Basophils Are Still Alive

Quality Assay & Sample Stability for Viable Basophils

Basophil Activation Testing (often referred to as BAT) is a flow-cytometry-based functional assay that assesses the degree of cell activation after exposure to stimuli.3

Are you approaching basophil activation test in your lab? Would you like to implement an allergy diagnostic BAT test service or basophils are the new biomarker in your research project?

You may face into the critical questions: how stable are basophils after the blood taking and which is appropriate anticoagulant that preserve viability.

If you search literature on the internet it will be difficult to retrieve precise and conclusive information regarding the acceptable timeframe for testing post-venipuncture.  In fact, you will find conflicting, confusing, and misleading data that may give the perception that basophils are very delicate cells that require testing as quickly as possible. It’s not uncommon to find statements like “BAT test must be performed immediately after venipuncture or “Whole blood BAT should ideally be performed within 4 h of blood collection to maximize viability and functionality of basophils1, but these statements are not accompanied by a reference to a clear analytical evaluation.

Have you found in your searches that this is in fact the case? If yes, then how do you know what data to trust? The exact knowledge of the viability of basophil cells in regards to their activation capability over time in different blood anticoagulant is essential to evaluate the quality of BAT data you are generating: it’s the key to setting up a reliable blood logistic for BAT test service in your clinical lab or for your research project.  Thus, what you can trust is data that you produce yourself or that is produced by experts in the field in a thorough pre-analytical study.

So you may design and perform an evaluation study to validate the viability of basophils for your testing or you may just review data already out there from an expert in the field.

BÜHLMANN Laboratories has extensive experience in BAT since the release of the first assay in 1992 (see assay timeline  below) and has performed numerous studies to support efficient, successful testing to fulfill the requirement of diagnostic use [see Figure 1 for a full timeline]. On October 18, 2018, at the EuroBAT2018 meeting, we presented data on a pre-analytical study on ‘Blood Stability for BAT test.

What BÜHLMANN did in this study you can also reproduce in your Lab:

  • 3 different blood anticoagulants were analysed: EDTA, heparin and citrate
  • 3 different temperatures of storage were considered to reproduce ideally real life conditions: 2-8°C (refrigerate), 20°C (room temperature), 28°C (non-controlled high temperature)

The results obtained were quite clear and comprehensive.  In fact, we found that viable basophils can be recovered not only immediately after blood collection but also over several days in any anticoagulant used.  However, the optimal efficiency of recovery was seen with EDTA blood stored at 2-8°C.  At this temperature, the loss of cells is minimized, with recovery of more than 500 basophils, which is the threshold to obtain a statistically significant number of cells for robust BAT data.  EDTA blood stored at 2-8°C was superior to heparin and citrate blood, where we only recovered a median of basophils between 300 and 500.

In terms of activation capabilities, basophils responded well to different stimuli. We confirmed the basophil activation by cross-linking the high affinity receptor for the IgE with a monoclonal antibody (the positive control included in any of our kits (anti FceRI Ab)) and several allergens.

We built very nice dose response curves, with a minimal loss of signal up to 24 hours after blood collection, slight increase at 48 hours but still making it feasible to obtain high quality BAT data (See Figure 1 below).

About the Author

michele-romano

Michele Romano, PhD

Michele Romano, PhD, is responsible at BÜHLMANN for the Product Management of the CAST® Assays for Basophil Activation Tests (BAT) in the field of in vitro allergy diagnosis as well as in the promising new application of drug candidate potency and efficacy testing.

He received his PhD degree in Cellular and Molecular Biology and Pathology at University of Verona, Italy and a degree in Industrial Biotechnology at University of Milano, Italy. He has joined BÜHLMANN in 2011, after a research career in the immunology field both in Industry and Academia, with the main focus in functional bioassay development and immune receptor characterization.

Dose Response Curves

Figure 1: Representative case of EDTA blood donors analysed with Flow CAST® for CD63 activation over a period of 3 day storage at 2-8° by  anti FceRI Ab (left) and specific allergen (right).  D0, D1, D2, D3 is representative of the days after venipuncture.

Our results suggest that whatever application of basophil activation is used, the cells are viable for up to 24/48 hours after blood collection, for drug and protein allergens, respectively.

We have reported the robustness of the BAT data in conjunction with time of storage demonstrating its dependence on the allergen used (You can find this information in the IFU of our CAST® assays and in the allergen booklets). For example, protein allergen typically gives very high responses, so BAT can be performed up to 48 hours after blood collection, since the loss of activation does not affect the results. Alternatively, drug allergens rarely give activations that exceed 20-30%, so we suggest to perform BAT testing within 24 hours to limit the potential of loss of reactivity and the generation of false negative results.

At the same time, when using the basophil sensitivity as a parameter of BAT, intended as the EC50 (Half maximal effective concentration of the allergen) of a dose response curve of your stimuli, you should take into account the limited variability in your data generated by the storage of blood.

Of course working with live cells is not the same as working with serum analytes, but the timeframe to obtain high quality data from a BAT test works well with sophisticated sample logistics of diagnostic labs. Therefore, a standardized basophil activation test can be successfully implemented in diagnostic labs as well as in is widely used in research.

If you want to get more details on this data, please find the list of additional resources below.

You will learn more on the stability of CD63 activation in different conditions (ie. anticoagulant, storage temperature, and stimulation buffer). In addition, you will discover differences with other activation markers such as CD203c.

BÜHLMANN BAT Assay Timeline

BÜHLMANN has been at the forefront of basophil activation test development for over 20 years, combining innovation with commitment to high quality standards.  The first assay was launched soon after Edward Knols‘ discovery of CD63 activation marker2 and next generation assays evolved thereafter following the first BAT publication.Please see the full assay evolution in timeline below.

References

  1. Hemmings, O., Kwok, M., McKendry, R. et al. Curr Allergy Asthma Rep (2018) 18: 77. 1007/s11882-018-0831-5
  2. Knol EF, Mul FP, Jansen H, Calafat J, Roos D. Monitoring human basophil activation via CD63 monoclonal antibody 435. J Allergy Clin Immunol 1991;88:328–338.
  3. McGowan, Emily C and Sarbjit Saini. “Update on the performance and application of basophil activation tests” Current allergy and asthma reports 13,1 (2013): 101-9. doi: 10.1007/s11882-012-0324-x PMID: 23188565.
  4. Sainte-Laudy J, Vallon C, Gu[1]erin JC. Analysis of membrane expression of the CD63 human basophil activation marker. Applications to allergologic diagnosis. Allerg Immunol (Leipz) 1994;26:211–214.
  5. Santos AF, Shreffler WG. Road map for the clinical application of the basophil activation test in food allergy. Clin Exp Allergy. 2017 Sep;47(9):1115-1124. doi: 10.1111/cea.12964. Epub 2017 Aug 1. Review. PubMed PMID: 28618090 DOI: 1111/cea.12964.