Didier G. Ebo

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, β-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.

The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease

The basophil activation test (BAT) has become a pervasive test for allergic response through the development of flow cytometry, discovery of activation markers such as CD63 and unique markers identifying basophil granulocytes. Basophil activation test measures basophil response to allergen cross‐linking IgE on between 150 and 2000 basophil granulocytes in <0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In addition to clinical history, skin prick test, and specific IgE determination, BAT can be a part of the diagnostic evaluation of patients with food‐, insect venom‐, and drug allergy and chronic urticaria. It may be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti‐IgE treatment or in the natural resolution of allergy. Basophil activation test may use fewer resources and be more reproducible than challenge testing. As it is less stressful for the patient and avoids severe allergic reactions, BAT ought to precede challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. In this EAACI task force position paper, we provide an overview of the practical and technical details as well as the clinical utility of BAT in diagnosis and management of allergic diseases.

Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy

Specific immunoglobulin E (IgE) and commercially available skin prick tests have been demonstrated to be unreliable methods to diagnose pollen‐associated food allergy. To evaluate the predictive value of the basophil activation test (BAT) in pollen‐associated food allergy, the apple‐mediated oral allergy syndrome (OAS) in patients with birch pollinosis was chosen as a representative model.

Contact allergic dermatitis and life-threatening anaphylaxis to chlorhexidine.

A 43-year-old nonatopic man was seen with a history of dyspnea, shock, and S-T segment elevations in the inferior leads of his electrocardiogram after disinfection of a drain insertion site with a chlorhexidine digluconate 2% solution. His personal history revealed a similar reaction 15 minutes after anesthesia was achieved with a preoperative administration of cephazolin; application of povidone-iodine; and the use of propofol, atracurium, and sufentanil. Macromolecular plasma expanders were not infused, and exposure to chlorhexidine was not mentioned. He also recalled two episodes of a rash at surgical incision areas, which, in the presence of an infiltrated, papular, and vesicular patch test for chlorhexidine digluconate 0.5% solution and Hibitane, was diagnosed as contact dermatitis to the antiseptic. Total serum IgE was 480 kU/L. Serum-specific IgE test results for latex, ethylene oxide, chloramine, penicilloyl G, penicilloyl V, and amoxicillin (Upjohn-Pharmacia CAP System, Brussels, Belgium) were negative. Lymphocyte transformation test results with chlorhexidine (10 mg/ ml), sufentanil (0.005 mg/ml), vecuronium (10 mg/ml), atracurium (10 mg/ml), propofol (10 mg/ml), and cephazolin (10 mg/ml) were negative (stimulation index ,2). Skin prick test (SPT), intradermal test (IDT), and subcutaneous test results with nonammoniated latex (IDT, 10–3 mg/ml), alcohol 70% (SPT, undiluted), 100 mg/ml povidone-iodine (SPT, undiluted), 50 mg/ml cephazolin (SPT, undiluted), 2.5 mg/ml bupivacaine (subcutaneous test, undiluted), 10 mg/ml propofol (IDT, 10–2 mg/ml), 2 mg/ml etomidate (IDT, 10–2 mg/ml), 5 mg/ml sufentanil (IDT, 10–2 mg/ml), 4 mg/ml vecuronium (IDT, 10–2 mg/ml) and 10 mg/ml atracurium (IDT, 10–2 mg/ml) were negative. A SPT result with chlorhexidine digluconate 2% in alcohol 70% was positive (SPT 10–4, wheal/flare, 5/13 mm). Patch test results showed a delayed hypersensitivity to cetrimide 0.1%, chloroxylenol 1%, chloramine 0.5%, and iodine 0.25%, but they were negative for hexamidine 0.15%, thiomerosal 0.1%, Mercurochrome 2%, eosine 50%, and Chlorophen. DISCUSSION

Increased IL-17 production by peripheral T helper cells after tumour necrosis factor blockade in rheumatoid arthritis is accompanied by inhibition of migration-associated chemokine receptor expression

