Basophils Are Still Alive

BUHLMANN Basophil Banner- Technical Post

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

Q: Do you know which BAT application makes sense for your lab? Are you thinking about implementing an allergy BAT?  Or are you planning on using basophils as the new biomarker in your research project?

A: If you are considering either of the BAT applications mentioned above, you may need to answer critical questions, such as: how stable are basophils after the blood collection and which anticoagulant is appropriate to 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.

Q: Have you found in your searches that this is in fact the case? If yes, then how do you know what data to trust?
A: 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.
Q: So now you are thinking, what type of study should we do to validate the viability of basophils for our testing? Or is there any data already out there from an expert in the field that we can review?
A: 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.

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.

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.

 

2019
April 10

2019

166 CAST® Allergens available in our allergy product pipeline to date with continual development over the past several years. Download WordPress ThemesDownload WordPress Themes FreeDownload WordPress ThemesDownload Best WordPress Themes Free Downloadudemy free downloaddownload lava firmwarePremium WordPress Themes Downloadfree online course
March 7

2011

Rebranding of the CAST® product: Flow CAST®, CAST® ELISA and CAST® Allergens Download Nulled WordPress ThemesDownload Best WordPress Themes Free DownloadDownload Premium WordPress Themes FreePremium WordPress Themes Downloadudemy paid course free downloaddownload karbonn firmwarePremium WordPress Themes Downloadudemy course download free
March 7

2008

Launch of Flow2-CAST (FK-CCR), second generation Download Premium WordPress Themes FreeDownload WordPress ThemesDownload Nulled WordPress ThemesDownload WordPress Themesfree download udemy coursedownload intex firmwareDownload WordPress Themes Freeudemy course download free
March 7

2002

Launch of Flow-CAST (FK-BAT), the first generation Download WordPress ThemesDownload Nulled WordPress ThemesDownload WordPress Themes FreeDownload WordPress Themesudemy free downloaddownload mobile firmwareDownload WordPress Themes Freelynda course free download
March 7

1994

The first publication for basophil activation: 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. (Independent of BÜHLMANN Laboratories AG) Download WordPress ThemesDownload Premium WordPress Themes FreeDownload Best WordPress Themes Free DownloadDownload Premium WordPress Themes Freeonline free coursedownload mobile firmwareDownload
March 7

1992

Launch of CAST-ELISA (EK-SLT), the first generation Download Nulled WordPress ThemesDownload Premium WordPress Themes FreeDownload Best WordPress Themes Free DownloadDownload Best WordPress Themes Free Downloadfree download udemy coursedownload karbonn firmwareDownload WordPress Themes Freefree download udemy course
March 7

1991

The Discovery of CD63 activation marker: 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. (Independent of BÜHLMANN Laboratories AG) Premium WordPress Themes DownloadFree Download WordPress ThemesDownload Nulled WordPress ThemesDownload WordPress Themes Freefree download udemy coursedownload lenevo firmwareDownload WordPress Themes
What BUHLMANN 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).

Results

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).

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.

Conclusion:

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 obtain more details on this data, please contact us for list of additional resources.  These resources will provide information on the stability of CD63 activation in different conditions (ie. anticoagulant, storage temperature, and stimulation buffer) as well as differences with other activation markers such as CD203c.

Learn More
Learn more on sample logistics, share your experience, obtain additional resources, and gather some tips on how to implement BAT testing.

Flow CAST® is for Research Use Only in the US. Not for use in diagnostic procedures.
Health Canada Licence: 101781

CAST® ELISA is for Research Use Only.  Not for use in diagnostic procedures.

CAST® allergens are for Research Use Only in the US. Not for use in diagnostic procedures.

Many of BÜHLMANN’s Allergens are Health Canada licensed.

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.