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.

Erroneous production of PAPP-A kits: the impact of a downward shift in PAPP-A concentration on the test performance of first-trimester combined screening for Down syndrome.

OBJECTIVE: To evaluate a 20% downward shift in the pregnancy-associated plasma protein A (PAPP-A) concentration on the test performance of first-trimester combined screening (FTS) for Down syndrome (DS) following a flaw in the production of PAPP-A kits on FTS for DS. METHODS: A retrospective re-evaluation of PAPP-A in stored sera. Inclusion criteria were a maternal weight-corrected PAPP-A multiple of the median value ≥ 0.9 and a biochemical risk of DS ≤ 1:200 at the time of testing. RESULTS: Of the 3100 women, 473 (15%) fulfilled the inclusion criteria. After combining the biochemical risk based on the incorrect PAPP-A values with nuchal translucency findings, an increased risk for DS was initially found in 107 women [false positive rate (FPR): 3.1]. Eighty-two (77%) of the 107 women opted for invasive testing. Following re-analysis of PAPP-A, the biochemical risk and the combined risk were statistically significantly different from the initial risk estimates (p < 0.001.). We noticed that 25 women (30%) had invasive testing, while this was unjustified given the re-analysed PAPP-A. CONCLUSION: Erroneous PAPP-A kits resulted in an increase in the FPR by 1.2%. There were no reports of iatrogenic miscarriage. The occurrence of this problem reaffirms the importance of continuous monitoring of quality in FTS.

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.

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.

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.

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.

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.

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.

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.

[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.

Standardization of basophil degranulation for pharmacological studies.

There is a need of a standard basophil degranulation test for diagnosis and for immunopharmacological studies. In order to obtain a sufficient concentration of basophils blood was sedimented at 1 g which gave a yield of 1500-3000 basophils/microliter instead of 50/microliter in whole blood. As the course of degranulation is exponential, it can be linearised using a logarithmic approximation Y = A0 + A1 log D where D is the allergen concentration. The application of this mathematical method permitted the collection of statistical information about the test itself. The coefficient of variation was less than 10%. The test can be used to compare the allergenic potency of different extracts of the same allergen.

Experimental autoimmune uveoretinitis in athymic rats: specific IgE response to retinal S-antigen and disease.

Experimental autoimmune uveoretinitis (EAU) is an ocular autoimmune disease induced in rats by immunization with retinal S-antigen. Athymic nude rats (rnu/rnu) have been previously shown to be refractory to EAU induction and antibody production to S-antigen, while heterozygous (rnu/+) are good responders. Increasing the antigen dose and adding pertussis adjuvant produced ocular disease in some nude rats, and antibody response in most of them. Specific IgE antibodies were demonstrated by ELI-SA only in the serum of nude rats presenting the disease. However, most immunized nude rats had evidence of mast cell sensitization to S-antigen (direct degranulation test) and of circulating specific IgE detected by passive sensitization of normal mast cells (indirect degranulation test). This positive response could be explained by an incomplete depletion of the different T lymphocyte subsets.

Comparison of effects of ioxaglate versus iomeprol on histamine and tryptase release in patients with ischemic cardiomyopathy.

We observed a release of histamine, but not of tryptase, in arterial blood from 64 patients with ischemic heart disease and 24 patients without coronary disease, which was provoked by ioxaglate, a ionic compound, but was not provoked by iomeprol, a non-ionic radiocontrast compound. The release of histamine in arterial blood after ionic contrast medium injection was higher in patients with ischemic heart disease compared with patients without coronary disease, suggesting that an increased release from heart mast cells previously observed exists also for systemic blood basophils.

Behavior-state-dependent changes in human fetal pulmonary blood flow velocity waveforms.

