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Background: The use of flow-cytometric basophil activation to different allergens has been recommended in recent years. In this study, we analyzed the diagnostic reliability of the flow-cytometric allergen stimulation test (FAST) after latex-specific stimulation in vitro. The diagnostic reliability of the technique was assessed as well as its correlation with other in vitro diagnostic parameters. Methods: 43 patients allergic to latex with a positive history and skin test participated in the study. Thirty subjects (20 of them exposed to latex) with a negative history, skin tests and serum-specific IgE determination to latex were used as controls. In FAST the percentage of basophils that express CD63 as an activation marker after in vitro stimulation with allergen (latex) is determined by flow cytometry, following double labelling with the monoclonal antibodies anti-CD63-PE and anti-IgE FITC. Results: Intraclass correlation coefficient in FAST with latex was 0.995 (p < 0.0001), which demonstrates the excellent reproducibility of this technique. Taking a cutoff point of 10% by means of ROC curves, FAST yields a sensitivity of 93% and a specificity of 100%. The FAST positive predictive value in latex allergy was 100% and the negative predictive value was 99.9%. We found a positive and significant correlation between FAST and specific IgE (CAP) with the histamine release test and specific sulphidoleukotriene production [cellular allergen stimulation test (CAST); p < 0.05]. Conclusions: FAST is a highly reliable technique (93% sensitivity and 100% specificity) in the in vitro diagnosis of IgE-mediated latex allergy.

Eosinophil granular proteins are a useful eosinophilic activation marker in asthmatic patients. In this study, the eosinophil peroxidase (EPO) levels were assessed in different stages of bronchial asthma, in 123 patients suffering from asthma, classified as mild (n =49), moderate (n = 49), and severe (n = 25), according to the International Consensus Report of Diagnosis and Treatment of Asthma, as well as in 27 healthy controls, with the aim of evaluating the importance of this protein as a severity marker in bronchial asthma, and its possible correlation with parameters such as anamnesis, respiratory function tests, and peripheral blood eosinophil count, and also with some allergologic diagnostic tests, both in vivo and in vitro. The geometric mean serum level of EPO was 9.3 +/- 11.3 ng/ml (median +/- SD) in controls, 28 +/- 37.8 ng/ml in the asthmatic patients. Depending on the asthma severity, the EPO levels were 25 +/- 30.5; 29 +/- 37.1, and 41 +/- 47.3 ng/ml in mild, moderate, and severe asthmatics, respectively, being the significant differences between the group of patients with mild and severe asthma (P < 0.001). The number of eosinophils (eos) in peripheral blood was 157 +/- 20 eos/mm3 in the controls, 334/35 eos/mm3 in mild asthmatics, 510 +/- 87 eos/mm3 in moderate asthmatics, and 658 +/- 72 eos/mm3 in severe asthmatics, with significant differences between all groups (from P < 0.05 to P < 0.001). Both the serum levels of EPO and the number of eosinophils were greater in patients with active asthma patients with inactive asthma (P < 0.001). Significant negative correlations (P < 0.001) were found between serum levels of EPO and FEV1 (rs = 0.30), MEF25-75 (rs = -0.33) and MEF50 (rs = -0.34), and a good positive correlation (rs = 0.80, P < 0.001) was found between EPO levels and the number of eosinophils in peripheral blood. We also found a significant positive correlation between eosinophil number and clinical score (rs = 0.54, P < 0.001) and between EPO levels and the mentioned score (rs = 0.46, P < 0.001).

Background In this study, we determined by flow cytometry the percentage of basophils activated after in vitro stimulation by allergens and expressing the CD63 marker. The diagnostic reliability of the technique was assessed as well as its correlation with other in vitro diagnostic parameters. Methods Fifty-three patients suffering from asthma and/or allergic rhinitis following sensitization to Dermatophagoides pteronyssinus and 51 patients sensitized to Lolium perenne were investigated. Twenty-four atopic patients not sensitive to these allergens and 38 healthy subjects were also selected as controls. The basophil activation test determines the percentage of basophils which express CD63 as an activation marker, by means of flow cytometry, after in vitro stimulation with allergen, using double labelling with monoclonal antibody anti-CD63-PE and anti-IgE-FITC. Results No differences in basal values (non-activated control) were found between sensitized patients, atopic controls and healthy controls. On the other hand, sensitized patients showed a significantly higher percentage of activated basophils after stimulation by allergens in vitro than both control groups (P < 0.001). We found a significant correlation between skin tests and basophil activation tests (r = 0.72, P < 0.001). We also found a positive and significant correlation between basophil activation tests and histamine release tests (r = 0.80, P < 0.001), allergen-specific sulphidoleukotriene production (r = 0.7, P < 0.001) and the occurrence of serum allergen-specific IgE (r = 0.71, P < 0.001). Conclusion The basophil activation test is a highly reliable technique in the diagnosis of allergy to inhalant allergens. The sensitivity of the basophil activation test was 93.3%, and its specificity 98.4%, when using a cut-off point of 15% activated basophils as positive result.

