Buffer for Basophil Histamine Release

1. 10X PIPES buffer, pH 7.4: 250 mM PIPES (Sigma), 1.10 M NaCl, and 50 mM KCl.

2. PAG buffer: 10% 10X PIPES, 0.003% human serum albumin (Calbiochem~Bering Corp., La Jolla, CA), and 0.1% D-glucose.

3. PAG-EDTA buffer: PAG containing 4 mM EDTA.

4. PAGCM buffer: PAG containing 1 mM CaCl2 and 1 mM MgCl2.

5. Isotonic Percoll: nine parts Percoll (Pharmacia, Piscataway, NJ) plus one part 10X PIPES.

5.3. Going With the Flow

In the last decade, basophiles have been found to upregulate cell surface markers (CD45, CD63, CD69, and CD203). CD63 is a transmembrane marker of basophiles, mast cells, macrophages, and platelets. The flow cytometry assay measures upregulation of CD63 concentration on basophiles after incubation of specific allergens (drug, food, or pollen) with heparinized blood supplemented with the cytokine IL-3. The relative upregulation of CD63 on basophiles is then determined by flow histograms after stimulation with allergen, compared to

Fig. 2. Representative increased expression of CD203c after fMLP or allergen challenge. Gated cells are presented on the basis of CD203c-IgE staining: before stimulation (upper left dot-plot), after fMLP challenge (upper right), and after latex challenge at two different concentrations (lower dot-plots). (Reproduced with permission from ref. 12.)

Fig. 2. Representative increased expression of CD203c after fMLP or allergen challenge. Gated cells are presented on the basis of CD203c-IgE staining: before stimulation (upper left dot-plot), after fMLP challenge (upper right), and after latex challenge at two different concentrations (lower dot-plots). (Reproduced with permission from ref. 12.)

appropriate controls. In one study of penicillin allergy, about 50% of the patients had a positive CD63 increase compared to controls.

CD203 is a neural cell surface biomarker found on IgE-bearing basophiles and mast cells. The advantage is that it is found primarily on basophiles. In a study of allergy to Hymenoptera (honeybee) venom, CD203 was found to be upregulated from 4.2- to 13.5-fold in patients relative to normal controls. The CD203 flow assay was found to confirm the presence of venom-specific IgE in 91% (20/22) of skin test positive patients who had clinical symptoms of honeybee sensitivity (12). In another study, latex-specific sensitivity was 63% and CD203 upregulation was better than CD63 (13) (Fig. 2).

A limitation of CD203 studies with specific allergens is the viability of basophiles in human blood. Some investigators estimate that basophile function may last 24-36 h in heparinized blood. For transport of blood to a distant reference laboratory, the ability to receive the sample within a short time is a limiting factor. Partial separation of basophiles from other blood products could prolong the basophile viability. With certain classes of specific allergen reactivity

such as drug and food allergy, where skin testing may be inadequate, flow cytometry may provide valuable clues for patients sensitive to these allergens.

5.4. Microarray-Bound Allergens

The availability of recombinant allergens and recent advances in chip technologies have led to the development of new systems for the identification of specific IgE antibody. The ordered array with micrometer spacing could accommodate more than 1000 allergens and controls bound to the microchip surface (14).

Jahn-Schmid and colleagues in Vienna studied IgE binding to recombinant ryegrass and birch allergens linked to silica chips. In a study of 51 sera from atopic patients, the results obtained with microarray technology correlated well with those of the ImmunoCAP assay (15). An advantage of microarray methodology is the relatively small serum specimen required (<40 pL). Fig. 3 shows the relative activity of microarray vs skin testing and immunoblot techniques. Because the current validated ImmunoCAP tests have over 400 specific allergen extracts available and each extract may contain five or more major allergens, validating and comparing hundreds of recombinant allergens will await future comparative studies.

6. Conclusion

In the last 40 yr we have witnessed amazing advances in the science of identifying allergy antibodies and in the understanding of the immunology of allergic diseases. Since the discovery of IgE and the development of recombinant technologies, the in vivo and in vitro diagnosis of allergic reactivity has rapidly progressed. In vitro techniques are currently approaching in vivo (skin test) accuracy and time frames. Skin tests take 15-20 min from application of the allergen extract to final reading of the wheal and flare endpoint. Because skin tests are usually performed with nonstandardized extracts, the results may vary from lot to lot of the tests performed. Also, many of the tests, especially for environmental allergens such as house dust, molds, and animal dander, may show positive wheal swelling and negative IgE-specific results with ImmunoCAP. This discordance could be due to greater sensitivity of the skin tests or false-positive results secondary to an irritant response. In any event, the ImmunoCAP and other IgE tests are highly specific for the allergen tested. The specific IgE

Fig. 3. Allergen microarray. (A) Scheme of environmental allergens spotted in triplicate in vertical columns. (B,C) Scanned images from a microarray chip-based IgE assay obtained with serum from an allergic (Fig. 2) (B) and nonallergic (Fig. 2) (C) individual. (D) Dot-blot IgE reactivity to the same allergens bound to nitrocellulose. (E) Skin reactivity to allergens and histamine (H6; wheal circled for visualization). (Reproduced with permission from ref. 14.)

tests may, however, be too sensitive: patients with class 3 or higher specific IgE (3.5-17.5 kU/L) may have food-related anaphylaxis (16) to important food allergens, whereas class 1 or 2 specific IgE (0.35-3.50 kU/L) to food allergens may not correlate with skin test positivity and or a history of food allergy (16). Thus, the relative clinical importance of skin testing, relevant clinical history and provocative tests, and specific IgE testing needs to be sorted out.

Current in vitro testing takes 2-4 h from the time serum is added to the analytical system until readout of the quantitative results. Patient time and discomfort can be minimized by a single venipuncture, and the time required to obtain results from in vitro tests may be similar to that for in vivo (skin) tests. It is not unreasonable to assume that allergy laboratory testing will take less time in the future. Laboratory testing is likely to be more accurate and nearly as quick, if not quicker than current skin test procedures. Currently, skin tests for drug allergy are not reliable because of the relative absence of purified drug metabolites that may be relevant in causing drug allergies. The lack of positive control subjects also limits the validity of drug testing. Flow cytometry may provide a highly specific and sensitive methodology to test relative drug sensitivity. In the future, a combination of biological testing (skin tests and or provocative tests) and specific IgE testing should provide important information for therapeutic intervention. However, the new technologies require careful validation to develop paradigms that encompass relevant clinical scenarios.

7. Notes

1. Date and initial all reagents upon opening. Each container should be labeled with substance name, lot number, date of preparation, expiration date, and any special storage instructions.


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Food Allergies

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