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.

[The wasp-mosquito syndrome: extension of cross-allergenicity to the horsefly]. / Le syndrome guêpe/moustique: extension de l'allergénicité croisée au taon.

The crossed allergenicity between wasp venom and mosquito extract was shown during recent work based on clinical observations and correlation studies between different biological parameters, indicating an IgE-dependent biological mechanism. A common protein was identified by Immunoblot. From observation of one of our patients involved in this work, we examined the possibility of the extension of crossed reactivity between wasp, mosquito and horsefly. In effect, our patient presented an anaphylactic reaction with neurological complications from attack on the central grey nucleus, shown by IRM; the immunological study showed a common protein between wasp venom and the total extracts of mosquito and horsefly.

[The wasp/mosquito syndrome]. / Le syndrome guêpe/moustique.

The authors describe for the first time the Wasp/Mosquito syndrome. The allergenic cross-reactivity between Vespula wasp venom and mosquito extract and the reverse rest objectively on several criteria. The frequency of the association between sensibilization to Vespula wasp venom and mosquito: In 10 subjects who had specific IgE to Vespula wasp venom 3 also had mosquito-specific IgE (30%), but out of 11 subjects with positive mosquito-specific IgE 10 also were positive with Vespula wasp venom specific IgE (about 91%). Three observations were documented showing a clinical and biological relationship between sensitisation to mosquito and wasp venom. A first patient had an anaphylactic reaction after a dozen mosquito bites. The allergy assessment confirmed sensitisation to the mosquitos, but also to Vespides (wasp, hornet) venoms. A second patient had an anaphylactic reaction after a wasp sting in 1997. From that date, he had local reactions, indeed loco-regional, to mosquito bites. The allergy assessment confirmed the double sensibilization to mosquito and wasp venom. Finally, a third patient at first had local reactions, also loco-regional, to mosquito bites, then two systemic reactions after mosquito bites. A first allergy assessment, made exclusively to hymenoptera venoms (not to mosquitos), showed a sensitivity to wasp venom and the patient, by mistake, was desensitised to wasp venom, which did not prevent recurrence after mosquito bites. After a second time, the allergy assessment for hymenoptera and mosquito, confirmed a double sensitisation (mosquito and Vespula wasp). Indirect criteria such as parallel increase in specific IgEs to Vespula wasp venom and mosquito in a patient who was desensitised only to mosquito and who had not been re-stung by wasps. Finally on direct criteria, such as: The inhibition technique by CAP RAST, which was positive in two ways: Vespula wasp--specific IgE was inhibited by mosquito extract, and vice versa. Electrophoresis, which showed a protein common to Vespula wasp venom and mosquito extract of M.W. of the order or 42KD and isoelectric point between 4.5 and 5. An immunoblot study identified this common protein with a M.W. of 44KD which is the same as hyaluronidase.

[Apropos of the evaluation and validation of in vitro tests for measurement of specific IgE]. / A propos de l'evaluation et de la validation des tests in vitro des dosages des IgE specifiques.

The performances of the reagents for IN VITRO TESTS in Allergy, are very variable, in spite of the existence of registration procedures as well as those of national quality control. Most of the discrepancies have their origin in the choice of technologies used by the manufacturers (reaction support, raw materials, calibration, vigour of the process of execution). It follows from this that the characteristics must be understood and the limitations appreciated of the tests that are used, as well as their interpretation. Of such information that is accessible in the records of the National Control of Quality of the Medicines Agency, as well as in the studies that are done with the aim of evaluating analytical and/or clinical performance of available tests (this work is a review of the performances of most of the tests sold in France today). It stands out that the best borders on very mediocre and that it is indispensable, like the planned nomenclature of the medical biological consultations, to take into consideration the technique used to obtain a result for interpretation of the latter.

[Level of histamine in supernatants from the basophil activation test: applications to hymenoptera allergy and drug allergy--preliminary study]. / Dosage de l'histamine sur les surnageants du test d'activation des basophiles: applications a l'allergie aux venins d'hymenopteres et a l'allergie medicamenteuse--étude préliminaire.

Histamine Release technic consists in calculating histamine liberated by blood cells in touch with an allergen. To this day, this method is only used in Hymenoptera venom allergy diagnosis. The principle of this study is to measure histamine released by activated basophils in surnageons of Basophil Activation Test (BAT) for different allergens: Hymenoptera venoms: Bee, White Faced Hornet, Vespula Wasp. Drugs: Cefaperos, Clamoxyl, Alfatil, Rapifen, Diprivan, Nesdonal, Mivacron. A threshold of positivity (amplification factor in comparison with the control) is determined for these two classes of allergens: 45 for Hymenoptera venoms and 9 for drugs. These results, compared to the other diagnosis technics (Histamine Release, Basophil Activation Test, Prick Tests) discloses very high correlation rates in each case. This method seems to be a reliable method for Hymenoptera venom allergy diagnosis and for drugs allergy diagnosis too. However, this study is based on a few number of patients, so a significant statistic conclusion can't be expressed but it opens an interesting way of research.

