Separation of the four most important latex allergens from latex gloves: A potential tool for diagnosis and immunotherapy purposes

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Evaluation and Comparison of Commercially Available Latex Extracts for Skin Prick Tests

Background: Crude latex extracts are commonly used in skin prick tests (SPT) for the diagnosis of natural rubber latex (NRL) allergy. Nevertheless, variations in protein and allergen composition between latex extracts from different manufacturers can hamper a correct diagnosis. Objectives: To analyze the heterogeneity of proteins and allergens in latex extracts from 7 different manufacturers and to assess its relevance in the diagnosis of latex allergy. Methods: Seven latex SPT extracts were analyzed for protein content using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The 4 major allergens Hev b 1, Hev b 3, Hev b 5, and Hev b 6.02 were also quantified using enzyme immunoassay. All commercial extracts were tested for their in vitro allergenic capacity using microarray inhibition assays and for their ability to induce biological reactivity in latex-allergic patients undergoing SPT. Results: The protein content of the extracts varied widely from 8.0 μg/mL to 526.5 μg/mL. SDS-PAGE revealed broad differences in protein profiles between the extracts. Marked variability in the contents of all 4 major allergens was observed, and Hev b 3 and Hev b 5 were undetectable in some extracts. Microarray inhibition assays and SPT demonstrated relevant differences in allergenic capacity between the extracts. Conclusions: The marked heterogeneity in protein and allergen content of latex extracts from different manufacturers could explain the broad spectrum of SPT results recorded. Our fi ndings suggest that the extracts used for the diagnosis of latex allergy should be improved and standardized (AU)

Antecedentes: En el diagnóstico de la alergia a látex natural se utilizan habitualmente extractos crudos de látex en técnica de puntura. No obstante la variación que existe en el contenido proteico y de los distintos alérgenos entre los extractos comerciales procedentes de distintos fabricantes podría afectar al correcto diagnóstico de la alergia. Objetivos: Analizar la heterogeneidad proteica y de alérgenos entre siete extractos de látex de distintos fabricantes y comprobar las posibles implicaciones clínicas en el diagnóstico de la alergia al látex. Métodos: Se analizó el contenido proteico de siete extractos de látex y también el perfil mediante la técnica de electroforesis en gel de poliacrilamida (SDS-PAGE). Además, se cuantificaron mediante enzimo-inmunoensayo (EIA), los cuatro alérgenos principales de látex Hev b 1, Hev b 3, Hev b 5 y Hev b 6.02. También se estudió en los siete extractos comerciales su capacidad de inhibición "in vitro", del ensayo de micromatrices y su capacidad para inducir respuestas biológicas "in vivo" en pacientes con alergia al látex, mediante pruebas cutáneas en puntura (SPT). Resultados: Los extractos presentaban una amplia variación en el contenido proteico que oscilaba entre 8.0 y 526.5 μg/mL de extracto. También se observaron importantes diferencias en el perfil proteico mediante la técnica de SDS-PAGE. El contenido de los principales cuatro alérgenos fue también muy variable, de forma que en algunos extractos los contenidos de Hev b 3 y Hev b 5 fueron prácticamente indetectables. Tanto la técnica de inhibición de micromatrices como las pruebas de puntura mostraron diferencias notables en la capacidad alergénica de los distintos extractos. Conclusiones: Los extractos de látex provenientes de distintos fabricantes presentan una importante heterogeneidad en contenido proteico y de alérgenos que podría claramente explicar las notables diferencias observadas en los resultados de las pruebas cutáneas en puntura que presentan los pacientes. Nuestros resultados apoyan la necesidad de mejora de la estandarización de los extractos de látex habitualmente utilizados en el diagnóstico clínico de la alergia al látex (AU)

Evaluation and comparison of commercially available latex extracts for skin prick tests.

BACKGROUND: Crude latex extracts are commonly used in skin prick tests (SPT) for the diagnosis of natural rubber latex (NRL) allergy. Nevertheless, variations in protein and allergen composition between latex extracts from different manufacturers can hamper a correct diagnosis. OBJECTIVES: To analyze the heterogeneity of proteins and allergens in latex extracts from 7 different manufacturers and to assess its relevance in the diagnosis of latex allergy. METHODS: Seven latex SPT extracts were analyzed for protein content using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 4 major allergens Hev b 1, Hev b 3, Hev b 5, and Hev b 6.02 were also quantified using enzyme immunoassay. All commercial extracts were tested for their in vitro allergenic capacity using microarray inhibition assays and for their ability to induce biological reactivity in latex-allergic patients undergoing SPT. RESULTS: The protein content of the extracts varied widely from 8.0 microg/mL to 526.5 microg/mL. SDS-PAGE revealed broad differences in protein profiles between the extracts. Marked variability in the contents of all 4 major allergens was observed, and Hev b 3 and Hev b 5 were undetectable in some extracts. Microarray inhibition assays and SPT demonstrated relevant differences in allergenic capacity between the extracts. CONCLUSIONS: The marked heterogeneity in protein and allergen content of latex extracts from different manufacturers could explain the broad spectrum of SPT results recorded. Our findings suggest that the extracts used for the diagnosis of latex allergy should be improved and standardized.

