between sagging index and RRT values were found for 110-170° and 290-350° only at the center of the cheek. Significant differences in RRT values were noted for 110-150° and 300-350° at this site between subjects with and without the use of YAC extract. A similar trend was found in sagging index for this site alone between subjects with and without YAC extract. CONCLUSION: The use of non-invasive procedures to measure skin mechanical parameters on the face in all directions may evaluate aging and effective preventive and restorative support.

Heterogeneity of commercial timothy grass pollen extracts.

BACKGROUND: The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources. OBJECTIVE: To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity. METHODS: Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients. RESULTS: The allergen extracts showed broad variations in protein compositions and amounts (24.1-197.7 microg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32-384 ng/mL; Phl p 2: 1128-6530 ng/mL, Phl p 5: 40-793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients. CONCLUSIONS: Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.

European allergen extract units and potency: review of available information.

BACKGROUND: There is considerable variability in how allergen extract potency is measured and reported worldwide. In Europe, where many sublingual immunotherapy studies have been conducted, manufacturers report allergen extract potency as units based on an in-house reference, making it difficult to understand the exact doses used and to compare studies. OBJECTIVES: To describe the various methods of expressing extract potency that European allergen extract manufacturers use and to gather reports on the micrograms of major allergen of the in-house units of European allergen extract manufacturers. METHODS: Information was derived from 3 sources: data on extract potency in micrograms of major allergen in articles on sublingual immunotherapy found by PubMed (references through October 2005) and in reference articles, brochures on allergen extracts from the manufacturers, and information provided by structured questionnaires e-mailed to the manufacturers. RESULTS: All but 1 of the European allergen extract manufacturers use in-house reference standards that are based on titrated skin prick testing of allergic patients. Subsequently, in vitro tests compare the potency of commercial batches with the in-house reference and potency is assigned as arbitrary units. Most manufacturers measure major allergen content of their standardized products but do not release this information with the package insert. Diversity in major allergen content was found. CONCLUSIONS: Micrograms of major allergens given in articles on sublingual immunotherapy to express the dose administered cannot be used to translate the dose to US extracts. Extract potency can only be compared if uniform test methods and reference extracts are used.

Can we manage demand for allergy testing by restricting requests to a small number of prime target allergens?

BACKGROUND: Demand for expensive tests such as allergen-specific IgE is expanding far faster than for cheaper tests: at Burton Hospital the annual growth rate is 24%. Different hospitals have different policies on allergen testing. We report a comparison of the effect of requesting policy on diagnostic yield. METHODS: All results from five years of allergen testing were downloaded from the data warehouse at Burton, and a representative sample of recent results was evaluated from Ipswich Hospital. Statistical analysis by chi(2) test and significance tests for differences of proportions were carried out. RESULTS: Ipswich hospital used a standard four-allergen panel for respiratory patients and demonstrated a statistically significantly lower positivity rate for three of those four allergens. No relationship between the number of allergens tested and the probability of a positive result was shown - the probability of a positive result was approximately 0.3. Number of allergen-specific IgE tests requested/patient have remained roughly constant over 5(1/2) years but total demand has increased. CONCLUSIONS: Selective requesting for allergen-specific IgE testing may be more effective than use of a standard panel but this cannot be conclusively proven. It is not appropriate to attempt to limit workload by specifying a maximum number of tests that are allowed for any individual patient.

The effect of montelukast (10mg daily) and loratadine (10mg daily) on wheal, flare and itching reactions in skin prick tests.

UNLABELLED: Antileukotriene agents are widely used for the treatment of allergic conditions including bronchial asthma and allergic rhinitis. The influence of montelukast on skin reactivity has not been clearly evaluated. The aim of this study was to determine the effect of montelukast on wheal, flare and itching in skin prick tests (SPTs). METHODS: Fifteen atopic patients (5 women and 10 men) with average age 28.04 (SD+/-8.24) were tested with histamine, codeine, negative control solution and allergen extract (grasses). Montelukast (10mg), loratadine (10mg) or placebo were given to the volunteers for 5 days in a double-blind manner, followed by SPT, with 14 days of wash-out period. RESULTS: There was no differences in wheal, flare and itching (p=0.205; 0.086 and 0.069, respectively, Wilcoxon rank-sum test) between SPT performed after placebo and wash-out period. The analysis revealed a statistically significant suppression of wheal and flare by loratadine (p<0.05 for all tested solutions). Pre-treatment with montelukast did not influence wheal size (p=0.099, 0.21, 0.066 for histamine, codeine and allergens, respectively), but significantly reduced flare (p=0.005; 0.003; 0.02 for histamine, codeine and allergens, respectively). We found a significant suppression of itching produced by montelukast (p=0.02) and loratadine (p=0.03) as compared to placebo (p=0.068 vs. wash out). CONCLUSIONS: Our data show a tendency to suppressive effect of montelukast on flare and itching but not on wheal which is basic for SPT interpretation. We conclude that found suppression have little impact on clinical effectiveness of SPT as a diagnostic tool.