Background Allergic reactions to lettuce (Lactuca sativa) are not too frequent and few cases of systemic adverse reactions after its ingestion have been described. Objective We report a case of clinical sensitization to lettuce on a patient who presented mucocutaneous manifestations after its ingestion, with positive skin tests, histamine release test and serum specific-IgE to lettuce. The allergens responsible for this sensitization were also characterized by means of SDS-PAGE immunoblotting. Materials and methods We performed skin tests, histamine release test, serum specific IgE determination and CAP inhibition with lettuce and mugwort (Artemisia vulgaris) extracts. An aqueous and enriched lettuce (from loose leaf type) extract was subjected to SDS-PAGE immunoblotting for determination of its IgE-binding components. Results and Conclusions CAP inhibition showed antigenic community between lettuce and mugwort. Four protein bands from the lettuce extracts with molecular weights of 50, 43, 39 and 16 kDa exhibited IgE-binding properties.

In recent years, eosinophil cationic protein (ECP) has been considered as a useful eosinophilic activation marker in asthmatic patients. In this study, serum ECP levels in different stages of bronchial asthma were evaluated. We studied 123 patients suffering from asthma, which was classified as mild (n = 49), moderate (n = 49), severe asthma (n = 25), and also 31 healthy controls. Serum ECP levels were 13.22 +/- 1.11 ng/mL (mean +/- s.e.m.) in controls, and 30.15 +/- 2.38 ng/mL in asthmatic patients. By subgroups, ECP levels were 24.23 +/- 3.37 ng/mL, 31.69 +/- 4.21 ng/mL and 37.61 +/- 4.52 ng/mL, in mild, moderate and severe asthmatic patients, respectively, being the differences among the three groups statistically significant (P < 0.01-P < 0.001). Peripheral blood eosinophil numbers were 157 +/- 20 eos/mm3 in controls, and 334 +/- 35 eos/mm3, 510 +/- 87 eos/mm3, and 658 +/- 72 eos/mm3, in mild, moderate, and severe asthmatic patients, respectively, with significant differences among all groups (P < 0.05-P < 0.001). The serum ECP levels as well as the eosinophil numbers were higher in symptomatic patients than in the asymptomatic ones (P < 0.01). Moderate negative correlations, although highly significant (P < or = 0.001), were found between serum ECP levels and forced vital capacity (FVC) (rs = -0.27), FEV1, MEF25-75 (rs = -031), and MEF50 (rs = -0.32). There was also a good positive correlation between ECP levels and peripheral blood eosinophil numbers (rs = 0.67, P < 0.001).

(3). The patient responded positively to prick testing with ceftriaxone (wheal 20 · 25 mm) at a concentration of 2 mg/ml, 10 min after application. Urticarial rash appeared on her arm, face and neck, which required immediate administration of an antihistamine. She also responded positively to skin testing with ampicillin at a concentration of 20 mg/ml (wheal 8 · 10 mm). Prick and intradermal histamine test produced wheals with maximum diameters of 5 and 10 mm respectively. Intradermal test with the above cephalosporins and semisyntetic penicillins were negative in 10 healthy control subjects, who had previously tolerated those drugs. All assays for specific IgE were negative. Challenges were positive only to cephalexin. Within 20 min of receiving the second dose of the drug, she began to experience facial flush with intensive itching in the throat and eyes, swelling of the lip, and urticarial rash on her trunk. She was immediately treated with anti-shock therapy. Clinical findings and positive skin tests with ceftriaxone and ampicillin, as well as positive challenges with cephalexin imply that the child developed type-I hypersensitivity. The observed cross-reactivity among drugs suggested presence of antigenic determinants unrelated to b-lactam ring but at the same time not completely side-chain specific. The patient reacted to cefalexin and ampicillin but not to cefaclor, the drugs with the identical side-chain. In fact, the difference between cephalexin and cefaclor is in only one functional group (methyl group instead of chlorine at R2 position of the thiazolidine ring). To our knowledge, this is the first case of immediate hypersensitivity to ceftriaxone and cross-reactivity with cefalexin and ampicillin demonstrated in children.

