Diagnosis of venom allergy by flow cytometry. Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release.

BACKGROUND: Potent allergens such as hymenoptera venoms are capable of inducing severe and life threatening clinical reactions. Percentage of false negative results obtained by the usual diagnostical methods is comprised between 10 and 25%. OBJECTIVE: Evaluation of the sensitivity and the specificity of cellular tests and particularly evaluation of a new flow cytometric method. METHODS: Forty-five allergic patients having experienced a local, a systemic reaction or an anaphylactic shock and 10 controls having undergone hymenoptera stings without clinical reactions were selected on the basis of the clinical history, skin tests and specific IgE. Three cellular tests were performed on the same cell suspensions and in the presence of 2 ng/mL of rIL3: histamine release (RIA), leukotriene C4 release (ELISA) and basophil activation test (flow cytometry after double anti-IgE FITC, anti-CD63 PE labelling). RESULTS: As compared to the clinical history, sensitivities of skin tests, specific IgE, flow cytometry, histamine release and leukotriene release were, respectively; 85%, 88%, 100%, 89% and 100%. Flow cytometric analysis of basophil activation showed a significant decrease of the mean fluorescence density and number of IgE positive cells and a significant increase of the number of CD63 positive cells. The 10 controls tested by flow cytometry were negative. CONCLUSION: As compared to the clinical history and to the other parameters tested here, flow cytometry showed a high sensitivity and a high specificity. The excellent correlation observed between this method and the other cellular tests such as histamine and leukotriene release are in favour of the specificity of flow cytomery and in favour of the use of this method for venom allergy diagnosis.

[Search for a threshold to distinguish between locoregional and systemic reactions using the histamine liberation value and the LTC4 level]. / Recherche d'un seuil permettant de différencier réaction loco-régionale et réaction systémique à partir de la valeur de l'histamine-release et du dosage des LTC4.

Allergy to Hymenoptera venom (VH) effects more and more patients in France. It is manifest in two main forms, which are Loco-regional (RLR) and Systemic reactions (SR). This allergy is detectable amongst others by the techniques of histamine release (HR) and release of leucotrienes C4 (LTC4). The aim of this work has been to fix a threshold that gives differentiation of RLR ad RS by the two techniques. We found in a positive population a threshold histamine release (HR) value of 25% and a concentration of LTC4 of 600 ng/ml. These are the thresholds above which a patient would be at around 70% risk of RS. This study has a predictive value for patients who are suspected of allergy of VH or who have already had clinical reactions and risk sensitization.

[Basophil activation test using flow cytometry in Hymenoptera venom allergy]. / Test d'activation des basophiles par cytométrie en flux dans l'allergie aux venins d'hyménoptères.

The basophil activation test (TAB) clearly shows evidence to Hymenoptera venoms. Comparison of the TAB value with the clinical history allows fixation of a threshold that will anticipate the type of clinical reaction by the TAB value. For the technique of TAB 3 dilutions are used. The basophil activation values produce a threshold for each dilution. For the first dilution the threshold is 50% activation: below this threshold, 70% of patients would have a local or systemic reaction and above this threshold, 70% of patients would have a systemic allergic reaction.

[Allergic immunobiology and anesthesiology]. / Immuno-biologie allergique et anesthésiologie.

A protocol has been produced for the study of anaphylactic accidents that occur post-operatively that allows definition of the anaphylactic origin, and so reactions that are mediated by IgE in post-operative accidents. This protocol occurs in two stages, the first is done in the minutes and hours that follow the anaphylactic accident, and the second a month or 6 weeks afterwards. At first, we evaluate the sequential study of the liberation of the mediators of anaphylaxis, plasma histamine, serum tryptase, urinary methylhistamine and, more recently, leucotriene E4. The second study is devoted to reactions that are mediated by IgE, essentially, specific serum IgE, tests of activation of basophils by flow cytometry, measurement of leucotriene C4 and skin tests. A study on 16 subjects has evaluated and validated the protocol and shown a significant level of correspondence of results between the sequential measurement of mediators on one hand and on the other the search for IgE-mediated reactions every time that there was an anaphylactic reaction.

[Testing basophil activation by flow cytometry in the diagnosis of allergy to hymenopteran venom]. / Le test d'activation des basophiles par cytométrie en flux dans le diagnostic de l'allergie aux venins d'hyménoptères.

