Leading Edge 2022 Issue 2

8 LEADING EDGE - 2022-2 Novel Changes to the Flow CAST® Allergy Assay Improving Stability and Optimising Laboratory Workflow Basophil Activation Tests (BAT) can be used to reliably diagnose allergies and are gaining increasing importance in this field. Basophil Activation assays, such as the Flow CAST® assay manufactured by BÜHLMANN, offer a very high specificity, and therefore a very good ability to distinguish those who are truly allergic from those who are not. Reduce the Use of Challenge Tests There is a growing body of evidence to suggest that Basophil Activation Tests can offer a higher degree of accuracy and clinical relevance compared to other allergy tests, and they have the potential to significantly reduce the number of challenge tests needing to be performed. In a study conducted by Santos et al. 20141, looking at peanut allergy in children, using BAT on its own as a single test led to a reduction in oral food challenges (OFC) by two thirds, and when using BAT as a second line test performed after equivocal skin prick tests (SPT) or Arah2 specific IgE (sIgE) tests, a 97% reduction in oral food challenges was observed. Basophil Activation Testing offers a safer alternative to a challenge test, which can help to reduce both the waiting time for a diagnosis, and the costs associated with performing challenge tests. BÜHLMANN has recently made changes to its Flow CAST Basophil Activation Test. The kit now includes a new version of the wash buffer which contains a stabiliser. The wash buffer is used to reconstitute the cell pellet immediately after red blood cell lysis. The stabiliser that is now included within the wash buffer allows for the cells to be fixed, with an additional thirty-minute incubation of the samples at room temperature. Extended Sample Stability - Improved Workflow Previously, the samples needed to be analysed using flow cytometry immediately following completion of the Flow CAST protocol. The added stabiliser now enables the samples to be analysed up to five days after completion of the protocol, if stored at 2-8C and protected from light. This change will significantly help to improve workflow in laboratories, allowing for samples to be reliably fixed, stored and then analysed on subsequent days, in case instrumentation is overwhelmed. This gives the laboratory additional time to analyse these samples, making it easier for them to plan instrumentation and staffing resources. Stability Studies BÜHLMANN performed stability studies using the anti-FcƐRI mAb stimulation control (included in the Flow CAST assay) on EDTA whole blood from four normal blood donors. This assessed the stability of the processed samples after cell stimulation and fixation with the new wash buffer, and determined the specimen stability of unprocessed EDTA whole blood samples before performing the Flow CAST assay. After completion of the Flow CAST protocol using the new wash buffer to fix the cells, the stability of the processed samples was assessed at 2-8C and 28C (whilst protected from light) and measured at several time points from 0 to 10 days. A decay over time of a maximum of 20% from the baseline according to results at time 0 were accepted to be 0. The results show that, for the short-term storage of stimulated and subsequently fixed cells, all test results remained above the 80% recovery criteria for the full study duration (10 days) when stored at 2-8C and protected from light (Figure A), and for 2 days when stored at 28C and protected from light (Figure B). Flow Cytometry Analysis In addition to this, during analysis by flow cytometry of the processed cells, similar analysis patterns can be observed between day 0 and day 5 (Figure C; opposite). Stability of Fixed Cells