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.

Haemostatic Platelet Plug Formation in the Isolated Rabbit Mesenteric Preparation - An Analysis of Red Blood Cell Participation

1980 ◽  
Vol 44 (01) ◽  
pp. 006-008 ◽  
Author(s):  
D Bergqvist ◽  
K-E Arfors
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
In Vitro Test ◽  
Pharmacological Agents ◽  
Vitro Test ◽  
Releasing Effect ◽  
Plug Formation

SummaryIn a model using an isolated rabbit mesenteric preparation microvessels were transected and the time until haemostatic plugs formed was registered. Perfusion of platelet rich plasma gave no haemostasis whereas whole blood did. Addition of chlorpromazine or adenosine to the whole blood significantly prolonged the time for haemostasis, and addition of ADP to the platelet rich plasma significantly shortened it. It is concluded that red cells are necessary for a normal haemostasis in this model, probably by a combination of a haemodynamic and ADP releasing effect.The fundamental role of platelets in haemostatic plug formation is unquestionable but there are still problems concerning the stimulus for this process to start. Three platelet aggregating substances have been discussed – thrombin, adenosine diphosphate (ADP) and collagen. Evidence speaking in favour of thrombin is, however, very minimal, and the discussion has to be focused on collagen and ADP. In an in vitro system using polyethylene tubings we have shown that "haemostasis" can be obtained without the presence of collagen but against these results can be argued that it is only another in vitro test for platelet aggregation (1).To be able to induce haemostasis in this model, however, the presence of red blood cells is necessary. To further study this problem we have developed a model where haemostatic plug formation can be studied in the isolated rabbit mesentery and we have briefly reported on this (2).Thus, it is possible to perfuse the vessels with whole blood as well as with platelet rich plasma (PRP) and different pharmacological agents of importance.

IN VITRO DEMONSTRATION OF "HEPARIN REBOUND"

1987 ◽  
Author(s):  
C Taylor ◽  
R F Baugh
Keyword(s):  
Whole Blood ◽  
Cardiovascular Surgery ◽  
Anticoagulant Activity ◽  
Blood Levels ◽  
In Vitro Test ◽  
Neutralizing Activity ◽  
Vitro Test ◽  
The Stability

"Heparin rebound", the in vivo appearance of measurable heparin anticoagulant activity following theapparent neutralization of heparin by protamine, hasbeen a problem sporadically associated with the use of heparin in cardiovascular surgery. A number of mechanisms have been proposed to explain rebound, and to some extent each may contribute to the phenomena. As yet no reliable, predictable method has been demonstrated for measuring, reproducing or quantifying "heparin rebound".We have demonstrated and measured the appearance of heparin anticoagulant activity following neutralization with protamine in citrated whole blood. The reappearance of heparin anticoagulant activity was associated with a rapid loss of protamine. The loss of protamine followed 1st order enzyme kinetics, and was indicative of the action of an enzyme. The anticoagulant activity which eappeared could be titrated againwith protamine. The loss of protamine neutralizing activity, in whole blood, could be followed by titration with heparin using a recalcified activated clotting time. The rate of loss varied with both individual blood donors and with the type and source of protamine. The rate of loss of protamine was great enough to influence in. vivo heparin/protamine neutralization ratios, i.e. at 4 units of heparin/ml, 1 unit/ml anticoagulant activity was routinely recovered within 30 minutes following initial neutralization. The indications for cardiovascular surgery are:1)the in vivo neutralization ratio should be adjusted to account for loss of protamine activity, 2) the higherthe blood levels of heparin used during surgery, themore significant the potential for heparin rebound, and 3) protamines may be evaluated in an in vitro test which measures the stability of protamine neutralizing activity in whole blood.

