P. Stahl Skov

Sensitive glass microfibre-based histamine analysis for allergy testing in washed blood cells. Results compared with conventional leukocyte histamine release assay

The new microfibre method for allergy testing is based on basophil histamine release after challenge with suspected allergens in samples of 50 μl washed blood cells. Released histamine is bound to microfibres and measured after removal of interfering substances by washing. The microfibre method was compared with the conventional leukocyte histamine release assay in 18 allergic patients tested with 10 different allergens. It was found that the same individuals responded with histamine release to the same allergens in both assays, and the number of responders was almost identical. Also the dose‐response curves and the cell sensitivity were almost identical, which further substantiated identity between the results obtained by the new microfibre method and the conventional assay. A comparison between the microfibre method and in vivo provocation tests showed good agreement when comparing the number of positive and negative responses in these test. The new method overcomes the problems in allergy testing, where only small amounts of blood are available and many tests have to be carried out.

A new method for detecting histamine release

Glass microfibres have been found to bind histamine with high affinity and selectivity. A new test for measuring basophil hisamine release has been developed using the glass microfibres as a solid phase. Glass microfibres are crushed and fixed to the bottom of microtitre plates with a water-soluble glue. Histamine release is performed in the glass microfibre-prepared microtitre plates by challenging 100 μl washed blood with 20 μl antigen per well for 90 min at 37°C. Released histamine is bound with high affinity to the glass microfibres, since 90% of histamine in the solution is adsorbed to the fibres. After incubation the microtitre plate is washed with H2O to remove cells and interfering substances. Fibre-bound histamine is detected by the fluorometrico-phthaldialdehyde method. The sensitivity of the assay is 0.63 ng histamine, 2 HCl and the histamine standard curve is linear up to at least 5 ng histamine, 2 HCl. Optimal conditions for the new assay are described. After challenge with anti-Ige a comparison with the conventional histamine release from Ficoll-Hypaque-isolated leukocytes showed almost identical results.

Histamine is released in the wheal but not the flare following challenge of human skin in vivo: a microdialysis study

Background The mediator mechanisms of the cutaneous wheal and flare response, which underlies allergic skin and urticarial conditions, are controversial. The wheal results primarily from a direct effect of histamine on the local vascular bed, but to what extent does histamine diffuse within the wheal? The flare is neurogenic in origin, being disseminated within the dermis by axon reflexes, but do the neuropeptides released from the nerve endings cause the vasodilatation directly or do they induce the further release of histamine which then transduces the fiare?

A SIMPLIFIED METHOD FOR MEASURING BASOPHIL HISTAMINE RELEASE AND BLOCKING ANTIBODIES IN HAY FEVER PATIENTS. BASOPHIL HISTAMINE CONTENT AND CELL PRESERVATION

A simplified method for measuring basophil histamine release in grass pollen hay fever patients has been developed. Leukocytes were challenged in vitro with extracts of Phleum pratense (timothy) and the release of histamine was determined indirectly as the residual histamine in the cell sediment. Several steps to purify histamine thus became superfluous and histamine was directly conjugated with o-phthaldialdehyde to form a fluorophore. The simplified method showed a basophil histamine content which was in accordance with results obtained by more specific methods. No difference in basophil histamine content was found between normal and allergic persons. For the histamine liberation assay blood could be adequately preserved for transport for 48 h at room temperature by adding cell culture medium. Basophil histamine release technique allows evaluation of cell sensitivity for determination of the degree of allergy as well as the level of blocking antibodies.

High dose grass pollen tablets used for hyposensitization in hay fever patients. A one-year double blind placebo-controlled study.

Previous, plaeebo‐controlled clinical trials with oral hyposensitization in grass pollinosis have been disappointing. Since the results possibly could be explained by too low doses of ingested allergens, the present study was initiated to evaluate the effect of high doses of allergens. Forty‐two adults with symptoms in the grass pollen season and with grass pollen sensitivity demonstrated by skin prick test and conjunctival provocation test were included. Enterosoluble tablets were administered daily for 1 year. Twenty‐two patients, who completed the study, received placebo and 17 mixed grass pollen allergens from rye grass, timothy grass, cultivated rye and velvet grass. Evaluated either by self‐assessment or by symptom and medicine score before and after treatment, the group receiving pollens did not improve clinically compared the controls. During the study, conjunctival sensitivity decreased equally in the two groups, and changes in specific IgE, allergen‐induced histamine release from blood cells and skin prick test were insignificant and with no difference between groups. Five patients, who received pollen allergens, had episodes of urticaria or angioedema, and a furter three patients on the same treatment had slight gastrointestinal side effects. In conclusion, enterosoluble grass pollen allergens in contrast to birch pollens did not have any therapeutic effect, even in doses more than 4,000 times higher than those used for subcutaneous hypersensitization. The reason may be degradation of allergens before the immune system is reached.

