Purification of Basophils from Peripheral Human Blood

Falcone, Franco H.; Gibbs, Bernhard F.

doi:10.1007/978-1-0716-0696-4_2

Cited by 6 publications

(5 citation statements)

References 39 publications

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“…In cancer patients, the interval to perform this diagnostic procedure is usually two weeks, a short interval that can interfere with BAT-FC results (basophils hyporeactivity response or anergy) [27]. Due to the low frequency of the human peripheral blood basophils (less than 1%) [14] and the difficulty of their purification [28], several rat basophilic leukemia (RBL) cell lines transfected with human FcεRI have been developed to be used as a diagnostic tool for detection of allergen-sIgE and as surrogate for patient basophils [12,29]. Among them, RBL-30/25 and RBL SX-38 cell lines presented an optimal IgE-mediated degranulation [19,29].…”

Section: Discussionmentioning

confidence: 99%

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

Fernández-Lozano,

Rita,

Barra-Castro

et al. 2024

Preprint

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Drug hypersensitivity reactions (DHR) to platinum-based compounds (PC) are on the rise, and their personalized and safe management is essential to enable first-line treatment of these cancer patients. This study aimed to evaluate the usefulness of basophil activation test by flow cytometry (BAT-FC) and the newly developed sIgE-microarray and BAT-microarray in diagnosing IgE-mediated hypersensitivity reactions to PC. 24 patients with DHR to PC (20 oxaliplatin and four carboplatin) were evaluated: 13 patients diagnosed as allergic with positive skin tests (STs) or drug provocation tests (DPTs), six patients diagnosed as non-allergic with negative STs and DPTs, and five patients classified as suspected allergic because DPTs could not be performed. In addition, four carboplatin-tolerant patients were included as controls. BAT-FC was positive in 2 of 13 allergic patients, with a sensitivity of 15.4% and specificity of 100%. However, the sIgE- and BAT-microarrays were positive in 11 of 13 DHR patients, giving a sensitivity of over 84.6% and a specificity of 90%. Except for one patient, all samples from the non-allergic and control group were negative for the sIgE- and BAT-microarrays. Our experience indicates that sIgE- and BAT-microarray could be helpful in the endophenotyping of IgE-mediated hypersensitivity reactions to PC and may provide an advance in decision-making for drug provocation testing.

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Section: Discussionmentioning

confidence: 99%

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

Fernández-Lozano,

Rita,

Barra-Castro

et al. 2024

Preprint

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“…Basophils were obtained from buffy coats, following ethical approval from the National Health Service (NHS) Research Ethics Committee (reference number 07/Q1206/3), purchased from the NHS Blood and Transfusion service. Basophils were first isolated by Ficoll-density centrifugation (using Ficoll-Paque Plus, GE Healthcare, Uppsala, Sweden) and purified further by immunomagnetic cell selection (negative selection) using commercial isolation kits (EasySep Human Basophil Enrichment Kit, STEMCELL Technologies, Grenoble, France) as previously reported ( 33 , 34 ). Basophil numbers and purities were verified by light microscopy using alcian blue staining and a Fuchs-Rosenthal haemocytometer.…”

Section: Methodsmentioning

confidence: 99%

IgE-dependent human basophil responses are inversely associated with the sarcoplasmic reticulum Ca2+-ATPase (SERCA)

2023

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Basophils crucially contribute to allergies and other Th2-driven diseases by rapidly releasing inflammatory and immunomodulatory mediators following high-affinity IgE-receptor crosslinking. Although these basophil-mediated responses depend on sensitization with antigen-specific IgE, this does not necessarily predict clinical symptom severity. It is thought that the balance of early stimulatory (e.g. SYK) and inhibitory (e.g. SHIP-1) intracellular signals are associated with basophil responsiveness, which is also critically dependent on calcium mobilization. Previous studies suggest that the sarcoplasmic reticulum Ca2+-ATPase (SERCA2), which regulates cytosolic calcium levels, may be inversely associated with airway smooth muscle reactivity in asthma. Since basophils are implicated in asthma severity, our aims were to address whether SERCA2 is implicated in human basophil responses, especially following IgE-mediated activation. Human basophils were obtained from buffy coats, following research ethics approval, and further purified by immunomagnetic cell sorting. Expressions of SERCA2, and other isoforms, were determined by Western blotting in parallel to measuring IgE-dependent histamine releases from the same donors. The effects of a SERCA-activator and inhibitor were also assessed on their abilities to modulate basophil histamine release. We observed an inverse correlation between basophil responsiveness to IgE-dependent stimulation and SERCA2 expression. Thapsigargin, a highly-specific SERCA inhibitor, stimulated basophil histamine release and potentiated IgE-dependent secretion of the amine. Conversely, disulfiram, a SERCA activator, inhibited IgE-dependent basophil activation. The results obtained from this exploratory study indicate that SERCA2 may be an additional regulator of basophil reactivity alongside early excitatory or inhibitory signal transduction pathways.

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“…The BAT-microarray was performed using rat basophil leukemia cells (RBL-30/25) transfected with the human FcεRIα receptor, produced at the Paul-Ehrlich-Institut (Langen, Germany; kindly donated by Stefan Vieths and Lothar Vogel) [28]. Cells were cultured in MEM (GIBCO)-10% fetal bovine serum (Thermo Scientific) at 37 • C in a 5% CO 2 atmosphere.…”

Section: Basophil Activation Test On Microarray Support (Bat-microarray)mentioning

confidence: 99%

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

Fernández-Lozano,

Rita,

Barra-Castro

et al. 2024

IJMS

View full text Add to dashboard Cite

Drug hypersensitivity reactions (DHRs) to platinum-based compounds (PCs) are on the rise, and their personalized and safe management is essential to enable first-line treatment for these cancer patients. This study aimed to evaluate the usefulness of the basophil activation test by flow cytometry (BAT-FC) and the newly developed sIgE-microarray and BAT-microarray in diagnosing IgE-mediated hypersensitivity reactions to PCs. A total of 24 patients with DHRs to PCs (20 oxaliplatin and four carboplatin) were evaluated: thirteen patients were diagnosed as allergic with positive skin tests (STs) or drug provocation tests (DPTs), six patients were diagnosed as non-allergic with negative STs and DPTs, and five patients were classified as suspected allergic because DPTs could not be performed. In addition, four carboplatin-tolerant patients were included as controls. The BAT-FC was positive in 2 of 13 allergic patients, with a sensitivity of 15.4% and specificity of 100%. However, the sIgE- and BAT-microarray were positive in 11 of 13 DHR patients, giving a sensitivity of over 84.6% and a specificity of 90%. Except for one patient, all samples from the non-allergic and control groups were negative for sIgE- and BAT-microarray. Our experience indicated that the sIgE- and BAT-microarray could be helpful in the endophenotyping of IgE-mediated hypersensitivity reactions to PCs and may provide an advance in decision making for drug provocation testing.

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Purification of Basophils from Peripheral Human Blood

Cited by 6 publications

References 39 publications

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

IgE-dependent human basophil responses are inversely associated with the sarcoplasmic reticulum Ca2+-ATPase (SERCA)

Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

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