Standardization of an ash (Fraxinus excelsior) pollen allergen extract.

BACKGROUND: Ash tree (Fraxinus excelsior) is the main representative of the Oleaceae family in temperate zones. Diagnosis of ash pollen allergy is made difficult due to (1) an overlapping pollinization period with Betulaceae, (2) non-inclusion in current diagnostic assays, and (3) some cross- reactivity with minor allergens from Betulaceae. The aim of this study was to calibrate an ash pollen in-house reference preparation (IHRP) in allergic patients in order to produce standardized products for diagnosis and immunotherapy purposes. METHODS: Ash pollen IHRP was extracted, ultrafiltered and freeze dried. Allergens in the extract were detected after 2-dimensional PAGE using specific sera and a monoclonal antibody. The Fra e 1 content of IHRP was evaluated by quantitative immunoprint. Forty-eight subjects from the North-East of France exhibiting clinical symptoms, a positive skin test and specific IgE levels > or =class 2 to ash pollen were recruited. IgE immunoprints were performed to select patients sensitized to the ash Fra e 1 allergen as opposed to cross-reacting allergens. Serial 10-fold dilutions of the IHRP were tested by skin prick tests in order to determine the concentration inducing a geometrical mean wheal diameter of 7 mm, said to correspond to an index of reactivity (IR) of 100 per millilitre. RESULTS: IgE-reactive molecules in IHRP comprise Fra e 1, Fra e 2, a 9-kDa molecule (presumably Fra e 3), as well as a doublet at 15 kDa and high molecular weight allergens. The 100 IR concentration of IHRP inducing a geometrical mean wheal diameter of 7 mm in 22 patients sensitized to Fra e 1 corresponds to the 1/126 (w/v) extraction ratio (i.e. 259 microg/ml of protein by Bradford) and contains 17 microg/ml of Fra e 1. The variability in total activity of 5 batches of standardized extracts was found to be significantly reduced when compared with 7 non-standardized extracts. CONCLUSION: An ash pollen IHRP was defined and molecularly characterized. Its successful standardization at 100 IR/ml in patients specifically sensitized to Fra e 1 allowed a skin reactivity-based calibration in properly diagnosed patients. Such a standardized ash pollen extract is a reliable tool to support immunotherapy of ash pollen allergy.

[Allergy to cypress pollen: preparation of a reference and standardization extract in vivo]. / Allergie au pollen de cypres: preparation d'un extrait de référence et standardisation in vivo.

Development of Cypress allergy frequency led to the standardization of commercial cypress extract used for diagnosis and immunotherapy. Previous in vitro studies on two cypress pollen species (Cupressus sempervirens and Cupressus arizonica) allowed us to produce an allergenic solution composed by a mixture of both extracts for in vivo standardization. Dilutions of this allergenic solution were tested by prick-test on 44 patients with clinical allergy to cypress pollen to define the dilution that corresponds to a 6 mm wheal conformed to the definition of 100 IR. The mixture of the two major species found in France is justified by the in vitro study results. Extracts revealed complementary allergenic composition: Cup sempervirens showed a wider diversity of allergens whereas Cup arizonica showed a higher content of the major 43 kDa allergen. Thus, according to in vivo analysis, we are able to produce a standardized extract of Cypress pollen expressed in IR.

Determination of the allergenic activity of birch pollen and apple prick test solutions by measurement of beta-hexosaminidase release from RBL-2H3 cells. Comparison with classical methods in allergen standardization.

BACKGROUND: A murine in vitro model of the allergic type I reaction was set up to determine the biologic activity of extracts without involvement of human beings. It is based on beta-hexosaminidase release from passively sensitized RBL cells after allergen challenge. The intended application of this RBL cell assay in the field of quality control of allergenic extracts requires its comparison with established methods. METHODS: The activity of five standardized birch-pollen prick test solutions was determined in parallel by RBL assay, direct IgE binding, IgE-binding inhibition, major allergen content, histamine-release assay, and skin testing. RESULTS: The RBL cell-release assay corresponded well to other methods if a reagin raised against natural birch-pollen extract was used for passive sensitization. However, in the case of a reagin against recombinant Bet v 1, only a decreased activity was observed, presumably because a reduced number of epitopes were recognized by the monospecific reagin. In contrast to standardized birch-pollen extracts, nonstandardized apple extracts showed poor activity in all assays. CONCLUSIONS: This murine model might be a useful tool in the quality control of allergenic extracts. It combines properties of assays based on standardized antisera and of assays that consider IgE cross-linking properties.