[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.

[Comparison of the measurements of total IGE using the CAP-RIA technique and the UNICAP-FEIA technique]. / Comparaison du dosage d'IgE totales par la technique CAP-RIA et et par la technique UNICAP-FEIA.

The contribution of a new method of measurement of total IgE by the Fluoro-Enzymo-Immuno-assay (FEIA) with the assistance of Pharmacia UNICAP automation is very reliable since, when compared with the measurement by the CAP SYSTEM RIA on 144 patients who were clinically allergic (84 with a raised and 60 with a normal IgE titre) the result of a comparative study between the two techniques is statistically highly significant. The two methods were also compared by age groups and these results were also statistically highly significant.

[Cross-reactivity between cow's milk and goat's milk]. / Etude de la réactivité croisée entre le lait de vache (LV) et le lait de chèvre (LC).

Tolerance of LC may be explained by a great intra and interspecific genetic diversity that leads to a great variety of proteins. In contrast, industrial treatment (UHT sterilisation) introduces configural modifications that may provoke a very great structural homology between the proteins of the two milks. Casein is in high concentration in both milks and shows some homology of the AA sequences between both and it makes a common allergen for a cross- reaction. It has been observed that a good IgE inhibition in vitro does not automatically form a clinical cross-allergenicity. Therefore there may exist an immunological crossed reaction between LC and LV without production of a real crossed allergenicity.

[Western blotting or immunoblotting: application of the Alastat-Alablot to the study of cross reactions between cow's milk and goat's milk]. / Westernblot ou immunoblot: application à l'Alastat-Alablot pour la recherche de la reactivite croisée entre lait de vache (LV) et lait de chèvre (LC).

The Alastat-Alablot was used as immunoblot technique for detecting cross-reactivity between CM and GM. The study was conducted on 9 patients presenting with a food allergy well-documented on the basis of clinical history and elimination diet and re-exposure tests. The Alastat-Alablot technique proved to be sensitive in detecting low levels of specific IgE not detectable by the Cap System. The most frequently encountered proteins that are shared between CM and GM have a molecular weight corresponding to beta-lactoglobulin and casein in the majority of patients.

[Histamine liberation (or histamine release HR): study of the positivity threshold in hymenoptera venom allergy]. / Histamino-libération (ou histamine release HR): étude au seuil de positivité dans l'allergie aux venins d'hyménoptères.

Dialysis of Hymenoptera venoms for measurement of HR by the radioimmunoassay technique (RIA, Immunotech) lowers the threshold of positivity of the HR titre from 30% to 12%. This modification of dialysis of the venoms significantly improves the sensitivity and specificity of the technique.

[Preliminary study of the basophil activation test for drug allergy using anti-membrane antibodies and flow cytometry]. / Etude préliminaire du test d'activation des basophiles à l'aide d'anticorps membranaires par cytométrie de flux dans l'allergie médicamenteuse.

This study deals with the basophil activation test (BAT) in drug allergy, basophil activation being analysed by flow cytometry. In the seven cases studied here (pollen, mite and drug hypersensitivity), we shown that BAT was a reliable test which presented interesting future prospects.

[Total serum IgE levels. Comparative study of the Kallestad and Pharmacia techniques]. / Dosages des IgE totales sériques. Etude comparative des techniques "Kallestad" et "Pharmacia".

The Kallestad technique for measurement of total IgE is based on a "sandwich" technique that uses a solid phase (wells in a sensitised microplate) as a separator. The samples that contain the IgE and monoclonal anti-IgE antibodies (AcM anti-IgE) are incubated in the wells sensitised with goat anti-mouse IgE. The AcM anti-IgE binds the IgG and the complex is then bound to the wells by the anti-mouse IgG. After washing, anti IgE marked with alkaline phosphatase is added and this binds to the previously linked IgE. The intensity of colouration is directly proportional to the concentration of IgE in the sample and is measured by spectrophotometry. The Kallestad technique has very great sensitivity (> 1 KUI/L) for a serum sample of 20 microliter. The measurement is quick, with 2 h incubation. There was good intra-assay reproducibility for values 2 to 1100 KUI/L with CV of 7.6. This good reproducibility was also found in inter-assays for values between 2 and 10 KUI/L. The Kallestad technique was evaluated firstly against the CAP RIA System of Pharmacia and secondly, for values against the kit RIA Ultra. For a population labelled "all comers" with values between 2 to 1100 KUI/L the statistical analysis showed no significant difference between the two techniques. The statistical analysis of the study of low values, made with the RIA Ultra kit showed that there was a significant difference between the two techniques. Exchange of standards between the two methods showed a certain over-evaluation of the values of Pharmacia whilst measurements with the Kallestad standards showed a relative under-evaluation. It should be noted that the comparison has been made between a RIA (Pharmacia) technique and one of EIA (Kallestad).