Different in vivo reactivity profile in health care workers and patients with spina bifida to internal and external latex glove surface-derived allergen extracts.

BACKGROUND: Allergy to natural rubber latex is a well-recognized health problem, especially among health care workers and patients with spina bifida. Despite latex sensitization being acquired in health institutions in both health care workers and patients with spina bifida, differences in allergen sensitization profiles have been described between these two risk groups. OBJECTIVE: To investigate the in vivo reactivity of health care workers and patients with spina bifida to extracts of internal and external surfaces of latex gloves and also to specific extracts enriched in major allergens for these risk groups. METHODS: Gloves from different manufacturers were used for protein extraction, and salt precipitation and hydrophobic interaction chromatography (HIC) were applied to obtain the enriched latex extracts. The major latex allergens were quantified by an enzyme immunoassay. The extracts obtained were tested in 14 volunteers using skin prick tests (SPT). RESULTS: Latex glove extracts enriched in the hydrophobic allergens that are most often seen in patients with spina bifida were obtained by selective precipitation, whereas HIC produced extracts enriched in the hydrophilic allergens commonly found in health care workers. The health care workers had positive SPTs to glove extracts from internal surfaces and to the hydrophilic allergen-enriched extracts. By contrast, patients with spina bifida had larger skin reactions both to external glove extracts and to the extracts enriched with the hydrophobic major allergens for this risk group. Despite the protein concentration of these extracts being less than half the concentration of the commercial extract, the weal-and-flare reactions were of similar magnitude. CONCLUSION: Using novel latex extracts, our study showed a different in vivo reactivity pattern in health care workers and in patients with spina bifida to extracts of the internal and external surfaces of gloves, which suggests that sensitization may occur by different routes of exposure, and that this influences the allergen reactivity profiles of these risk groups.

Latex allergy: new insights to explain different sensitization profiles in different risk groups.

BACKGROUND: Differences in latex allergen sensitization profiles have been described between children subjected to repetitive surgical interventions and health care workers (HCW). 'Major' allergens for patients with spina bifida are Hev b 1, 3 and 7, while for HCW, 'major' allergens are Hev b 2, 5, 6.01 and 13. The reason for these differential sensitization profiles is currently unknown. OBJECTIVES: To investigate latex allergen profiles on internal and external surfaces of natural rubber latex gloves. METHODS: Eighty-two samples of commonly used surgical gloves (41 glove brands) were used for analysis. Specific allergen levels of Hev b 1, 3, 5 and 6.02 on both surfaces of the gloves were quantified using an enzyme immunometric assay, a FITkit (FIT Biotech, Tampere, Finland). RESULTS: Differences in allergen levels were observed between internal and external surfaces of all glove types. Concentrations of Hev b 1 and Hev b 3 were significantly higher on external surfaces, while internal surfaces had higher allergen levels of Hev b 5 and Hev b 6.02. Analysis of surgical and examination gloves, powdered and nonpowdered gloves also showed that the content of Hev b 5 and Hev b 6.02 was significantly higher on internal surfaces while that of Hev b 1 and Hev b 3 was higher on external surfaces. CONCLUSIONS: Our study showed different allergen profiles on internal and external surfaces of natural rubber latex gloves. These results may suggest a relationship between latex allergen localization and sensitization routes in different risk groups.

Therapeutic monitoring of warfarin: the appropriate response marker.

Warfarin is a 4-hydroxycoumarin anticoagulant drug used for the prevention and management of thromboembolic and vascular diseases. It acts through the inhibition of the vitamin K-dependent transcarboxylation reactions that convert precursors of clotting factors into their active form. Appropriate use of warfarin requires patient monitoring and dosage adjustments, to ensure its safety and efficacy. The aim of this work was to clarify the relationship between traditional (prothrombin time, usually expressed as the international normalized ratio; INR) and alternative (clotting factors II and X) warfarin response markers to establish their usefulness for therapeutic drug monitoring. Seventy adult outpatients, aged between 31 and 86 years old, were involved in the study. All subjects received warfarin in a monotherapy regimen and had been on a stable dosing schedule for at least two weeks to assure a steady-state condition. A total of 81 prothrombin times (expressed as INR), and factor II and factor X activity were simultaneously determined. Eleven patients presented repeated measurements at different time periods under the same dosing regimen. The results obtained from regression and cluster analysis showed a close relationship between factors II and X (r = 0.73), a weak correlation between INR and both factor II (r = -0.35) and factor X (r = -0.36), and a very slight dependency between warfarin and the response markers used. In addition, it seems that independent of the selected response marker, in long-term warfarin therapy, reproducible responses can be obtained over time if a steady-state condition is achieved. The coefficients of variation for factors II and X were greater (35.44 and 37.93%, respectively) than INR (14.50%), indicating that INR is a more precise measure than either factor II or factor X. In conclusion, INR appears to be the most appropriate warfarin response marker for therapeutic drug monitoring due to its universality, objectivity as a direct physiological effect measurement, and the available information regarding appropriate endpoints. However, when INR values are not in accordance with patient response therapy, factor II and factor X should be considered as an alternative to optimize warfarin therapy.