Background: We assessed the reliability of basophil activation test (FAST) and sulphidoleukotriene production (CAST) in the in vitro diagnosis of allergy to metamizol, evaluating its sensitivity and specificity. Methods: Twenty-six patients allergic to metamizol and 30 control individuals were studied. Skin tests with metamizol, FAST, and CAST were performed. Results: FAST sensitivity was 42.3% and specificity 100%. The PPV of FAST is 100% and the NPV 99.4%. The likelihood ratio for a positive value cannot be calculated because the specificity is 100% and the likelihood ratio for a negative value is 0.58. CAST sensitivity was 52%, and specificity 90%. The PPV of the test is 5% and the NPV 99.5%. The likelihood ratio for a positive result was 5.2 and that for a negative result 0.53. FAST detects a larger number of cases when patients are studied within the first 6 months after the clinical reaction (χ = 4.2, P = 0.04) than later. Together with skin tests, FAST allowed detection of 69.2% patients allergic to metamizol, the same as CAST 76%. The joint use of the three techniques allowed identification of 76.9% of cases. Conclusions: FAST and CAST are useful for the diagnosis of allergy to pyrazolones. Its usefulness clearly increases when recent reactions are studied.

Subjective complaints and reactions after placebo administration during food challenges (FC) may make their outcome difficult to interpret. We determined serum ECP and tryptase as tryptase in saliva during FC, looking for markers to support challenge outcomes.Twelve patients with systemic reactions after food intake and nine presenting oral allergy syndrome (OAS) underwent skin tests; total and specific IgE determination; double-blind, placebo-controlled FC (DBPCFC); and open challenges. Blood samples were collected before and 1, 2, and 5 h after challenge and saliva before and 5, 30, and 60 min after challenge. ECP and tryptase were quantified by ImmunoCAP (Pharmacia-Upjohn, Sweden). Serum tryptase of10 microg/l was considered positive.After positive DBPCFC (n = 8), ECP rose significantly (P0.05) at 1-h - 16.03 (12.8) microg/l (mean [standard deviation]) - and 2-h intervals - 17.56 (10.7) microg/l - compared to basal level of 9 (6.4) microg/l. After negative DBPCFC (n = 6), ECP increased from basal 9.63 (3.9) microg/l to 24.84 (14.17) microg/l at the 2-h time point. There were nonsignificant differences in ECP between patients with positive and negative FC. Two patients with positive challenge showed a tryptase level of10 microg/l and only one patient with OAS showed 5.6 microg/l of tryptase 5 min after FC.ECP and tryptase in serum and saliva were not useful markers for FC outcomes.

Introduccion. Los errores congenitos del metabolismo (ECM) se consideran dentro del grupo de las enfermedades raras por su baja incidencia y su forma de presentacion. Pueden manifestarse ya en el periodo neonatal. Los progresos en su conocimiento y el disponer de metodos bioquimicos mas precisos permiten diagnosticar muchos ECM que hace un tiempo pasaban desapercibidos. Pacientes y metodos. Hemos revisado los 52 pacientes con ECM de debut neonatal diagnosticados en los ultimos 25 anos en nuestro centro y hemos valorado su forma de presentacion, sus manifestaciones neurologicas, los examenes complementarios y su evolucion. Resultados. La edad media de inicio de los sintomas fue de 5 dias, y la de confirmacion del diagnostico, de 88 dias. Destacamos un primer grupo formado por 36 pacientes con defectos del metabolismo intermediario, en los que predominaba la hipotonia, el estancamiento ponderal y las convulsiones; un segundo grupo formado por ocho pacientes con defectos del metabolismo energetico, que manifestaban trastornos del ritmo respiratorio e hipotonia, y, finalmente, 8 pacientes dentro del subgrupo de los trastornos de las moleculas complejas, en los que destacaban la hipotonia y las cataratas como sintomas mas frecuentes de debut. Los sintomas neurologicos mas comunes en este periodo fueron la hipotonia (60%), la afectacion del sensorio (35%) y las convulsiones rebeldes (23%). Los examenes complementarios de laboratorio en las primeras fases de la enfermedad orientaron hacia un ECM especialmente en los trastornos del metabolismo intermediario y energetico. Por su parte, el registro electroencefalografico y el estudio del LCR fueron importantes para el diagnostico en muchos trastornos del metabolismo intermediario. La neuroimagen inicial fue menos orientativa, pero permitio descartar otras patologias. Mas de la mitad de los pacientes con ECM de debut neonatal habian fallecido ante de los 12 meses de vida. Conclusion. Es importante sospechar estas enfermedades en el periodo neonatal para mejorar la precocidad diagnostica y terapeutica y reducir la morbimortalidad.