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays

Background Peanuts are often consumed after roasting, a process that alters the three‐dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity.

Quantitative and Qualitative Estimations of IgE Bound to Basophil Leukocytes from Hay Fever Patients

IgE was removed from human basophils of 4 nonatopic persons and 10 hay fever patients allergic to timothy grass pollen by treating the cells with buffer to adjusted pH 4. IgE could be removed and refixed to the same cells. Refixation was demonstrated by immunofluorescence and by the ability of basophils to release histamine on exposure to timothy pollen. Removed total IgE and specific IgE directed against timothy pollen were estimated, and a linear correlation to the level of total IgE and specific IgE in serum was found. The total number of IgE molecules per basophil was calculated to be in the range of 30,000 to 300,000, and timothy‐specific IgE constituted 4%‐15% of the total IgE molecules on the cells. It was furthermore established that specific cell‐bound IgE was linearly correlated to the pollen concentration releasing 20% of the histamine contents of the basophils. Separated IgE from sensitized and nonsensitized basophils could be bound to basophils from other patients, resulting in a change in cell sensitivity. This could be ascribed to additional binding to free cell receptors as well as to a partial replacement of bound IgE. Basophils from nonatopic persons could not be sensitized by incubation with surface IgE from atopic persons. The results indicate that acid treatment is a simple method suitable for removing IgE from basophils. This IgE is intact and can be quantitated.

Double‐blind, placebo‐controlled food challenge with apple

The aim of the study was to develop and evaluate different methods of double‐blind, placebo‐controlled food challenge (DBPCFC) with apple. Three different DBPCFC models were evaluated: fresh apple juice, freshly grated apple, and freeze‐dried apple powder. All challenges were performed outside the pollen season and took place from 1997 to 1999. The freeze‐dried apple material was characterized by means of leukocyte histamine release (HR), skin prick test (SPT), and immunoblotting experiments. The study population consisted of birch pollen‐allergic patients with a history of rhinitis in the birch‐pollen season and positive specific IgE to birch. For comparison of the DBPCFC models, 65 patients with a positive open oral challenge with apple were selected. In the characterization of the freeze‐dried apple material, 46 birch pollen‐allergic patients were included. The IgE reactivity to apple was evaluated by measurement of specific IgE, HR, and SPT. Golden Delicious apples were used in all experiments. The results of this study showed that it was possible to perform DBPCFC with apple in birch pollen‐allergic individuals. The model with freshly squeezed apple juice had a low sensitivity and displayed a high frequency of reactions to placebo, probably due to the ingredients used for blinding. The sensitivity of the models with freshly grated apple and freeze‐dried apple powder was 0.74/0.60. An increase in sensitivity is desirable. The freeze‐dried apple powder proved to be useful for SPT, HR, and oral challenges, but further investigation of the stability and the allergenic profile of the material is needed.

Diagnostic value of scratch-chamber test, skin prick test, histamine release and specific IgE in birch-allergic patients with oral allergy syndrome to apple

Background: The aim of the study was to examine the diagnostic value of skin prick test (SPT), scratch‐chamber test (SCT), histamine release (HR) and specific immunoglobulin E (IgE) in birch‐allergic patients with oral allergy syndrome to apple.

A new glass microfibre-based histamine analysis for allergy testing in children. Results compared with conventional leukocyte histamine release assay, skin prick test, bronchial provocation test and RAST

A new microfibre method for allergy testing measuring histamine release from human basophil leukocytes is described. Samples of 50 μl washed blood are challenged with the suspected allergens. Released histamine is bound to microfibres and measured by a spectrofluorometrical method after removal of interfering substances by washing. The microfibre method (HR‐MM) was compared to the conventional histamine release assay using the Ficoll‐Hypaque gradient method (HR‐FH) in 19 allergic children tested with one of three allergens. In addition, a comparison was made between the microfibre method and in vivo provocation tests, i.e. skin prick test (SPT), bronchial provocation test (BPT) and allergen specific serum IgE (RAST). It was found that the same individuals responded with histamine release to the same allergens in both histamine release assays, and the dose‐response curves were almost identical. A positive correlation was found between the in vivo and in vitro tests. Thus it is concluded that the new method can provide reproducible, analytically precise (at the nanogram level) histamine release results in pediatric cases where: 1) a positive SPT does not correlate with case history; 2) BPT may be considered too hazardous or inconvenient; 3) confirmation of negative or inconclusive SPT or RAST is needed. In contrast to other histamine release assays it is a convenient diagnostic tool in children since only small amounts of blood are needed and at least 96 tests can be carried out in 21/2 h