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PubMedDate not supplied

Introduction: basophil histamine release and the diagnosis of food allergy.

The glass fiber-based automated chlorometric microtiter plate whole blood leukocyte histamine release test (LHRT) may provide an in vitro correlate to in vivo allergic responses. Depending upon the high affinity of histamine for glass fibers, the fluorometric assay for histamine has a high correlation with radioimmunoassay for native histamine. For the diagnosis of respiratory allergy, up to 90% concordance between basophil histamine release results and skin test results to common inhalant allergens has been noted, although up to 96% concordance of basophil histamine release results in bronchoprovocation tests may occur. In general, the concordance between LHRT results and either skin test results or allergen inhalation challenge results is greater than the concordance between the LHRT results and radioallergosorbent (RAST) results. The LHRT may be a valuable tool for the investigation of drug allergy and food sensitivity. The ability of the LHRT to detect both IgE-mediated and non-IgE-mediated basophil responses is a potential advantage over RAST testing in situations in which the presence of specific IgE directed toward a potential allergen has not as of yet been demonstrated. False-negative LHRT results may occur in 10 to 20% of subjects who have basophils that fail to respond to anti-IgE as a positive control. Advances in both sample and blood and constitution of sample media may minimize this percentage of nonresponding basophils. Differences between mast cell and basophil responses may occur due to functional heterogeneity between basophils and mast cell subtypes.

Adult↗

PubMedDate not supplied

[Induction of reaginic hypersensitivity to tartrazine in the rabbit immunization by ingestion of the covalent conjugate tartrazine-human serum albumin (author's transl)].

Sensitization to simple chemical substances acting as haptens is not well known in food allergy. For that purpose, a study was made in rabbit using tartrazine alone with adjuvant, or mixed with human serum albumin (HSA), or conjugated by covalent BOP bonds with HSA. Hypersensitivity was evaluated by rabbit basophil degranulation test which would offer more specificity through cell membrane IgE-antibody reactions, and thus, more accuracy than classical passive cutaneous anaphylaxis. The data showed that sensitization could not be induced by ingestion of tartrazine with adjuvant or mixed with a protein. But covalent conjugate T-HSA mixed with adjuvant sensitized 8 of the 9 rabbits studied. It seems that preceding irritation of intestinal mucous membrane by acetylsalicylic acid enables one to obtain a more long lasting sensitization.

Animals↗

PubMed1983

[Immediate hypersensitivity to lacrimal gland proteins in sicca syndromes].

A biological study was conducted on tears from 20 subjects with a clinical sicca syndrome. Electrophoresis of lacrymal proteins, determination of lacrymal and salivary immunoglobulins G, and a human basophil degranulation test (HBDT) with a lacrymal gland extract as antigen were performed in each patient. Three different groups could be distinguished: a group with normal electrophoresis and normal HBDT corresponding to hyposecretion; a second group with abnormal electrophoresis and negative HBDT corresponding to an inflammatory state of the lacrymal gland; and a third group with abnormal electrophoresis and positive HBDT corresponding to a deficit in synthesis function of the lacrymal gland. These findings demonstrate that HBDT with lacrymal gland extract as antigen is a reliable means of diagnosis of the sicca syndrome.

Adult↗

PubMed1982

Basophil sensitisation in idiopathic nephrotic syndrome.

The degranulation of basophils in the presence of a specific allergen (human basophil degranulation test, HBDT) is an index of IgE-dependent cellular response and it was tested in 46 unselected idiopathic nephrotic syndrome (INS) patients with or without atopic manifestations. 28 of the INS patients had "minimal change" nephrotic syndrome (MCNS) and 18 had focal segmental glomerular sclerosis (FSGS). 29 patients with different types of primary glomerulonephritis (GN) and 11 healthy donors acted as controls. The HBDT was positive in 16 (57%) of 28 MCNS and in 14 (77.8%) of 18 FSGS patients. Only 5 (17.2%) of 29 GN and 1 (6%) of 11 blood donors exhibited positive HBDT. House dust was the commonest allergen to which MCNS and FSGS subjects were sensitive, whereas sensitivity to grass pollen, moulds, and animal danders was less common. 8 MCNS and 8 FSGS subjects were sensitive to house dust plus another allergen. 4 MCNS and 1 FSGS patient had fewer than 5/microliters basophils; 4 of these 5 patients were having a relapse of their nephrotic syndrome at the time. The demonstration of enhanced IgE-basophil sensitisation in INS patients suggests than an anaphylactic process plays a part in the pathogenesis of INS.

