Ann Lab Med.  2016 Jan;36(1):28-35. 10.3343/alm.2016.36.1.28.

Basophil Markers for Identification and Activation in the Indirect Basophil Activation Test by Flow Cytometry for Diagnosis of Autoimmune Urticaria

• Kim Z
• Choi BS
• Kim JK
• Won DI

• KMID: 2373494
• DOI: http://doi.org/10.3343/alm.2016.36.1.28

Abstract

BACKGROUND
The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c).
METHODS
Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20).
RESULTS
For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity.
CONCLUSIONS
For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

Keyword

Chronic urticaria; Autoimmune urticaria; Basophil; Markers; Donor; Flow cytometry

MeSH Terms

Autoimmune Diseases/blood/*diagnosis/immunology
Basophils/*immunology/metabolism
Biomarkers/blood
Child
Flow Cytometry
Humans
Interleukin-3 Receptor alpha Subunit/blood
Male
Receptors, CCR3/blood
Urticaria/blood/*diagnosis/immunology
Biomarkers
Interleukin-3 Receptor alpha Subunit
Receptors, CCR3

Figure

• 

  Fig. 1 Data acquisition and analysis of indirect flow BAT for autoimmune urticaria diagnosis. On the FSC/SSC plot (left), the basophil scatter gate (BasoScatter) and leukocyte gate are defined to calculate the basophil percentage among total leukocytes. On the CCR3/CD123 plot (middle), three basophil gates, gates 1, 2, and 3, are defined. The upper left subset, indicated by a curved red arrow within gate 1 and just outside gate 3, appears to be monocyte doublets, an assumption based on their back-gated location on the FSC/SSC plot. On the CD63/CD203c plot (right), a quadrant is set to obtain the expression percentage of CD203c (two upper quadrants), DP (red-shaded quadrant, CD203c+CD63+), or CD63 (two right quadrants).Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PECy5, PE-cyanine 5; APC, allophycocyanine; FSC, forward scatter; SSC, side scatter; DP, double positivity; CCR3, eotaxin CC chemokine receptor-3.

• 

  Fig. 2 Correlation of the basophil percentage between flow cytometry using basophil gate 3 and automated CBC using Advia 2120i. CBC was more highly correlated with basophil counts using gate 3 (r=0.530) than with gates 1 (r=0.498) or 2 (r=0.195).Abbreviation: CBC, complete blood count.

• 

  Fig. 3 Comparison of basophil activation markers according to basophil variables. Indirect flow BAT was performed in positive control sera. The expression percentage for each activation marker (CD203c, DP, and CD63) is plotted according to the four basophil preparation methods (DNAUP, DNAP, DAUP, and DAP). To prepare artificial positive control serum, negative control serum was spiked with reconstituted monoclonal anti-FcεRIα antibody. The spiking volume (µL) per 100 µL of negative control serum is indicated by colored markings (see legend). All four types of basophils were tested in parallel by changing the volume of the monoclonal anti-FcεRIα antibody in series of batches. DAP basophils, however, were tested only with the minimum volume of 6 µL. Nevertheless, DAP basophils displayed the highest SNR and positivity rate (PR, assay sensitivity). Expression percentages of each activation marker were measured by using basophil gate 3.Abbreviations: FcεRIα, α-chain of IgE receptor; DP, double positive (CD63+CD203c+); SNR, signal to noise ratio; PR, positivity rate, DNAUP, non-atopic donor unprimed; DNAP, non-atopic donor primed; DAUP, atopic donor unprimed; DAP, atopic donor primed.

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