Fully Automated Enzyme Immunoassay System for the Determination of Activator-specific Histamine Release from Basophils in Whole Blood

¹ Hycor Biomedical Inc., Garden Grove, CA 92841;
² Becton Dickinson, San Jose, CA 95131;
^a author for correspondence: fax 714-895-6920, e-mail kjkontis{at}ix.netcom.com

Histamine release from washed leukocytes and/or whole blood has been extensively utilized for in vitro studies of allergy (1)(2)(3)(4)(5) . It has been shown that when the history and skin test of an individual are positive for a given activator, the probability is very high that the in vitro tests will also be positive for histamine release (6) . Throughout the past 10 years, numerous methods have been developed for the determination of histamine release. The commercially available RIA and enzyme immunoassay (EIA) kits for histamine release are not automated and require the investigator to provide the activators or compounds to be used in stimulating histamine release. This study describes a fully automated basophil histamine release (BHR) EIA method for the determination of histamine in whole blood samples after stimulation of histamine release with various cellular activators available from the manufacturer.

The assay is performed on the HY·TEC^TM System, a fully automated laboratory analyzer with existing methods for allergy and autoimmune diagnostics. All of the components, including the allergen extracts (activators), histamine-coated microtiter plates, buffers, and conjugate, are available from Hycor Biomedical Inc. The HY·TEC BHR^TM is a competitive assay based on a mouse monoclonal anti-histamine antibody-alkaline phosphatase conjugate.

Sixteen histamine analogs and other amines including spermidine and spermine were tested for cross-reaction in the assay system. Each compound was assayed by the histamine EIA at three concentrations: 10, 20, and 1000 mg/L. The histamine EIA result for each concentration, expressed as milligrams per liter, was divided by the corresponding concentration. For example, a compound at a concentration of 1000 mg/L with a histamine EIA result of 0.02 mg/L would have the following cross-reactivity:

All of the compounds had undetectable (<0.001%) or very low cross-reactivity (<0.2%).

The detection limit of this assay (mean of the zero calibrator plus two standard deviations, n = 20) was 10 µg/L.

The intraassay and interassay CVs were determined by assaying three buffer controls of low, medium, and high values of histamine in six replicates per assay with 11 assays. Histamine challenge and spontaneous and total release of whole blood samples were also determined in replicates. The intraassay and interassay CVs with buffer controls were determined to be 5.1–7.3% and 7.7–11.6%, respectively. The average intraassay variations for histamine challenge and spontaneous and total release of whole blood samples were 13.0%, 9.5%, and 9.7%, respectively.

Recovery of added histamine was studied in four freshly drawn whole blood samples, 1.0 mL each, with the addition of 20 µL of each of five histamine stock solutions, 0–8000 mg/L of histamine (blood samples were obtained from all subjects only after informed consent was documented on approved forms on file at Hycor Biomedical Inc., Garden Grove, CA). These samples were assayed with 50 µL of activator diluent in place of activator predispensed in microtubes. Recoveries of histamine were 97.6–132%.

Four whole blood samples with added histamine were serially diluted with sample and assayed by the HY·TEC System in the routine method but using 50 µL of activator diluent instead of activator predispensed in microtubes. The plot of the expected value at each dilution vs its corresponding measured value for all four samples with added histamine were demonstrated to be linear, with the slope, intercept, and correlation coefficient of 0.998, 1.78 µg/L, and 0.998, respectively. The observed values were 91.9–111% of those expected (mean, 102%).

Thirty-two samples with added histamine and 30 whole blood samples were assayed by the Immunotech Histamine RIA and the HY·TEC BHR EIA. The correlation between the two methods was determined for all 62 samples. The slope was 1.2, and the intercept was 3.5 µg/L (r = 0.97).

A preliminary clinical study was performed in six men and two women, of ages 28–50. Care was taken to test the subjects only after confirming that no antihistamines, beta-2-agonists, cromolyn, or theophylline were being taken. Heparinized whole blood samples were challenged with dilutions of seven activator extracts (five foods and two grasses), using HY·TEC BHR. A cutoff of 15 µg/L histamine observed at any dilution was used to define a positive response. Fig. 1 shows a representative plot of results from a blood sample from a test subject with a dramatic increase in histamine release at increased concentrations of June grass (G8). Dose-dependent release of histamine was also observed with increasing concentrations of anti-IgE. At higher concentrations of G8 or anti-IgE, histamine release diminishes, as expected for a typical IgE-dependent response. The response to cow milk extract (F2) was very weak. These results are in agreement with specific serum IgE determinations and skin prick tests performed on the sample donor. Concordance tables were constructed to compare the HY·TEC BHR with skin prick tests and specific IgE tests for all of the activators used in the study. Similar comparisons were done using only the data from the two inhalant activators, Timothy grass and June grass pollen extracts. Table 1 shows the concordance with skin prick testing, specific IgE measurements, and the Biomerica RIA method. When only the results for the grass activators are considered, the concordances are >90%. This high degree of concordance with inhalant activators is in agreement with similar comparisons made by other investigators using different histamine assay methods.

View larger version (22K): [in this window] [in a new window]   Figure 1. Histamine release from a whole blood sample stimulated with June grass pollen extract (G8), cow milk extract (F2), and anti-IgE. Histamine in micrograms per liter is plotted against the log of the dilution factor for each activator and anti IgE. The highest concentration of anti-IgE used was 10 mg/L.

The BHR method presented here is rapid, precise, specific, fully automated, and has potential uses in a number of clinical situations. We conclude that the many advantageous features of the HY·TEC BHR Assay will make it attractive to clinical investigators as a powerful adjunct for the diagnosis and treatment of allergy and hypersensitivity.

References

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