1Department of Clinical Pathology, School of Medicine, Kyungpook National University, Daegu, Korea.
2Division of Allergy and Infectious Diseases, Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu, Korea.
3Green Cross Reference Laboratory, Yongin, Korea.
IgG-mediated anaphylaxis occurs after infusion of certain monoclonal antibody-based therapeutics. New
Neutrophil OB was measured by dihydrorhodamine 123 flow cytometry using a leukocyte suspension spiked with a very small patch of the allergen crude extract,
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.
IgE-mediated anaphylaxis has been widely studied and is thought to be the main anaphylactic pathway. However, increasing evidence from animal studies supports the presence of a second pathway . Such IgE-independent mechanisms can be mediated by IgG antibodies or complement proteins . In the IgG-dependent pathway, macrophages and IgG, rather than mast cells and IgE, are the main immune components involved, and platelet-activating factor (PAF), rather than histamine, is the main mediator released .
In mice, the IgG-dependent pathway requires proportionately higher amounts of antibody and antigen than the IgE-mediated pathway . High doses of allergen can induce IgG-mediated anaphylaxis by forming complexes that activate macrophages and basophils through the Fcγ receptor (FcγR) III. In a mouse model, this mechanism acted in concert with neutrophil activation resulting from the interaction between allergen-specific IgG2 and FcγRIV on cells. PAF was also the predominant mediator .
In humans, IgG receptors can activate PAF secretion by macrophages and neutrophils  and PAF can activate mast cells
The IgE-independent mechanism is clinically indistinguishable from IgE-mediated anaphylaxis . To detect suspected drug-induced anaphylaxis,
Therefore, new tests for detection of the IgE-independent mechanism, particularly whether and when IgG-mediated anaphylaxis occurs, are urgently needed. We investigated the action of circulating neutrophils in peripheral blood. Neutrophils destroy microorganisms by producing reactive oxygen species as part of normal host defenses against infectious diseases .
The aim of this study was to develop an assay for diagnosis of IgG-mediated anaphylaxis. We investigated whether allergens trigger neutrophil oxidative burst (OB) and whether neutrophil OB occurs due to allergen-specific IgG (sIgG). Crude extracts of
A total of 31 subjects were selected according to the following criteria: 1) subjects who visited the allergy clinic of the Kyungpook National University Hospital (Daegu, Korea) and 2) subjects for whom multiallergen testing was conducted by the Department of Laboratory Medicine. The male to female ratio of the enrolled subjects was 16/15 and the median age was 44 years (3–76 yr). Lithium heparin tubes were used to collect whole venous blood samples for multiallergen testing. Samples that remained from these tests were used in the study. All participants provided informed written consent and the study was approved by the institutional review board. Experiments were performed within 4 h after blood collection.
A FACSCalibur flow cytometer equipped with the blue laser (488 nm) was calibrated daily using CaliBRITE beads and FACSComp software (BD Biosciences, San Jose, CA, USA). At acquisition, using CellQuest Pro software (BD Biosciences, San Jose, CA, USA), forward and side light scatter (FSC and SSC, respectively) were linearly amplified, whereas all fluorescence was logarithmically amplified.
Leukocyte suspensions were separated from whole blood using an overlay procedure . Briefly, 500 µL of whole blood was carefully overlaid onto 1.0 mL of Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) in a 1.5 mL microcentrifuge tube. The tube was left standing at room temperature for 20 min for red blood cell sedimentation. The supernatant (250 µL of plasma containing leukocytes) was carefully aspirated with a micropipette.
The isolated leukocyte suspension of each subject was divided into two fractions (“stimulated” and “unstimulated”), with a volume of 60 µL per tube, to which 2.0 µL of thawed and diluted DHR 123 (Sigma-Aldrich; final concentration = 375 ng/mL) were added . Both tubes were incubated at 37℃ for 5 min in shaking water bath to allow loading of DHR 123 into the cells, and the fraction to be stimulated (or the “stimulated” fraction) was spiked with a patch (diameter of −1 mm) of
The mean fluorescence intensity (MFI) of rhodamine 123 and the mean FSC of neutrophils were determined (Fig. 1). Two regional gates were used for neutrophils: one in the FSC/SSC plot and the other in the rhodamine 123/SSC plot. The MFI value and mean FSC value were expressed as the geometric means of the main portion (M1 or M2) of the peak in the rhodamine 123 histogram and in the FSC histogram of gated neutrophils, respectively. The change in MFI for unstimulated and stimulated neutrophils was expressed as the neutrophil oxidative index (NOI), which was calculated using Equation 1:
The change in FSC (ΔFSC) was calculated using Equation 2:
The cutoff value for distinguishing positive from negative results was 1.20 for NOI and 50 for ΔFSC.
A BAT was performed by three-color flow cytometry . For basophil priming, interleukin-3 (Sigma-Aldrich) was added to 100 µL of whole blood and incubated for 10 min at 37℃. This primed whole blood was spiked with a patch of
The level of
Statistical analyses were performed using SPSS Version 19.0 (SPSS, Inc., Chicago, IL, USA). The two groups were compared using regression analysis or Wilcoxon signed rank test.
The leukocyte suspension was isolated using an overlay method for minimal manipulation. In unstimulated samples (not spiked with
The stimulated samples (spiked with
A single subject (73-year-old woman) among 31 subjects enrolled showed a
Neutrophil OB was nearly absent (NOI= −1) in both cases of the lowest and highest
No significant relationship was found between
We selected four subjects who were previously positive for both
In this study, we verified that an allergen can trigger neutrophil OB via a preexisting allergen-sIgG immune complex, as measured by the DHR assay using leukocyte suspensions spiked with the allergen. This protocol is relatively simple and cost-effective.
In our protocol without red cell lysis, the leukocyte suspension was separated from red blood cells using an overlay method , which is suitable for rapid processing with minimal manipulation of leukocytes. We used
Shortly after adding a tiny patch of
In addition to the role of preexisting
We investigated the relationship between
Our protocol may be applicable for
We conclude that allergens can trigger neutrophil OB via preexisting allergen-sIgG and that neutrophil OB can be easily measured in a DHR assay using a leukocyte suspension spiked with the allergen. These findings can be used for
Flow cytometry data acquisition and analysis for oxidative burst in response to
Time course experiments showing effects of activation period on neutrophil reaction against
Blood levels of
Abbreviations: BAT, basophil activation test;