In vitro assessment of hypersensitivity to allergen before and after allergen immunotherapy with whole blood basophil histamine release assay

2018 ◽  
Author(s):  
Olga  Sidorovich
Keyword(s):  
Histamine Release ◽  
Whole Blood ◽  
Allergen Immunotherapy ◽  
Before And After ◽  
Blood Basophil ◽  
Release Assay ◽  
In Vitro Assessment

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

2017 ◽  
Vol 14 (2) ◽  
pp. 30-36
Author(s):  
A A Babakhin ◽  
V V Smirnov ◽  
I S Gushchin ◽  
N I Ilyina ◽  
M R Khaitov
Keyword(s):  
Histamine Release ◽  
Case History ◽  
Whole Blood ◽  
Birch Pollen ◽  
Specific Ige ◽  
In Vitro Test ◽  
Rp Hplc ◽  
Allergenic Extracts ◽  
Release Assay

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.

scholarly journals Inhibition of Stimulated Interleukin-2 Production in Whole Blood: A Practical Measure of Cyclosporine Effect

1999 ◽  
Vol 45 (9) ◽  
pp. 1477-1484 ◽  
Author(s):  
C Michael Stein ◽  
John J Murray ◽  
Alastair JJ Wood
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Major Effect ◽  
Biologically Relevant ◽  
Maximum Inhibition ◽  
Before And After ◽  
Dose Dependent Fashion ◽  
The Relationship

Abstract Background: Prediction of cyclosporine (CSA) efficacy and toxicity in individual patients is difficult. There is no practical, biologically relevant, pharmacodynamic measure of CSA effect. A major effect of CSA is to decrease interleukin-2 (IL-2) production; however, measurement of this effect in isolated lymphocytes as a marker of response to CSA has been problematic. Methods: CSA inhibition of phytohemagglutinin-P (PHA)-stimulated IL-2 production, measured by ELISA, was studied ex vivo in whole blood drawn before, and after subjects received 4 mg/kg oral CSA. Results: Four hours after CSA was administered, the mean (± SD) CSA concentration was 702 ± 196 μg/L and PHA-stimulated IL-2 production decreased by 68.7% ± 17.2% (P <0.0001; n = 17). Twenty-four hours after CSA was administered, concentrations were low (64 ± 24 μg/L), with no inhibition of IL-2 production. A rapid, concentration-dependent response occurred. Maximum CSA concentrations (944 ± 187 μg/L) and maximum inhibition of IL-2 production (86.9% ± 13.7%) occurred 90 min after subjects received CSA. In vitro, 32.5–1200 μg/L CSA also inhibited PHA-stimulated IL-2 production in whole blood in a dose-dependent fashion with a similar IC50 (∼300–400 μg/L) ex vivo and in vitro. Conclusion: In the search for a pharmacodynamic marker to better guide immunosuppressive therapy, the relationship between this simple, biologically relevant measure of CSA effect and clinical outcome should be determined.

Histamine Release Factor from Dermanyssus gallinae (De Geer): Characterization and in vitro assessment as a protective antigen

2009 ◽  
Vol 39 (4) ◽  
pp. 447-456 ◽  
Author(s):  
Kathryn Bartley ◽  
Alasdair J. Nisbet ◽  
Jill E. Offer ◽  
Nicholas H.C. Sparks ◽  
Harry W. Wright ◽  
...  
Keyword(s):  
Histamine Release ◽  
Protective Antigen ◽  
Dermanyssus Gallinae ◽  
Release Factor ◽  
In Vitro Assessment

scholarly journals Hot-Stage Microscopy for Determination of API Particles in a Formulated Tablet

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Michal Šimek ◽  
Veronika Grünwaldová ◽  
Bohumil Kratochvíl
Keyword(s):  
Active Pharmaceutical Ingredient ◽  
Assessment Method ◽  
Hot Stage Microscopy ◽  
Tablet Disintegration ◽  
Before And After ◽  
In Vitro Assessment ◽  
Active Substances ◽  
Good Agreement