OBJECTIVES The contribution of IL-17-producing Th17 cells to the pathogenesis of T-cell-mediated inflammatory disorders such as RA and atopic dermatitis (AD) has to be viewed in relation to the role of Th1/Th2 cells and long-recognized key cytokines like TNF. We aimed to study the frequency and migration-associated phenotype of peripheral Th17, Th1 and Th2 cells in healthy individuals, RA and AD patients, and to study the influence of anti-TNF therapy in RA. METHODS Intracellular IL-17, IFN-γ and IL-4 production and CC-chemokine receptor CCR4 and CCR6 expression were analysed flow cytometrically in peripheral memory Th cells from healthy individuals, AD and RA patients. The latter were grouped by disease activity and presence or absence of adalimumab therapy. In RA patients initiating anti-TNF therapy, cytokine production by in vitro-stimulated peripheral mononuclear cells was measured by cytometric bead array. RESULTS The peripheral Th17 cell frequency is elevated in AD but not in RA. In RA, Th17 cells and IL-17 production increase after anti-TNF therapy, irrespective of disease activity. Th1 cells and IFN-γ production are elevated in remission and under anti-TNF therapy. CCR6 expression is up-regulated in Th17 cells, but RA patients in remission under anti-TNF therapy have significantly lower expression than those with active disease. CONCLUSIONS The increase in peripheral Th17 cells in RA patients after anti-TNF therapy is accompanied by a decrease in Th17-specific CCR6 expression, which might prevent homing of these potentially pro-inflammatory cells to the synovium.

Latex-specific IgE, skin testing, and lymphocyte transformation to latex in latex allergy

BACKGROUND This study was designed to determine the discriminative value of latex-specific IgE tests, latex skin tests, and lymphocyte transformation tests (LTTs) to latex in 38 patients with latex allergy (12 nonatopic and 26 atopic) and 44 control subjects (24 nonatopic and 20 atopic). We also evaluated the recommended positive cutoff (i.e., 0.35 kU/L) of both in vitro latex-IgE tests. METHODS Latex-specific IgE levels were determined by the Immuno-CAP (Upjohn-Pharmacia) and the ALaSTAT-RIA (Diagnostic Products Corp.) assays. Skin tests and LTFs were performed with a nonammoniated latex extract (DPC). Sensitivities and specificities were defined according to the 95th percentile value of nonatopic control subjects. For the in vitro IgE tests, sensitivity and specificity were also calculated by using the proposed positive threshold of 0.35 kU/L. Sensitivities and specificities of both cutoffs were compared. RESULTS Compared with a clinical history of latex allergy and according to the 95th percentile value of nonatopic control subjects (0.44 kU/L), latex-specific IgE determined by the Immuno-CAP assay achieved a sensitivity of 97% and a specificity of 86%. For the ALaSTAT-RIA assay, with 0.54 kU/L as the 95th percentile threshold value in nonatopic control subjects, sensitivity was 100%, and specificity was 83%. According to the threshold value of 0.35 kU/L, a sensitivity of 97% and a specificity of 83% for the Immuno-CAP assay and a sensitivity of 100% and a specificity of 33% for the ALaSTAT-RIA assay were observed. The latex skin test reached a sensitivity of 97% and a specificity of 100%. The LTF to latex showed a sensitivity of 39% and a specificity of 95%. No relation between symptoms and latex-specific IgE tests, latex skin tests, or LTTs was found. CONCLUSIONS Our results confirm that latex skin tests and latex-specific IgE assessments are sensitive and specific methods for establishing the diagnosis of latex allergy, although the specificity of the ALaSTAT-RIA assay was very low when interpreted according to the threshold of 0.35 kU/L. The LTT to nonammoniated latex is too insensitive for diagnosis of allergy to latex. This reemphasizes that in order to evaluate the sensitivity and specificity of diagnostic procedures, one should always include an appropriate control group.

Activated T cells complicate the identification of regulatory T cells in rheumatoid arthritis.