Diagnosis of allergy to hymenoptera venom must be precise and depends on indisputable bio-clinical criteria, because of specific immunotherapy for indications such as systemic and/or anaphylactic reactions. Until nowadays, diagnosis was by specific IgE, histamine release and skin tests, most often done for the venoms of wasp, honey bee and hornet at the same time since, in 7-8 cases in 10 the patients had not identified the responsible insect. Basophil activation test (TAB) by Flux cytometry and measurement of leukotriene C4 (LTC4) are new techniques of great reliability. The work shows the correlations between the different immunobiological parameters by reference to TAB by CAF and measurement of LTC4. When the overall results for mixed venoms or those for single venoms are considered, the correlations between TAB, LTC4 and the other parameters are highly significant. It can now be considered objectively that TAB by CTF and measurement of LCT4 are the two highest-performing techniques for diagnosis of hymenoptera venom allergy and so validates them.

[Immuno-biological diagnosis of food allergy]. / Diagnostic immuno-biologique de l'allergie alimentaire.

Food allergy is becoming more frequent, with 6% of asthmatics reporting an isolated food allergy, and 5 to 6% of atopic dermatitis patients also have either a single or multiple true food allergy. There is value in immuno-biological diagnosis by: Measurement of total serum IgE. Measurement of mono-allergen-specific IgE, following a measurement by a multi-allergen of the Trophatope type. A study of elimination of foods for 2 or 3 months followed by their re-introduction. Oral provocation tests in a hospital environment under clinical control and subsequent measurement of the mediators:-Plasma histamine, tryptase, and urinary methylhistamine to give proof of responsibility of the food allergen. Nowadays, it is perfectly possible to include in diagnosis the new technologies of the test of activation of basophils/or lymphocytes by means of flow cytometry.

[Allergy to mosquitoes and use of new immunobiological parameters]. / A propos de l'allergie aux moustiques et de l'utilisation des nouveaux paramètres immuno-biologiques.

With regard to an observation of anaphylaxis after a mosquito bite, we have shown by new immunobiological parameters and clinical investigations: Skin tests, specific IgE, Tests of Activation of basophils by flow cytometry (on basophils and total blood) and measurement of Leucotriene LTC4, the objective reality of the mechanism of IgE-dependent hypersensitivity (HS). This observation is correlated with rare work in support of true IgE-mediated allergy to mosquito 9 authors having used skin tests, PK in 1984 and immunoblot.

[Immunobiologic diagnosis of food allergy]. / Diagnostic immuno-biologique de l'allergie alimentaire.

Food allergy is becoming more frequent, with 6% of asthmatics reporting an isolated food allergy, and 5 to 6% of atopic dermatitis patients also have either a single or multiple true food allergy. There is value in immuno-biological diagnosis by: Measurement of total serum IgE. Measurement of mono-allergen-specific IgE, following a measurement by a multi-allergen of the Trophatope type. A study of elimination of foods for 2 or 3 months followed by their re-introduction. Oral provocation tests in a hospital environment under clinical control and subsequent measurement of the mediators: Plasma histamine, tryptase, and urinary methylhistamine, to give proof of responsibility of the food allergen. Nowadays, it is perfectly possible to include in diagnosis the new technologies of the test of activation of basophils/or lymphocytes by means of flow cytometry.

[Anaphylactic or anaphylactoid reactions to paracetamol. Report of 3 cases]. / Réactions anaphylactiques ou anaphylactoïdes au paracétamol. A propos de 3 cas.

Anaphylaxis to paracetamol exists and perhaps nowadays diagnosis is not confined to clinical evidence of: The repeatability of clinical symptoms. Skin tests. Provocation tests. But also by immuno-biological evidence. Application of the test of Activation of basophils by Flow Cytometry and measurement of leukotriene C4 have been nowadays largely validated and constitute irrefutable proof of the responsibility of the drug as the origin of the clinical symptoms.

[Contribution of flow cytometry to allergologic diagnosis]. / Apport de la cytométrie en flux dans le diagnostic allergologique.

The technique of Flow Cytometry for activation of basophils (TAB), expressed as the marker CD63, is at present one of the pathways of research applied to drug allergy. In this work are reported the results of TAB of Pneumoallergens and Hymenoptera venoms. TAB can define better than total IgE the atopy of the subject: in effect the fluorescence of the basophils is emphasized in comparison with non-atopic subjects. This hypothesis has been confirmed by a study of three groups of subjects. With regard to drug allergy, it is important to study patients in whom the observations have been documented very objectively by clinical history and positive skin tests to the drug, compared with a negative reference to the same drug. So, TAB has been shown to be very useful in diagnosis of allergy to certain drugs, such as the Myorelaxants.