OBJECTIVE: To establish the influence of fetal behavior states on venous and arterial pulmonary blood flow velocity waveforms in the normally developing term fetus. METHODS: The relation between venous and arterial pulmonary blood flow velocity waveforms and fetal behavior states was investigated in 18 normal term fetuses. Recordings of the venous pulmonary blood flow velocity waveforms were obtained just proximal to the entrance in the left atrium, and the arterial pulmonary blood flow velocity waveforms were taken from the most proximal branch of the pulmonary artery in the same lung using color Doppler imaging. Time-averaged peak systolic, peak diastolic, and end-diastolic flow velocity; peak systolic to peak diastolic ratio; pulsatility index; and fetal heart rate were calculated from both venous and arterial Doppler recordings obtained during behavior states 1F (quiet sleep) and 2F (active sleep). Fetal behavior states were determined from combined recordings of fetal eye and body movements. RESULTS: Recordings of sufficient quality for analysis were obtained from ten fetuses. Venous pulmonary blood flow velocity waveforms demonstrated a statistically significant increase in time-averaged peak diastolic and end-diastolic velocity during fetal behavior state 2F. No behavior-state-related changes were observed for the arterial pulmonary blood flow velocity waveform. CONCLUSION: The data suggest an increased pressure gradient between the pulmonary venous system and the left atrium during behavior state 2F. Flow velocity waveforms from the proximal arterial pulmonary branch are independent of behavioral state.

Severe retinochoroidopathy: variations of humoral and cellular immunity to S-antigen in a longitudinal study.

Ten patients with birdshot retinochoroidopathy, six with isolated retinal vasculitis and eight with Behçet's disease were treated with cyclosporine for one to three years. Autoantibodies to several retinal proteins, circulating lymphocyte subsets and cellular reactivity to S-antigen were evaluated repeatedly during this period. Autoantibody titers were similar in patients and in controls. However the serum content of antibodies to IRBP or S-antigen was lessened during inflammatory periods in some patients. In some sera, antibodies reacted with enzyme digested S-antigen preparations by immunoblot, whereas the same sera were negative for the native protein. A decrease of the CD4+ subpopulation of peripheral blood lymphocytes was associated with relapses of ocular inflammation in birdshot retinochoroidopathy. In this disease and in idiopathic retinal vasculitis, the positive lymphocyte stimulation test and basophil degranulation test with S-antigen were significantly most frequent in the period preceding a relapse of ocular inflammation. These tests could therefore be of predictive value for relapses occurring within the next few months.

Pharmacological modulation of IgE-dependent mast cell degranulation in experimental autoimmune uveoretinitis.

Immediate hypersensitivity phenomena have been shown previously to occur at the beginning of experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by immunization with purified S-antigen from bovine retina. The onset time and severity of the disease were modified by modulating the mast cell (MC) function. Drugs blocking the release of mediators from MC, disodium cromoglycate and ketotifen, given as eyedrops, slightly delayed the onset and decreased the severity of inflammation. Compound 48/80, a drug effective in depleting MC of their inflammatory mediators, delayed significantly the onset, decreased and sometimes suppressed EAU, when given by the subconjunctival or intraperitoneal routes. No modification of the IgG and the IgE circulating anti-S-antibody level was demonstrated in both types of treatment, whereas in vitro reagin-dependent degranulation of peritoneal MC in the presence of the antigen was decreased in both cases. Identification of MC-bound reagins as IgE was strongly suggested by the blocking effect of anti-IgE antibodies on MC degranulation. These data confirm the link between MC activation and the onset and severity of EAU.

Allergy to nickel in dental alloys.

A case of oral allergy to dental alloys is presented, highlighting the interest of dosage of salivary nickel and of flow cytometry showing a selective CD4+ activation. The discrepancy between the rarity of oral allergy to dental alloys and the frequency of nickel sensitization and nickel-induced contact dermatitis leads to discuss the mechanisms of oral tolerance.

[Interpretation of results on a human basophil degranulation test (author's transl)]. / Interprétation du résultat d'un test de dégranulation des basophiles humains.

A statistical analysis of a result on a human basophile degranulation test was performed. The shape of the curve representing the number of basophiles, as a function of the logarithm of the allergenic dosage, was parabolic, in the case of degranulation. This curve permits a calculation of the dosage necessary to provoke a degranulation of 10 per cent, in comparison with the mean of the two control test tubes. The authors also determined the significant degranulation threshold to be 5 per cent, as regards the mean number of cells counted for the controls (example: for 50 control cells counted, the threshold was equal to 28 per cent degranulation). This statistical analysis largely facilitates the interpretation of the granulation test by basophiles, by assuring the value of observed degranulations, and by allowing a better quantification of the phenomenon. The latter might permit a comparison of different batches of allergen, that is, the standardisation of allergens. These calculations can be easily programmed, using a currently available calculator.