Adolescent↗

PubMed1999

[Basophil activation test using flow cytometry in Hymenoptera venom allergy].

The basophil activation test (TAB) clearly shows evidence to Hymenoptera venoms. Comparison of the TAB value with the clinical history allows fixation of a threshold that will anticipate the type of clinical reaction by the TAB value. For the technique of TAB 3 dilutions are used. The basophil activation values produce a threshold for each dilution. For the first dilution the threshold is 50% activation: below this threshold, 70% of patients would have a local or systemic reaction and above this threshold, 70% of patients would have a systemic allergic reaction.

Adult↗

PubMed1996

[Importance of the leukotriene C4 liberation test for the diagnosis of drug allergy (preliminary results)].

Pharmacological or immunological activation of human basophil induces the expression of membrane markers such as CD63 and the release of histamine and leukotriene C4 (LTC4). LTC4 release test has the advantages of a cellular test which is obviously closer to the physio-pathological reaction than the direct determination of circulating antibodies. Moreover, the determination of a newly formed mediator eliminates the spontaneous release of mediator by simple cellular damage. We present here, after having controlled the reliability of the method for aero-allergens, preliminary results of LTC4 release obtained for haptens (ampicillin, methylprednisolone and acetylsalicylic acid) which are in favour of the use of this method in the complex field of drug allergy diagnosis.

Adult↗

PubMed2002

Flow cytometric basophil activation test: a review.

Flow cytometry is a technique enabling the analysis of physical and biological characteristics of cells or other biological particles when labeled with antibodies coupled to fluorochromes or other dyes. The basophil activation test (BAT), also called flow-cytometric allergen stimulation test (FAST) [commercially available under the name of Flow CAST (Bühlmann Laboratories) or BASOTEST (Beckton-Dickinson)] is based on the in vitro allergen-induced specific activation of basophils. This assay rests on the demonstration of some membrane protein markers that appear after exposure to the allergen. This paper reviews some of the literature about the use of this technique in the investigation of immediate-type allergies to inhalant allergens, drugs, and foods, as well as our own experience with this diagnostic technique. Flow cytometry is a reliable method for the quantification of basophil activation after allergenic stimulus in vitro and in vivo. It also enables us to detect allergic and pseudoallergic reactions because of other mechanisms than allergen-specific IgE antibodies. Further clinical evaluation of this technique will allow validation and better establishment of its diagnostic value in allergy.

Adult↗

PubMed2005

Functional assessment of pathogenic IgG subclasses in chronic autoimmune urticaria.

BACKGROUND: Chronic urticaria is caused by a complement fixing, IgG antibody directed to the alpha-subunit of the IgE receptor, which is present in 35% to 45% of patients. This autoimmune subgroup can be identified by an autologous skin test or histamine release from human basophils or cutaneous mast cells. However, binding assays do not correlate with these functional assays. We considered the possibility that pathogenic antibody may be present within particular IgG subclasses, which might facilitate development of a binding method that can reliably screen patients. OBJECTIVE: To determine the subclass distribution of IgG antireceptor antibodies on the basis of histamine release, and to assess the possibility that a subclass specific ELISA binding method could be used to screen patients. METHODS: We isolated patient IgG by protein G affinity chromatography and then isolated patient IgG subclasses 1, 2, 3, and 4 by a combination of antibody affinity chromatography and protein A affinity chromatography. The ability of each subclass to activate basophils was assayed by histamine release. RESULTS: Patient IgG subclasses IgG 1 , IgG 3 , and to a lesser degree IgG 4 have antibody capable of activating basophils to release histamine, whereas IgG 2 is inactive. Immunoblot or RAST assay that is subclass-specific does not correlate with histamine release as a result of nonfunctional but binding antibody within IgG subclasses 1, 3, or 4, and complement activation by IgG 1 and IgG 3 . CONCLUSION: Purification of IgG subclasses from patients with chronic urticaria demonstrates functional antibody in IgG 1 and IgG 3 and occasionally IgG 4 . Nonfunctional antibody within IgG 2 plus nonfunctional antibody mixed with functional antibody within IgG 1 , IgG 3 , and IgG 4 and effects of complement are responsible for a lack of correlation of histamine release with binding assays even if subclass-specific.

Autoimmune Diseases↗

PubMed2007

Flow-assisted quantification of in vitro activated basophils in the diagnosis of wasp venom allergy and follow-up of wasp venom immunotherapy.

