Limits...
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 - Ann Lab Med (2016)

Bottom Line: CD203c).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).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697340&req=5

Figure 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.

Mentions: The three activation markers, all originating from the atopic donor, that attained 100% assay sensitivity were DAUP CD203c, DAP DP, and DAP CD63 (Table 2, Fig. 3). In the unprimed basophil CD203c, DNAUP CD203c (95.2%, 20/21) was not different from DAUP CD203c (100%, 21/21; P=1.000). A single false-negative DNAUP CD203c case displayed an expression percentage of 0.74%, and a stimulation index of 1.05. DNAP CD63 (76.2%, 16/21) was significantly higher than DAUP CD63 (14.3%, 3/21; P=0.001), while DAP CD63 (100%, 21/21) was significantly higher than DNAP CD63 (75%, 18/24; P=0.023).


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 - Ann Lab Med (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697340&req=5

Figure 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.
Mentions: The three activation markers, all originating from the atopic donor, that attained 100% assay sensitivity were DAUP CD203c, DAP DP, and DAP CD63 (Table 2, Fig. 3). In the unprimed basophil CD203c, DNAUP CD203c (95.2%, 20/21) was not different from DAUP CD203c (100%, 21/21; P=1.000). A single false-negative DNAUP CD203c case displayed an expression percentage of 0.74%, and a stimulation index of 1.05. DNAP CD63 (76.2%, 16/21) was significantly higher than DAUP CD63 (14.3%, 3/21; P=0.001), while DAP CD63 (100%, 21/21) was significantly higher than DNAP CD63 (75%, 18/24; P=0.023).

Bottom Line: CD203c).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).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Korea.

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.

No MeSH data available.


Related in: MedlinePlus