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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

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.
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Figure 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.

Mentions: A FACSCalibur flow cytometer with CellQuest Pro software was calibrated daily by using CaliBRITE beads and FACSComp software (all from BD Biosciences). All the available cellular events were acquired with an unlimited target event count (Fig. 1). For the data analysis, leukocyte and potential basophil events were defined by using "Leukocyte" and "BasoScatter" gates, respectively, on the forward/side scatter (FSC/SSC) plot (Fig. 1, left plot). All potential basophil events defined by the BasoScatter gate were purified further by three variations of basophil gates set on the CCR3/CD123 plot (Fig. 1, middle plot). Finally, the double-gated basophils were analyzed on the CD63/CD203c plot. The expression percentage of each activation marker (CD203c+, double positive DP [CD203c+CD63+], or CD63+) was determined by setting previously defined quadrants (Fig. 1, right plot) such that CD63 or CD203c positive events would be less than 1.0% in the negative control tube. To measure CD203c expression (an ectoenzyme expressed constitutively even at a resting state), the stimulation index was calculated as the ratio of mean fluorescence intensity (MFI) of total gated basophils to MFI of sample/MFI of negative control (cutoff=1.38) [11]. To compare diagnostic performance among basophil markers, the signal to noise ratio (SNR) was calculated as the ratio of its expression percentage to the average of expression percentages/corresponding cutoff.


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)

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

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

Figure 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.
Mentions: A FACSCalibur flow cytometer with CellQuest Pro software was calibrated daily by using CaliBRITE beads and FACSComp software (all from BD Biosciences). All the available cellular events were acquired with an unlimited target event count (Fig. 1). For the data analysis, leukocyte and potential basophil events were defined by using "Leukocyte" and "BasoScatter" gates, respectively, on the forward/side scatter (FSC/SSC) plot (Fig. 1, left plot). All potential basophil events defined by the BasoScatter gate were purified further by three variations of basophil gates set on the CCR3/CD123 plot (Fig. 1, middle plot). Finally, the double-gated basophils were analyzed on the CD63/CD203c plot. The expression percentage of each activation marker (CD203c+, double positive DP [CD203c+CD63+], or CD63+) was determined by setting previously defined quadrants (Fig. 1, right plot) such that CD63 or CD203c positive events would be less than 1.0% in the negative control tube. To measure CD203c expression (an ectoenzyme expressed constitutively even at a resting state), the stimulation index was calculated as the ratio of mean fluorescence intensity (MFI) of total gated basophils to MFI of sample/MFI of negative control (cutoff=1.38) [11]. To compare diagnostic performance among basophil markers, the signal to noise ratio (SNR) was calculated as the ratio of its expression percentage to the average of expression percentages/corresponding cutoff.

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