BACKGROUND: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy (VIT). Despite the efficacy of VIT, issues as how to monitor treatment and when to discontinue maintenance therapy remain to be established. METHODS: To evaluate diagnostic performances of the basophil activation test (BAT) in wasp venom allergy, 80 patients with a definite history of wasp venom anaphylaxis (systemic reactors) and 14 wasp-stung asymptomatic controls (stung controls) were enrolled. Venom-induced basophil activation was analyzed flow cytometrically by double-labeling with anti-IgE and anti-CD63. Results were compared to wasp IgE levels and results of a venom skin test (VST). To establish whether the BAT constitutes a candidate marker to monitor VIT, the BAT was repeated in 22 patients on the 5th day of a build-up course and after 6 months of maintenance VIT. Whether the BAT could contribute in the decision of discontinuing VIT was assessed in a cross-sectional analysis in 30 patients receiving treatment for 3 years. RESULTS: Comparison between systemic reactors and stung controls revealed a sensitivity of 86.4% and specificity of 100% for venom IgE, and sensitivity of 81.8% for VST, respectively. In contrast to stung controls, patients demonstrated dose-dependent venom-induced basophil activation. The BAT attained a sensitivity of 83.8% and specificity of 100%. At the end of the build-up course, no effect of VIT on the BAT was demonstrable. When the BAT was repeated after 6 months of treatment, submaximal stimulation of the cells demonstrated a significant decreased CD63 expression (P < 0.04). Patients having VIT for 3 years also demonstrated significantly lower venom-induced CD63 expression (P < 0.001). After 3 years, 60% of the patients had a negative BAT for submaximal stimulation of the cells whereas only 17.9% of the patients had negativation of wasp IgE. CONCLUSIONS: The BAT is a reliable instrument for the diagnosis of wasp venom anaphylaxis and might constitute an instrument to monitor wasp VIT.

Adolescent↗

PubMed2004

The diagnostic interpretation of basophil activation test in immediate allergic reactions to betalactams.

BACKGROUND: Basophil activation by allergens, including drugs, has been used to determine sensitivity and to study IgE recognition and cross-reactivity. OBJECTIVE: We sought to determine the sensitivity and specificity of a basophil activation test (Basotest) in patients with immediate allergic reactions to betalactams, with a later comparison between patients who were selective (those recognizing the culprit drug excluding benzylpenicillin (BP)) and cross-reactors (those recognizing several penicillin determinants including BP). METHODS: Basotest to different haptens was performed in 70 patients with immediate allergic reactions to betalactams, classified into three groups: (A) skin test positive independently of CAP/RAST immunoassay value, (B) skin test negative and CAP/RAST positive, and (C) skin test and CAP/RAST negative but drug provocation test positive. Basotest was carried out by flow cytometry following the manufacturer's instructions using different betalactam determinants and results expressed as a stimulation index. RESULTS: Of the 70 patients, 34 (48.6%) were positive to Basotest (sensitivity: 48.6%), 31 (44.3%) to CAP/RAST and 46 (65.7%) to either one or the other. Considering the different groups, Basotest was positive in 50.9% of patients in Group A, 60% in Group B and 14.3% in Group C. The specificity was 91.3%. Positivity to the haptens was 28.6% to amoxicillin (AX), 21.7% to BP, 20% to benzylpenicilloyl-poly-l-lysine, 12.5% to ampicillin and 2.2% to minor determinant mixture. In patients with cephalosporin reactions, Basotest to the culprit cephalosporin was positive in 77.7%. There were differences between the two reactor groups in the sensitivity of Basotest (selective to AX=50%, cross-reactors=28.6%; chi(2)=10.809, P=0.004) and in the CAP/RAST (selective to AX=28.6%, cross-reactors=61.9%; chi(2)=8.944, P=0.011). CONCLUSIONS: The sensitivity of Basotest is similar to immunoassays (CAP/RAST). Sensitivity is improved when used in combination. Although further studies are required, Basotest results for cephalosporin allergy seem very promising. This technique does not help differentiate between selective reactors and cross-reactors.

Adult↗

PubMed2001

Differential mediator release from basophils of allergic and non-allergic asthmatic patients after stimulation with anti-IgE and C5a.

