Blood Res.  2018 Dec;53(4):299-306. 10.5045/br.2018.53.4.299.

Neutrophil oxidative burst as a diagnostic indicator of IgG-mediated anaphylaxis

• Won DI
• Kim S
• Lee EH

• KMID: 2429304
• DOI: http://doi.org/10.5045/br.2018.53.4.299

Abstract

BACKGROUND
IgG-mediated anaphylaxis occurs after infusion of certain monoclonal antibody-based therapeutics. New in vitro tests are urgently needed to diagnose such reactions. We investigated whether allergens trigger neutrophil oxidative burst (OB) and if neutrophil OB occurs due to allergen-specific IgG (sIgG).
METHODS
Neutrophil OB was measured by dihydrorhodamine 123 flow cytometry using a leukocyte suspension spiked with a very small patch of the allergen crude extract, Dermatophagoides farinae (Der f). The mean fluorescence intensity ratio of stimulated to unstimulated samples was calculated as the neutrophil oxidative index (NOI).
RESULTS
The Der f-specific NOI (Der f-sNOI) showed a time-dependent increase after Der f extract addition. At 15 min activation, higher Der f-sIgG levels were associated with lower Der f-sNOI values in 31 subjects (P < 0.05). This inverse relationship occurs due to the initial blocking effect of free Der f-sIgG. Additionally, neutrophil OB was nearly absent (Der f-sNOI of −1) in two cases: a subject with undetectable Der f-sIgG levels and washed leukocyte suspensions deprived of Der f-sIgG.
CONCLUSION
Allergens can trigger neutrophil OB via preexisting allergen-sIgG. Neutrophil OB can be easily measured in a leukocyte suspension spiked with the allergen. This assay can be used to diagnose IgG-mediated anaphylaxis.

Keyword

Neutrophils; Oxidative burst; Anaphylaxis; Immunoglobulin G; Dihydrorhodamine 123

MeSH Terms

Allergens
Anaphylaxis*
Dermatophagoides farinae
Flow Cytometry
Fluorescence
Immunoglobulin G
In Vitro Techniques
Leukocytes
Neutrophils*
Respiratory Burst*
Suspensions
Allergens
Immunoglobulin G
Suspensions

Figure

• 

  Fig. 1 Flow cytometry data acquisition and analysis for oxidative burst in response to Der f extract. Calculations of NOI and ΔFSC are described in the Materials and Methods section.Abbreviations: Der f, Dermatophagoides farinae; FSC, forward scatter; MFI, mean fluorescence intensity; NOI, neutrophil oxidative index; SSC, side scatter.

• 

  Fig. 2 Time course experiments showing effects of activation period on neutrophil reaction against Der f extract. Measurements were performed from 0 min (just prior to addition) to 40 min after adding Der f extract in 10 min intervals (total of five times). The typical results of four separate experiments are shown. (A) Raw data of FSC and MFI values. The difference between stimulated values and unstimulated values was significant (P<0.05) starting at 10 min activation for both FSC and MFI. Asterisks indicate significant stimulated values. (B) Calculated indices (ΔFSC and NOI). The linearity of Der f-sNOI values according to the activation time was significant (P<0.01, asterisk), which was not observed for the ΔFSC values.Abbreviations: Der f, Dermatophagoides farinae; FSC, forward scatter; MFI, mean fluorescence intensity; NOI, neutrophil oxidative index; SSC, side scatter.

• 

  Fig. 3 Correlation between Der f-sNOI vs. ΔFSC measured at 15 min activation in the 31 enrolled subjects. Correlation between Der f-sNOI and ΔFSC measured at 15 min after activation in the 31 enrolled subjects. ΔFSC was significantly correlated with Der f-sNOI (P<0.00005).

• 

  Fig. 4 Blood levels of Der f-sIgG (total) in 31 subjects and their correlation with (A) subject age (P<0.05); (B) Der f-sIgE level; and (C) Der f-sIgG4 level. In the panel C, IgG4 levels of five subjects with less than the limit of detection (0.1 µg/mL) were assumed to be 0.05 µg/mL for convenience. The red and blue arrows indicate extreme cases of Der f-sIgG levels (highest and lowest, respectively). In panel A, the case with the lowest (undetectable) Der f-sIgG levels (73-year-old woman, potential non-responder against Der f) was excluded from the regression analysis.

• 

  Fig. 5 Der f-sNOI and Der f-sΔFSC according to the Der f-sIgG level. The relationship between Der f-sIgG levels and (A) NOI; (B) 1/NOI (P<0.05); and (C) ΔFSC (P=0.201). The red and blue arrows indicate extreme cases of Der f-sIgG levels (highest and lowest, respectively). In panel B, the case with the lowest (undetectable) Der f-sIgG levels (73-year-old woman, potential non-responder against Der f) was excluded from the regression analysis.

• 

  Fig. 6 Der f-sNOI and its relationship with (A) Der f-sIgG4; (B) Der f-sBAT; (C) Der f-sIgE; and (D) subject's age (P=0.157). No significant relationships were found. The red and blue arrows indicate extreme cases of Der f-sIgG levels (highest and lowest, respectively).Abbreviations: BAT, basophil activation test; Der f, Dermatophagoides farinae; NOI, neutrophil oxidative index.

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