Most cell surface markers for CD4(+)CD25(+) regulatory T cells (Tregs) are also expressed by activated non-regulatory T cells. Recently, CD127 down-regulation was found to identify functional Tregs in healthy individuals, but there are no data from patients with inflammatory conditions. We examined peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis patients with active inflammation and from healthy controls, and found that CD4(+) T cells contained an equal proportion of CD25(+)CD127(-)/low cells in both groups. In patients, not all these cells expressed intracellular FOXP3. Upon activation by anti-CD3/anti-CD28, PBMC rapidly down-regulated CD127, while FOXP3 up-regulation was transitory and occurred in fewer cells. The activated cells were not anergic to restimulation and had no suppressive effects. The distinct kinetics indicate that the FOXP3(-)CD127(-)/low cells in rheumatoid arthritis patients most likely represent activated non-regulatory T cells. This complicates the use of CD127 for identification of Tregs in inflammatory diseases.

The in vitro diagnosis of drug allergy: status and perspectives

To cite this article: Ebo DG, Leysen J, Mayorga C, Rozieres A, Knol EF, Terreehorst I. The in vitro diagnosis of drug allergy: status and perspectives. Allergy 2011; 66: 1275–1286.

Immunoglobulin E antibodies to rocuronium : a new diagnostic tool

Background: Diagnosis of allergy from neuromuscular blocking agents is not always straightforward. The objectives of the current study were to investigate the value of quantification of immunoglobulin E (IgE) by ImmunoCAP (Phadia AB, Uppsala, Sweden) in the diagnosis of rocuronium allergy and to study whether IgE inhibition tests can predict clinical cross-reactivity between neuromuscular blocking agents. Methods: Twenty-five rocuronium-allergic patients and 30 control individuals exposed to rocuronium during uneventful anesthesia were included. Thirty-two sera (total IgE > 1,500 kU/l) were analyzed for potential interference of elevated total IgE titers. Results were compared with quantification of IgE for suxamethonium, morphine, and pholcodine. Cross-reactivity between drugs was assessed by IgE inhibition and skin tests. Results: Sensitivity of IgE for rocuronium, suxamethonium, morphine, and pholcodine was 68, 60, 88, and 86%, respectively. Specificity was 100% for suxamethonium, morphine, and pholcodine IgE and 93% for rocuronium IgE. ROC analysis between patients and control individuals changed the threshold to 0.13 kUa/l for rocuronium, 0.11 kUa/l for suxamethonium, 0.36 kUa/l for morphine, and 0.43 kUa/l for pholcodine. Corresponding sensitivity was 92, 72, 88, and 86%, respectively. Specificity was unaltered. Interference of elevated total IgE with quantification of IgE was demonstrated by the analysis in sera with a total IgE greater than 1,500 kU/l. IgE inhibition did not predict clinical relevant cross-reactivity. Conclusions: The rocuronium ImmunoCAP constitutes a reliable technique to diagnose rocuronium allergy, provided an assay-specific decision threshold is applied. IgE assays based on compounds bearing ammonium epitopes are confirmed to represent reliable tools to diagnose rocuronium allergy. High total IgE titers were observed to affect specificity of the assays.

Haemodialysis‐associated anaphylactic and anaphylactoid reactions

Anaphylactic and anaphylactoid reactions related to haemodialysis have been increasingly described for almost 3 decades. The majority of these cases used to occur with ethylene oxide sterilized, and complement‐activating cellulose membranes. However, a considerable number of publications have focused on polyacrylonitrile AN69® high flux membranes, angiotensin converting enzyme inhibitors and iron as other important causes of potentially severe haemodialysis‐related anaphylactoid reactions. Clinical manifestations vary considerably and generally do not allow differentiation between IgE‐mediated anaphylaxis and anaphylactoid reactions (e.g. from nonspecific mediator release). Successful management of these patients requires multidisciplinary approach and involves prompt recognition and treatment by the attending physician, and identification of the offending agent(s) with subsequent avoidance of the incriminated compound(s). This review focuses on some major causes of anaphylactoid and anaphylactic reactions during haemodialysis. Special consideration is given to the therapeutic and diagnostic approach.