Rapid in vitro assessment of hypersensitivity with whole blood leukocyte histamine release assay

Abstract

Background. Clinical application of leukocyte histamine release assay (HRA) can be enhanced through the development of automated reversed-phase high-performance liquid chromatography with mass spectrometry (RP-HPLC-MS/MS) employing of whole blood (WB) samples. The purpose of this study was to evaluate the possibility to use WB-HRA technology for the in vitro diagnosis of hypersensitivity employing RP-HPLC-MS/MS. Methods. Method principle: heparinized whole blood samples after substitution of plasma with PIPES buffer («reconstituted» blood) are incubated at 37 °C with different concentrations of substances (allergens, drugs, chemicals, food etc.) suspected in relation to hypersensitivity reactions. Release of histamine is occured mainly from basophilic granulocytes depending on their sensitivity to stimulating substances (allergens etc.). The released histamine is subsequently direct determined in the supernatant using RP-HPLC-MS/MS technology. Heparinized whole blood (8 ml) was drawn from patients with sensitivity to D. pteronyssinus (D1), birch pollen (T3) and peach (F95) confirmed by case history and results of skin prick tests or detection of specific IgE. After removing plasma and substitute it with PIPES buffer aliquots of «reconstituted» blood were put into separate tubes (300-450 μl for macro-method) or to wells of U-shape 96-well micro-titer plate (150-200 μl for micro-method) already contained different concentrations (dilutions) of histamine standard, anti-IgE and allergenic extracts D1, T3, F95 including their chemically modified forms sD1 and sF95. After 1 h incubation at 37 °C tubes or plates were centrifuged and supernatants from each tube (macro-method) or well of the plate (micro-method) were directly analyzed for histamine content by RP-HPLC-MS/MS. Results. It is shown that the levels of histamine released from leukocytes of whole blood of patient sensitized to D. pteronyssinus upon stimulation with non-modified D1 extract are much higher than that of upon stimulation with modified sD1 that means that chemical modification of allergen leads to suppress of B-cell epitopes. It seems that this method is suitable for evaluation of hypersensitivity to allergen, as well as for detection of allergenicity of modified allergens (allergoids). We also found a significant reduction (in 60%) of histamine release from blood leukocytes upon stimulation with T3 extract in patient with sensitivity to birch pollen after allergen-specific immunotherapy (ASIT) in compare to the level of histamine before ASIT. These data indicate that our method of histamine release assay can be convenient as in vitro test for monitoring and evaluation of ASIT efficacy. It was also studied histamine release from blood basophils of patient with sensitivity to peach, confirmed by case history and detection in serum specific IgE. Incubation of patient's whole blood with peach extract (F95) resulted a histamine release at the level comparable to that of stimulated with anti-IgE. This high level of histamine is correlated with the level of allergen-specific serum IgE. Besides the incubation patient's blood with modified peach extract sF95 induced histamine release two times less than that of non-modified F95 indicating substantial reduction of sF95 allergenicity. So this methodology for detection of histamine released from leukocytes of whole blood may be successfully applied for diagnosis of food allergy employing of laboratory made allergenic extracts. Conclusion. Speed and simplicity of performance including the requirement of small quantities of blood makes the WB-HRA employing RP-HPLC-MS/MS a useful laboratory tool not only of scientific interest (detection of allergenicity of modified allergens, peptides etc.) but also of practical significance for evaluation the degree of sensitivity of patients to allergens (including those who received anti-allergic medication or ASIT), analysis of pathophysiological responses to drugs, chemicals and other compounds suspected for their adverse side effects.