Although methods exist to readily determine the particle size distribution (PSD) of an active pharmaceutical ingredient (API) before its formulation into a final product, the primary challenge is to develop a method to determine the PSD of APIs in a finished tablet. To address the limitations of existing PSD methods, we used hot-stage microscopy to observe tablet disintegration during temperature change and, thus, reveal the API particles in a tablet. Both mechanical and liquid disintegration were evaluated after we had identified optimum milling time for mechanical disintegration and optimum volume of water for liquid disintegration. In each case, hot-stage micrographs, taken before and after the API melting point, were compared with image analysis software to obtain the PSDs. Then, the PSDs of the APIs from the disintegrated tablets were compared with the PSDs of raw APIs. Good agreement was obtained, thereby confirming the robustness of our methodology. The availability of such a method equips pharmaceutical scientists with an in vitro assessment method that will more reliably determine the PSD of active substances in finished tablets.

scholarly journals In Vitro Antiallergic Activity of Flavonoids in Histamine Release Assay Using Rat Basophilic Leukemia (RBL-2H3) Cells

1994 ◽  
Vol 35 (5) ◽  
pp. 497-503_1 ◽  
Author(s):  
Masaru KAWASAKI ◽  
Masatake TOYODA ◽  
Reiko TESHIMA ◽  
Jun-ichi SAWADA ◽  
Toshimitsu HAYASHI ◽  
...  
Keyword(s):  
Histamine Release ◽  
Antiallergic Activity ◽  
Release Assay ◽  
Basophilic Leukemia

In vitro influence of theophylline on anti-IgE-induced histamine release from leucocytes and whole blood in asthmatic children

1981 ◽  
Vol 11 (1-2) ◽  
pp. 109-110 ◽  
Author(s):  
P. Scheinmann ◽  
C. Burtin ◽  
J. Paupe ◽  
B. Lebel ◽  
J. Fermanian
Keyword(s):  
Histamine Release ◽  
Whole Blood ◽  
Asthmatic Children

scholarly journals In vitro assessment of interaction between lidocaine hydrochloride and sodium hypochlorite on root canal dentin before and after chemomechanical instrumentation procedures

2019 ◽  
Vol 22 (3) ◽  
pp. 255
Author(s):  
Balasubramanian Saravanakarthikeyan ◽  
Shari Devarajan ◽  
Kolli Sankeerthana ◽  
Venkatappan Sujatha ◽  
Sekar Mahalaxmi
Keyword(s):  
Sodium Hypochlorite ◽  
Root Canal ◽  
Lidocaine Hydrochloride ◽  
Before And After ◽  
In Vitro Assessment ◽  
Root Canal Dentin

Human whole-blood granulocyte aggregation in vitro

1989 ◽  
Vol 76 (2) ◽  
pp. 183-187 ◽  
Author(s):  
T. C. Fisher ◽  
J. J. F. Belch ◽  
J. C. Barbenel ◽  
A. C. Fisher
Keyword(s):  
Whole Blood ◽  
Cell Activation ◽  
Single Cells ◽  
Cell Types ◽  
Separation Procedure ◽  
Disease States ◽  
Before And After ◽  
Pure Cell ◽  
Cell Sizing

1. Aggregation assays are a commonly used technique for the study of granulocyte activation. These studies are usually performed using a pure cell suspension in buffer. This necessitates a separation procedure which is time-consuming and may modify the function of the cells. Interaction between different cell types is precluded. 2. To avoid these disadvantages a method was developed which quantifies granulocyte aggregation in whole blood. Samples drawn from an incubated vessel before and after the addition of a chemotactic stimulus were fixed with formaldehyde to prevent disaggregation. Erythrocytes were then removed by chemical lysis and using an electronic cell-sizing device the number of single cells and aggregates could then be easily measured. 3. Results from a group of volunteers showed a rapid and reversible response to a chemotactic tripeptide, with a fall in single granulocyte count and the appearance of doublets and triplets. Lymphocytes were unaffected. Intra-assay reproducibility was better than ± 5%. 4. Using this assay, a significant elevation in aggregability was observed in blood from patients after acute myocardial infarction. 5. This novel technique, by avoiding the separation step, is faster, simpler and more physiological than previous methods, and as such is useful for both assays of drug action in vitro and the study of cell activation in disease states.

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