In vitro testing for allergic and immunologic diseases

2019 ◽  
Vol 40 (6) ◽  
pp. 480-484
Author(s):  
Elisa N. Ochfeld ◽  
Melanie Makhija
Keyword(s):  
Immune Cell ◽  
Allergic Diseases ◽  
Basophil Activation Test ◽  
Specific Ige ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Ige Mediated ◽  
Lymphocyte Immunophenotyping ◽  
Immunologic Diseases

In vitro tests are used to assist in the diagnosis of both allergic and immunologic diseases. Unfortunately, there is no single test that is pathognomonic for most allergic diseases. The most commonly ordered in vitro test by allergists is allergen specific IgE (sIgE), which is used to help diagnose IgE mediated hypersensitivity to foods, aeroallergens and venoms. Multiple assays exist, although none of these assays have been adopted as the industry standard. Epicutaneous skin test is also a fundamental test in the diagnosis of IgE mediated hypersensitivity. In addition, total IgE, basophil activation test (BAT), and serum tryptase may also be useful in elucidating allergic diseases. Immunologists rely on laboratory testing to diagnose primary immunodeficiency diseases. These tests include serum quantitative immunoglobulins, lymphocyte immunophenotyping by flow cytometry and immune cell functional testing. Furthermore, genetic testing is invaluable in the diagnosis of many primary Immunodeficiencies.

Development Of An In Vitro Test For Triatoma Specific IgE

2010 ◽  
Vol 125 (2) ◽  
pp. AB202
Author(s):  
B.J. Goldberg ◽  
D. Lo ◽  
C. Weirauch ◽  
E. Macy ◽  
C. Lin
Keyword(s):  
Specific Ige ◽  
In Vitro Test ◽  
Vitro Test

scholarly journals Interleukin-4 (IL-4) enhances and soluble interleukin-4 receptor (sIL-4R) inhibits histamine release from peripheral blood basophils and mast cellsin vitroandin vivo

1997 ◽  
Vol 6 (2) ◽  
pp. 111-118 ◽  
Author(s):  
B. Niggemann ◽  
T. Zuberbier ◽  
U. Herz ◽  
K. Enssle ◽  
U. Wahn ◽  
...  
Keyword(s):  
Mast Cells ◽  
Histamine Release ◽  
Peripheral Blood ◽  
Specific Ige ◽  
Interleukin 4 ◽  
Effector Cells ◽  
Antibody Titres ◽  
Interleukin 4 Receptor

The aim of the study was to analyse the effect of interleukin-4 (IL-4) on allergen and anti-IgE mediated histamine release from basophils and human skin mast cells and to assess whether soluble recombinant interleukin-4 receptor (sIL4R) can inhibit these effects. Anti-IgE stimulated histamine release from peripheral blood basophils and mast cells of atopic donors was enhanced after preincubation with IL-4, whereas after preincubation with sIL-4R it was inhibited. These effects were even more pronounced when samples were stimulated with a clinically relevant allergen. In IL-4 preincubated skin mast cells, there was a similar enhancement of anti-IgE stimulated histamine release, which could again be inhibited by sIL-4R. The effects of IL-4 and sIL4R were dose- and time-dependent. Mice sensitized to ovalbumin and treated with soluble recombinant murine sIL-4R showed significantly reduced immediate-type cutaneous hypersensitivity responses compared with untreated mice. Thesein vivoeffects were IgE independent, since there were no significant differences in total and allergen specific IgE/IgG1 antibody titres between treated and untreated mice. This indicates that IL4 exerts priming effects on histamine release by effector cells of the allergic response and that these effects are potently antagonized by soluble IL-4R bothin vitroandin vivo.

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

scholarly journals An in vitro Test for Detection of Specific IgE-mediated Penicillin Reactions in Children

1970 ◽  
Vol 4 (5) ◽  
pp. 436-436
Author(s):  
Zack H Haddad ◽  
Joel Korotzer ◽  
John James
Keyword(s):  
Specific Ige ◽  
In Vitro Test ◽  
Vitro Test ◽  
Ige Mediated

scholarly journals Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

2002 ◽  
Vol 9 (3) ◽  
pp. 588-597 ◽  
Author(s):  
Yukari Nakagawa ◽  
Hideko Maeda ◽  
Toshimi Murai
Keyword(s):  
Blood Culture ◽  
Whole Blood ◽  
Proinflammatory Cytokine ◽  
Ex Vivo ◽  
Test System ◽  
In Vitro Test ◽  
Cytokine Release ◽  
Human Whole Blood ◽  
Culture Test

ABSTRACT The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I ·  C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I · C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.

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