The differentiation between allergic and non-allergic asthma is a common and important challenge for the clinician. Until now, no in vitro diagnostic characteristics have been described to distinguish between these types. To examine the diagnostic value of a basophil stimulation test, we compared anti-IgE- and C5a-induced mediator release from peripheral blood leucocytes in different types of bronchial asthma. Peripheral blood leucocytes (PBL) from 10 aspirin-sensitive asthmatics (ASA), 12 non-allergic asthmatics without aspirin intolerance (NAA), seven allergic asthmatics (AA), and nine healthy controls were prepared by dextran sedimentation. After priming with interleukin-3 (IL-3) PBL were stimulated with anti-IgE and C5a, and the release of histamine (HR) and sulfidoleukotrienes (LTR) in the supernatant was compared. Additionally, purified leucocyte fractions were studied to determine the cellular source of mediator release. Upon stimulation with anti-IgE LTR was slightly, but not significantly, lower in ASA and NAA compared to AA and controls. In contrast, C5a-triggered LTR was significantly higher in ASA (14.4 +/- 12.88 pg/105 cells) and NAA (22.9 +/- 22.61 pg/105 cells) than in AA (9.6 +/- 3.29 pg/105 cells) and controls (7.5 +/- 7.19 pg/105 cells) (P < 0.05). This difference between ASA and NAA vs. AA and controls was even more pronounced when determining the quotient C5a-/anti-IgE-induced LTR (P < 0.001). At an optimal cut-off point of 1.0, calculated by relative operating characteristics (ROC) analysis, the positive predictive value for a donor to belong to ASA or NAA was 0.94. No significant differences could be found in HR between the asthmatic patient groups and healthy controls in either condition. As cellular source of LTR and HR the basophil could be determined. Determination of anti-IgE- and C5a-induced LTR from basophils allows us to discriminate between allergic and non-allergic asthmatic patients. For diagnostic purposes the quotient C5a-/anti-IgE-induced LTR is more significant than considering a single parameter. ASA cannot be distinguished from NAA.

Adolescent↗

PubMed1990

Reactivity of human basophils to anti-IgE and protein A in atopic dermatitis.

We compared by basophil degranulation and histamine release the reactivity to protein A and anti-IgE of the basophils from 3 groups of patients: group I (blood donors), group II (rhinitis) and group III (atopic dermatitis). We demonstrated that histamine release and basophil degranulation gave tightly correlated results and that by both methods group III was significantly (p less than 0.01) more sensitive to the effectors than the two other groups. Protein A and anti-IgE stimulation curves showed a rebound effect at low concentrations of effectors. This was, however, only significant (p less than 0.01) for group III. Bacterial hypersensitivity may be IgE-mediated and may be related to the increased sensitivity to bacterial antigens and the severity of atopic dermatitis in patients infected by Staphylococcus aureus.

Basophil Degranulation Test↗

PubMed1990

High-performance liquid chromatographic method for the evaluation of possible interferences in basophil-histamine release measurements.

Basophil activation studies commonly rely on the measurement of histamine following extraction and condensation to o-phthalaldehyde (OPT) as a quantitative measurement of degranulation. Specificity has long been recognized as a problem with this method. We have described a new high-performance liquid chromatographic method that allows for both a qualitative and a quantitative check of the purity of the OPT-histamine adduct. This method was sensitive (limit of detection = 2.55 pmol) and linear over a wide range (5 to 1000 ng/ml).

Basophil Degranulation Test↗

PubMed1993

Purification of human IgG4 subclass with allergen-specific blocking activity.

Blocking antibodies (bAb) induced by allergen immunotherapy are restricted to the IgG1 and IgG4 subclasses, with IgG1 predominating early and IgG4 coming later. Study of IgG4 bAb has been limited, in part, by the absence of a method to purify IgG4. We describe a rapid immunoaffinity chromatographic method for the purification of that subclass from whole serum. Starting serum (TR) contained 90 micrograms/ml Dactylis glomerata (orchard grass) pollen (DGP)-specific IgG4, measured by indirect ELISA. The blocking activity of TR was assayed in vitro on IgE-sensitized human basophils. Immunoadsorption on a strong-binding anti-IgG4 monoclonal antibody (mAb) removed about 90% of the total and allergen-specific IgG4 and nearly all of the blocking activity from TR. An IgG4-rich fraction was then obtained by absorption of several small volumes of TR on a weak-binding anti-IgG4 mAb column at neutral pH followed by elution with glycine-HCl buffer. The pooled eluates contained 82% IgG4, amounting to a 65-fold purification of the serum IgG4; the yield was approximately 30%. Nearly all the DGP-specific antibody was in the IgG4 component of the eluate. The blocking activity of the eluate was approximately equal to that of TR. Immunoblot patterns with the eluate and with TR on SDS-PAGE of DGP were nearly identical. This method thus provides a fully active, relatively pure IgG4 blocking antibody. Moreover, the results reinforce the importance of using a well-chosen mAb when purifying proteins by immunoaffinity chromatography.

