Basophil activation test: Implementation and standardization between systems and between instruments

Abstract

The basophil activation test (BAT) is a good ex vivo alternative for measuring hypersensitivity to an allergen in sensitized patients but still lacks standardization. In this present study, we have implemented one of the systems and proposed inter-systems, inter-instrument standardization. Our method for basophil activation and labeling on whole blood: EDTA in one step using BasoflowEx and FlowCast . Setup on Navios and fluorescence targets converted to set up FACSCanto™ instrument. Our results: 1) A CD203c/CD63 (BasoflowEx) method was adapted for EDTA samples and simplified. 2) Final washing and concentration and use of time parameter help acquiring as many basophils as possible, spare acquisition time and noise. 3) The modified method was validated according to ISO15189 with a precision at 5.1% RCV, linearity between 1 and 1/8 of anti-IgE stimulation. Results were very close with CCR3/CD63 system (FlowCast). 4) Standardization, between systems and even between instruments. Mean Fluorescence Intensity targets are proposed using standard beads (Cytocal ) middle peak: FITC = 19.4; PE = 28.8 on Navios corresponding to FITC = 4,966; PE = 7,373 for FACSCanto. Data analyzed on common software (Kaluza ) were very closely correlated. 5) Co-labeling of B cells (CD20+) gives the possibility to monitor a significant drop of basophils under stimulation that could explain some underestimation in case of strong hypersensitivity. In conclusion, BAT would strongly benefit from easy implementation [EDTA, one step stimulation/labeling, wash, full sample analysis over time parameter, B cell relative basophil count] and standardization of instrument settings on MFI targets whatever system or instrument is used. © 2017 International Society for Advancement of Cytometry.

A novel tool for clinical diagnosis of allergy operating a microfluidic immunoaffinity basophil activation test technique

“…Since the background activation caused by CD203c crosslinking is quite low, it might be acceptable if it can be reduced in other steps during miBAT processing. Collecting blood in tubes containing EDTA instead of sodium heparin could possibly prevent degranulation prior to allergen incubation [169], as discussed above (paper III).…”

“…The basophil capture (crosslinking of CD203c) seems to be a minor factor, whereas washing likely impacts the background activation due to shear stress that will be active on the captured cells [152]. Using ethylenediaminetetraacetic acid (EDTA) as anticoagulant instead of sodium heparin might reduce background activation since degranulation is calcium dependent and EDTA has the ability to sequester calcium [168,169]. This would require additional calcium in the buffer used to dilute the allergen before basophil activation.…”

Basophil activation test: mechanisms and considerations for use in clinical trials and clinical practice

“…The use of the BAT to clinical practice requires analytical validation of the methodology, clinical validation of the test against patients' phenotype and continued quality assurance 57,[87][88][89] .…”

Tetraspanins in the regulation of mast cell function

“…It is expressed in small amounts also on the surface of non-activated MCs and basophils and similarly to CD63 significantly upregulated upon MCs and basophil stimulation [ 89 ]. However, the dynamics of CD203c and CD63 membrane up-regulation are different and probably induced by different signaling pathways [ 90 , 91 ]. Also the stability of CD63 and CD203c on plasma membrane depends on cell isolation and storage procedures [ 90 ].…”