Validation of inducible basophil biomarkers: Time, temperature and transportation.

BACKGROUND: The short stability window of several hours from blood collection to measuring basophil activation has limited the use of flow cytometry-based basophil activation assays in clinical settings. We examine if it is possible to extend this window to 1 day allowing for shipment of samples between laboratories. Several options exist for reporting the results including reporting all the measured values directly, calculating ratios and reporting a single value covering all measured results. Each of these options have different stability and value to the physician. METHODS: Whole blood samples from peanut allergic patients were stimulated with four different peanut concentrations at Day 0, Day 1, and Day 2. Samples were stored under temperature-controlled conditions. Flow cytometry was used to analyze the samples. The basophil activation and degranulation were measured as percentage of positive CD63 basophils and CD203c MFI fold change. Shipped samples were transported under ambient conditions. RESULTS: The results show that CD63 is a stable marker at Day 1. The CD203c ratio decreases significantly at Day 1. Calculating the CD63/IgE ratio proves to be more stable than CD63 alone. The most stable readouts are the semi-quantitative results and the trajectory of the dose response curve. Finally, we confirmed that the stability can be extended to samples shipped overnight to the laboratory. CONCLUSIONS: It is possible to extend the stability of the basophil activation assay to 1 day for samples stored at 18-25°C as well as samples shipped under ambient conditions as long as the temperature is within the 2-37°C range.

Systemic reactions with aeroallergen cluster immunotherapy in a clinical practice.

BACKGROUND: Subcutaneous allergen-specific immunotherapy is a proven, highly effective treatment for immunoglobulin E-mediated diseases. Despite its proven benefits, only a small percentage of patients with allergic disease use immunotherapy, in part because of the inconvenience associated with treatment. Cluster allergen immunotherapy may offer patients a more convenient treatment option but is prescribed infrequently because of the perception that accelerated immunotherapy buildup leads a higher rate of systemic reactions. OBJECTIVE: To examine the safety of cluster immunotherapy and identify risk factors for systemic reactions during cluster buildup. METHODS: A retrospective, observational review in a large, multicenter allergy practice group was conducted for patients receiving cluster immunotherapy between May 2008 and October 2010. RESULTS: Data from 441 patients receiving cluster immunotherapy were collected. Forty-eight patients (10.9%) experienced systemic reactions. Based on the World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System, 18 grade 1 reactions (38.3%), 23 grade 2 reactions (48.9%), 5 grade 3 reactions (10.6%), 1 grade 4 reaction (2.1%), and no grade 5 reactions were seen. Risk factors for a systemic reaction included: female sex, physician diagnosis of asthma, age 21 to 40 years, and inclusion of certain allergens in the immunotherapy vaccine. CONCLUSIONS: Cluster immunotherapy allows patients to reach their immunotherapy maintenance dose more rapidly and may lead to more rapid symptomatic improvement. However, the cluster buildup may lead to a higher rate of systemic reactions. Identifying risk factors for systemic reactions will help improve the safety of cluster immunotherapy.

Cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life.

Daycare attendance and siblings are associated with viral-induced wheezing in children. Preexisting immunologic factors may influence the expression of viral infections in infancy, and in turn, recurrent infections may influence the development of immune responses. A total of 285 children were enrolled in the Childhood Origins of Asthma Project at birth and followed for at least 1 year. Cord blood and 1-year mononuclear cells were stimulated with phytohemagglutinin, and cytokine-response profiles were measured by enzyme-linked immunosorbent assay. Nasal lavage was performed for moderate to severe respiratory illnesses. Daycare attendance and/or siblings significantly increased the likelihood of contracting respiratory syncytial virus (1.5-1.6-fold increase) and rhinovirus (1.8-2.1-fold increase), and increased the risk of rhinovirus-induced wheezing (14-18% vs. 2%, p = 0.011). Cord blood IFN-gamma responses were inversely related to the frequency of viral respiratory infections (r(s) = -0.11, p = 0.05), and more significant for subjects with high exposure to other children (r(s) = -0.27, p = 0.028). The interval change in infantile IFN-gamma responses correlated positively with the frequency of viral infections in infancy (r(s) = 0.12, p = 0.047). These data suggest that neonatal IFN-gamma responses may influence antiviral activity, or may represent a marker of antiviral immunity maturation. Conversely, the frequency of viral infections in infancy can influence IFN-gamma responses.