Adult↗

PubMed1995

Simultaneous initiation of degranulation and inhibition of leukotriene release by soman in human basophils.

Previous studies noted that the serine esterase inhibitor, soman, could induce histamine release from human basophils. To investigate the mechanisms by which soman causes histamine release (a preformed mediator), we also examined its ability to induce leukotriene release (a newly synthesized mediator) from basophils. We found that no leukotriene release followed activation with soman, while histamine release was usually greater than 70%. In addition, soman and diisopropyl-fluorophosphate were found actively to suppress low level spontaneous leukotriene release as well as ongoing leukotriene release induced by anti-IgE antibody. Soman (0.3 mM) was able to stop leukotriene release as rapidly as the calcium chelator, EDTA. In a series of control experiments, it was noted that soman did not influence the metabolism of LTC4 to LTD4 or LTE4 (for which little metabolism occurred), eliminating the possibility that reduced LTC4 release could have resulted from its enhanced metabolism. Therefore, using one compound (soman), basophils could be simultaneously activated to degranulate while having the pathway leading to leukotriene release actively suppressed. These results provide further evidence that histamine and leukotriene release are independent pathways resulting from the activation of basophils.

Basophil Degranulation Test↗

PubMed2006

Initial Fc epsilon RI-mediated signal strength plays a key role in regulating basophil signaling and deactivation.

BACKGROUND: Mediator releases after high-affinity IgE receptor (Fc(epsilon)RI) cross-linking in basophils and mast cells crucially govern the symptoms of allergic disease and amplify underlying T(H)2-type responses. Interestingly, the dose-response curve for Fc(epsilon)RI activation is bell-shaped, with supraoptimal stimulation leading to reduced mediator release. OBJECTIVE: We sought to characterize the mechanisms responsible for this control of Fc(epsilon)RI-triggered basophil activation. METHODS: Human basophils were purified by means of Ficoll density centrifugation, elutriation, and negative selection with immunomagnetic beads. Various intracellular signal protein activities were assessed by means of Western blotting, and mediator releases were analyzed either spectrofluorometrically (histamine) or by means of ELISA (IL-4 and IL-13). RESULTS: Supraoptimal anti-IgE concentrations led to lower mediator release than optimal concentrations but simultaneously to considerably faster histamine release kinetics. In parallel, basophil signaling proteins (Syk, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinases 1 and 2) were more rapidly phosphorylated at higher anti-IgE concentrations but more transiently activated in the supraoptimal range. This endogenous regulation most likely involved src homology 2 domain-containing inositol 5' phosphatase (SHIP), which was highly phosphorylated after supraoptimal anti-IgE triggering compared with lower stimulus concentrations. Conversely, N-formyl-methionyl-leucyl-phenylalanine-stimulated basophils failed to phosphorylate SHIP in the supraoptimal concentration range and did not display a bell-shaped dose-response curve. CONCLUSION: The kinetics of IgE-mediated signaling and mediator release in primary human Fc(epsilon)RI(+) cells varies substantially according to the magnitude of stimulation, and SHIP most likely plays an important role in terminating these events. CLINICAL IMPLICATIONS: The speed of allergic symptom generation depends on the degree of IgE receptor triggering, which is downregulated by SHIP, a potential target for allergy therapy.

Basophil Degranulation Test↗

PubMed2002

Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography-mass spectrometry method.

A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described. After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds. Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis. The reliability of the results is verified by use of two different ion pairs for detection. The method is validated according to DIN 38402. Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1). The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml. Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.

Allergens↗

PubMed1998

Cell biology of the basophil.

The cell biology of basophils, based on published studies spanning 1990-1997, is reviewed. These rarest cells of granulocyte lineages are now available in sufficient numbers for such studies to be done, based on new methods for isolating and purifying the cells from peripheral blood and organ sources and for their derivation in growth factor-containing cultures from their precursors de novo. These studies are dependent on electron microscopy for the accurate identification of basophils, studies which have recently established the presence of basophils in two new species--mice and monkeys. Secretory, endocytotic and storage properties of basophils constitute their mechanistic role(s) in human disease; their role(s) in health is, however, obscure. Development of immunoaffinity and enzyme-affinity ultrastructural labeling techniques to image the Charcot-Leyden crystal protein and histamine in human basophils, coupled with ultrastructural analysis of kinetic samples of cells obtained after stimulation with diverse secretogogues, has provided insight into the role of vesicles in secretory transport mechanisms in human basophils as well as the definition of key ultrastructural phenotypes of secreting basophils.

Animals↗