Analytical validation of the basophil activation test
Analytical validation determines the accuracy of the testing procedure
from the draw of the blood sample to the reporting of the results. There
are several important components of the analytical validation of a
basophil activation test90:
- Inter and intra-run precision : Inter-run precision analyses
samples at different time points, whereas intra-run precision assays
for repeats of samples at the same time point on the same day. The
precision analysis for the BAT shows good
correlation88.
- Analytical interferences : A given allergen does not
stimulate/induce basophils of non-allergic patients and basophil
activation in a given patient are specific to the allergen being
tested and the concentration of the allergen. A given concentration of
allergen does not induce the same basophil response in all patients,
hence the importance of clinical correlations for each allergen at a
number of concentrations.
- Stability of samples : The question of stability of the samples
before reaching the laboratory has mostly been
resolved91. When transported in heparin tubes,
samples can stay stable up to 24 hours even when shipped in ambient
conditions. EDTA is an alternative calcium chelating anticoagulant
that stabilises basophils before testing. Allergens should be prepared
freshly, even if previously stored frozen or lyophylised.
- Proficiency Testing: For a sustained high quality use of BAT in
the clinical setting, constant quality control is necessary. In 2017
the EU approved the in vitro diagnostic medical devices regulation
(IVDR), that has to be implemented by 202492. Since
BAT is not a widely available assay and regulatory bodies have not yet
established proficiency testing, laboratories have created
individualized quality control measures to assure that the validated
assays continue to perform accurately. RfB (www.rfb.bio) and INSTAND
(www.instand-ev.de) are planning to offer external quality assurance
systems. Standardization of BAT procedures, allergen preparations and
sharing databases in which annotated raw data can be deposited are
important as it allows comparison of results in different centers and
would ensure consistency.
It is important to note that regulations and reimbursement/coverage by
healthcare systems vary for flow cytometry based assays in different
parts of the world. In the United States, BAT is used as a diagnostic
test as a part of clinical decision making in allergy practices that has
the capabilities of a high-complexity flow cytometry laboratory93-95. At the time of this review there are such
set-ups in private clinical practice as well as academic institutions.
In Europe, the BAT is mostly used in research and has been adopted as a
clinical test in some countries, such as Sweden, Spain, Germany and
Italy. Basophil testing has gained acceptance throughout the world,
including South Africa, Eastern Europe and South America. Many allergy
clinics use in house procedures (also referred to as “Laboratory
Developed Tests”) detecting CD63, others use kits that are commercially
available.