The importance of EN ISO 15189 accreditation of allergen-specific IgE determination for reliable in vitro allergy diagnosis.

BACKGROUND: Allergen-specific serum immunoglobulin E detection and quantification have become an important step in allergy diagnosis and follow-up. In line with the current trend of laboratory test accreditation to international standards, we set out to design and assess an accreditation procedure for allergen-specific serum IgE. METHODS: Method validation according to the accreditation procedure under the EN ISO 15189 standard was carried out for allergen-specific immunoglobulin E determination using the fluoroimmunoenzymatic method ImmunoCAP(®) (ThermoFisher). Data were produced by 25 hospital laboratories in France. A total of 29 allergen specificities including mixes, extracts, and molecular allergens were assayed. Allergen-specific serum immunoglobulin E concentrations ranged from 0.1 to 100 kUA /l. RESULTS: Repeatability, reproducibility, and accuracy results fulfilled method validation criteria for automated laboratory tests and proved similar irrespective of the allergen specificity, allergen-specific serum immunoglobulin E concentration, or individual laboratory. CONCLUSION: Allergen-specific serum immunoglobulin E determination with the fluoroimmunoenzymatic method ImmunoCAP(®) is a highly repeatable, reproducible, and accurate method which may be considered as a single analyte assay in view of the EN ISO 15189 accreditation procedure.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome: a multicenter study. II. Basophil activation by nonsteroidal anti-inflammatory drugs and its impact on pathogenesis.

BACKGROUND: Patients who are clinically hypersensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) sometimes present basophil activation in vitro, and in 50% of cases a parallel response to release of sulfidoleukotrienes (cellular allergen stimulation test) is observed. These phenomena occur not only in clinically hypersensitive patients, but also in some healthy controls who tolerate NSAIDs. MATERIAL AND METHODS: We studied 16 clinically hypersensitive patients, 22 controls tolerating NSAIDs, and 29 healthy blood donors (clinical NSAID status unknown) using 2 different basophil isolation techniques (buffy coat or plasma leukocytes). RESULTS: In a population of 13 aspirin-tolerant healthy controls and 29 healthy blood donors, basophil activation with aspirin, diclofenac, and naproxen was analyzed at 4 different concentrations. The results in the 2 groups were quite similar in qualitative terms. Choosing a cutoff of 5% and a stimulation index >2, the proportion of positive results increased with the concentration. There were more positive results at all concentrations using the plasma leukocyte technique. CONCLUSIONS: The most important finding of this study is that basophil activation by NSAIDs occurs not only in clinically hypersensitive patients but also, to a very variable extent and on an individual basis, in apparently normal healthy individuals who tolerate NSAIDs. The phenomenon is clearly dose-related, and hypersensitive patients seem to react to lower NSAID concentrations.

Inhibition of basophil activation by histamine: a sensitive and reproducible model for the study of the biological activity of high dilutions.

BACKGROUND: At the beginning of this series of experiments we were looking for a model based on the use of purified commercially available compounds based on a fully described and accepted pharmacological model to study of the biological effect of high dilutions. Negative feedback induced by histamine, a major pro-inflammatory mediator, on basophils and mast cells activation via an H2 receptor me these criteria. The simplest way of measuring basophil activation in the early 1980's was the human basophil activation test (HBDT). OBJECTIVES: Our major goal was first to study the biological effect of centesimal histamine dilutions beyond the Avogadro limit, on the staining properties of human basophils activated by an allergen extract initially house dust mite, then an anti-IgE and N-formyl-Met-Leu-Phe (fMLP). Technical development over the 25 years of our work led us to replace the manual basophil counting by flow cytometry. The main advantages were automation and observer independence. Using this latter protocol our aim was to confirm the existence of this phenomenon and to check its specificity by testing, under the same conditions, inactive analogues of histamine and histamine antagonists. More recently, we developed an animal model (mouse basophils) to study the effect of histamine on histamine release. METHODS AND RESULTS: For the HBDT model basophils were obtained by sedimentation of human blood taken on EDTA and stained with Alcian blue. Results were expressed in percentage activation. Histamine dilutions tested were freshly prepared in the lab by successive centesimal dilutions and vortexing. Water controls were prepared in the same way. For the flow cytometric protocol basophils were first labeled by an anti-IgE FITC (basophil marker) and an anti-CD63 (basophil activation marker). Results were expressed in percentage of CD63 positive basophils. Another flow cytometric protocol has been developed more recently, based on basophil labeling by anti-IgE FITC (fluorescein isothiocyanate) and anti-CD203 PE (another human basophil activation marker). Results were expressed in mean fluorescence intensity of the CD203c positive population (MFI-CD203c) and an activation index calculated by an algorithm. For the mouse basophil model, histamine was measured spectrofluorimetrically. The main results obtained over 28 years of work was the demonstration of a reproducible inhibition of human basophil activation by high dilutions of histamine, the effect peaks in the range of 15-17CH. The effect was not significant when histamine was replaced by histidine (a histamine precursor) or cimetidine (histamine H2 receptor antagonist) was added to the incubation medium. These results were confirmed by flow cytometry. Using the latter technique, we also showed that 4-Methyl histamine (H2 agonist) induced a similar effect, in contrast to 1-Methyl histamine, an inactive histamine metabolite. Using the mouse model, we showed that histamine high dilutions, in the same range of dilutions, inhibited histamine release. CONCLUSIONS: Successively, using different models to study of human and murine basophil activation, we demonstrated that high dilutions of histamine, in the range of 15-17CH induce a reproducible biological effect. This phenomenon has been confirmed by a multi-center study using the HBDT model and by at least three independent laboratories by flow cytometry. The specificity of the observed effect was confirmed, versus the water controls at the same dilution level by the absence of biological activity of inactive compounds such as histidine and 1-Methyl histamine and by the reversibility of this effect in the presence of a histamine receptor H2 antagonist.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

Diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study.

INTRODUCTION: This multicenter study aimed to evaluate the diagnostic value of 2 cellular tests based on basophil reactivity--the basophil activation test (BAT, Flow-CAST) and the sulfidoleukotriene release assay (CAST-ELISA)--in immediate-type beta-lactam allergy, particularly in patients with a clinical history of allergy and a negative skin test result. MATERIAL AND METHODS: In a multicenter study encompassing 10 European centers, 181 patients with a history of immediate-type beta-lactam allergy, and 81 controls, we evaluated the diagnostic efficiency of specific IgE determinations and of 2 cellular tests based on basophil reactivity, the BAT and the sulfidoleukotriene release assay. RESULTS: With Flow-CAST, sensitivity varied for individual beta-lactam allergens from 16% for penicilloyl-polylysine to 33% for amoxicillin, reaching 50% when all 5 allergens were considered. In beta-lactam-allergic patients with negative skin test results (22.8%), Flow-CAST showed positive results for at least 1 of the 5 allergens in 37%. Specificity varied from 89% to 97%, depending on the allergens used. In CAST-ELISA, the overall sensitivity in skin test-positive patients was 41.7%; in patients with negative skin test results it was 27.9%. Both tests were not absolutely correlated, so that when all the results were considered together, sensitivity increased to 64.3% and specificity varied for both tests combined from 73% to 92%. In contrast, specific IgE determinations in the same population yielded a lower sensitivity (28.3%). CONCLUSIONS: A diagnostic algorithm including skin tests and specific IgE, followed by cellular tests in negative patients and controlled challenge enabled us to confirm beta-lactam allergy in 92% of cases. This procedure would also allow us to avoid two-thirds of the required controlled challenges.

Pre-lethal anaphylaxis to carboxymethylcellulose confirmed by identification of specific IgE--review of the literature.

BACKGROUND: Carboxymethylcellulose (CMC) is used extensively in the pharmaceutical and food industries on account of its various properties. Anaphylactic reactions are rare. It has been reported principally after intra-articular infiltration of sustained-release corticosteroids containing CMC and, very rarely, after barium enema. METHODS: A case of pre-lethal anaphylactic shock after barium enema was studied by prick-test, intra-dermal reaction (IDR), leukocyte histamine release test (LHRT), basophil activation test (BAT), cystein-leukotriene release test (CAST) and dot-blot analysis. RESULTS: IDR to CMC was positive at a concentration of 10 microg/ml. BAT and CAST were positive. Specific IgE were identified using dot-blot analysis. DISCUSSION: This is the third report of CMC-specific IgE and the second of anaphylaxis to CMC associated with a barium suspension in contact with GI tract mucosa. CMC as an excipient in medicinal products may therefore be a risk factor for severe anaphylaxis after injection or following contact with GI tract mucosa. Sensitization and allergic reactions by CMC in food additives have to be considered.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.

Cellular basophil activation tests (BAT) such as histamine or sulfidoleukotriene-release tests for allergy diagnosis have been available for some time, but expression of basophil-activation markers such as CD63 and CD203c detected by flow cytometry has attracted particular attention in recent years. Not only the potential but also the possible pitfalls of flow-cytometric BAT have been stressed recently. Some authors have suggested that the technical problems are still such that BAT should only be performed in specialist laboratories. In an earlier review based on our clinical experience obtained over several years, we showed that, even using different protocols, reproducible and meaningful clinical results can be obtained. In this paper, we review the current knowledge in relation to several technical issues and show that flow-cytometric BAT already represents a major advance in the field of in vitro allergy diagnosis. We conclude that there are no serious technical justifications for depriving allergic patients of clinically indicated BAT tests, which can be performed reliably by any laboratory with the appropriate experience in allergy diagnosis and flow cytometry.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, beta-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

Use of both CD63 up regulation and IgE down regulation for the flow cytometric analysis of allergen induced basophil activation. Definition of an activation index.

OBJECTIVE AND DESIGN: The aim of this study was to compare the use of a late (CD63) and an early (IgE) marker of basophil activation in the flow cytometric diagnosis of beta-lactam induced allergic hypersensitivity reactions. SUBJECTS: Twelve patients who had had a clear cut betalactam induced immediate reaction and 16 controls were selected, as well as 11 patients who had had an immediate reaction to bee or wasp stings. METHODS: Leukocyte suspensions were incubated with allergen dilutions as well as 2 positive controls (anti-IgE and NFormyl- Methionyl-Leucyl-Phenylalanine (fMLP)). Basophils were labelled with an anti-IgE FITC (fluorescein isothiocyanate) and an anti-CD63 PE (phycoerythrin). Results were expressed as percentage CD63 expression and index calculated according to a specific algorithm including the two activation markers. RESULTS: Significant CD63 expression (>5 %) was observed in 3/12 cases for the beta-lactam sensitized population, in 0/16 cases for the controls and in 11/11 cases for the venom sensitized population. A significant index (determined by a ROC analysis) was observed in 11/12 beta-lactam sensitized patients and in 0/16 controls. CONCLUSION: These results show that IgE (an early activation marker) is more sensitive than CD63 (a later activation marker) in the diagnosis of beta-lactam allergy.

Flow-assisted allergy diagnosis: current applications and future perspectives.

Physicians predominantly rely upon quantification of serum-specific immunoglobulin E (IgE) and/or skin test to confirm clinically suspected IgE-mediated allergy. However, for various reasons, identification of the offending allergen(s) and potentially cross-reactive structures is not always straightforward. Flow-assisted allergy diagnosis relies upon quantification of alterations in the expression of particular basophilic activation markers. Actually, upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. Currently, the technique has been applied in the investigation of IgE-mediated allergy caused by classical inhalant allergens, food, Hevea latex, hymenoptera venoms and drugs. It is also appreciated; the technique proves valuable in the diagnosis of non-IgE-mediated (anaphylactoid) reactions such drug hypersensitivity and the detection of autoantibodies in certain forms of chronic urticaria. This review will not address immunologic features, characteristics and general pitfalls of flow-assisted analysis of in vitro-activated basophils as summarized elsewhere. After a recapitulation of the principles and some specific technical issues of flow-assisted analysis of in vitro-activated basophils, we principally focus on the current clinical and research applications of the basophil activation tests. Personal experience of both research groups is provided, where appropriate. Finally, a viewpoint on how the field might evolve in the following years is provided.

Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c.

BACKGROUND: Histamine is known to elicit a negative feedback effect on anti-IgE and allergen-induced basophil activation. A series of experiments performed between 1981 and 1995 using a manual method showed biological activity of highly diluted histamine. Most of the experiments used histamine in the range 10(-30) (15C)-10(-36) M (18C). These results were confirmed by automated flow cytometry, but this method is based on the selection of basophils by anti-IgE and analysis of basophil activation by anti-CD 63, showing significant but relatively low inhibition (approximately 14%), insufficient to convince the scientific community of the reality of the phenomenon. OBJECTIVE: We investigated if the use of CD 203c, a basophil specific, earlier marker than CD 63 of the activation cascade, increased the sensitivity of the method, testing two target histamine dilutions, 10(-4) (2C) and 10(-32) M (16C). METHODS: Basophils, obtained from buffy coats, were pre-incubated with the histamine dilutions and activated by two agonists: anti-IgE and fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Basophil activation was stopped with EDTA. The cells were labelled with anti-IgE, anti-CD 13 and anti-CD 14 for basophil selection, and anti-CD 63 and anti-CD 203c for basophil activation. Results were expressed in up-regulation percentage for CD 63 or mean intensity of fluorescence (MFI) for CD 203c. RESULTS: Histamine 10(-4) M (2C) and histamine 10(-32) M (16C) were capable of inhibiting both IgE-dependent (anti-IgE) and IgE-independent (fMLP) basophil activation. The percentage inhibition depended on the activation marker used. The highest inhibition for histamine dilution 16C was observed with CD 203c (38%, P<0.001), approximately half the inhibition observed with histamine 2C (73%). CONCLUSION: These new flow cytometric protocols confirmed that high dilutions of histamine may inhibit basophil activation and that the inhibitory effect is not restricted to IgE-dependent activation. The use of CD 203c instead of CD 63 increased the magnitude of the response.

[Investigation of allergic reactions to platinum salts]. / Exploration d'une allergie aux sels de platine.

INTRODUCTION: We report two cases occurring in 2004 of patients being treated for pleural mesothelioma with a combination of cisplatin or carboplatin and pemetrexed. Investigation by skin tests and flow cytometry confirmed the clinical diagnosis in both cases. CASE REPORTS: Case 1: a man of 62 developed, after 12 courses of cisplatin-pemetrexed, an anaphylactic reaction 5 minutes after the infusion of cisplatin. Treatment was withdrawn permanently. Case 2: a man of 66 developed, after 7 courses of cisplatin-pemetrexed, an anaphylactic reaction within the first minute of the infusion of cisplatin. Subsequently, in March 2004, he received pemetrexed alone without any problems. In August 2004 he was prescribed carboplatin-pemetrexed. Within 5 minutes he developed urticaria, pruritus and abdominal pain. He was treated later with pemetrexed alone with no problems. CONCLUSION: Hypersensitivity to platinum salts usually occurs after several courses of treatment. Skin tests and flow cytometry are a simple, concordant, and reliable way of confirming the diagnosis.