Methods

The techniques described herein are, for the most part, used when histamine is analyzed by automated fluorimetry.

1. Blood is drawn into a plastic syringe, preferably using a butterfly infusion set with a needle size reflecting the desired volume required for the assay. Usually 1 mL of blood per reaction tube will provide total histamine levels of approx 20 ng with some variation occurring among donors.

2. Whole blood is immediately transferred to disposable 50-mL polypropylene centrifuge tubes, each containing 12.5 mL dextran, 5.0 mL 0.1 MEDTA, and 375 mg dextrose, which allows proper mixing for up to 20 mL of blood.

3. After sitting for 60-90 min at room temperature (23-25°C), or until a sharp interface develops between the plasma and erythrocyte pellet, the leukocyte-rich plasma layer is carefully removed using a disposable plastic pipet and transferred to a clean tube. The plasma is then centrifuged for 8 min at 110g in a refrigerated centrifuge at 4°C.

4. The upper plasma is carefully decanted (or aspirated) and discarded. The leukocyte pellet is gently resuspended and approx 40 mL (or twice the amount of blood used) of cold PAG buffer and centrifuged as previously listed.

5. The centrifugation and washing done in this manner are sufficient to remove most of the platelet contamination. It is sometimes useful to do one or more washes in PAG containing EDTA (~4 mM) because this reagent chelates calcium and prevents platelet clumping. However, it is important that the final wash be done in the absence of EDTA because histamine release requires calcium, and residual EDTA may prevent the reaction cascade. After the final wash, the cell pellet is resus-pended in PAGCM buffer.

6. Histamine release is performed in polystyrene test tubes (12 X 75 mm). Total reaction volumes usually range from 0.1 to 1.0 mL. In practice, most experiments are run in a total volume of 0.1 mL. Because the sample volume necessary for automated fluorimetry is 0.5-1.0 mL, reactions performed in smaller volumes are brought up to 1.0 mL with PAG buffer at the end of the reaction, prior to harvesting the supernatants for histamine analysis.

7. For simple histamine release experiments, 0.02 mL each of 5X concentrations of antigen, buffer, perchloric acid, or other reagents are added to test tubes and kept at 4°C.

8. The washed leukocytes in PAGCM buffer and the reaction tubes are then warmed separately by incubating them for 5 min in a 37°C water bath before adding 0.08 mL of the cell suspension to each tube.

9. The total histamine content (called "completes") is obtained by lysing the cells in a duplicate set of reaction tubes using perchloric acid at a final concentration of 1.6%. It is also important that tubes containing only cells and buffer (blanks) be included in each experiment as a measure of the spontaneous release of histamine (usually less than 5% of the total).

10. All conditions are tested in duplicate. During the reaction, the tubes are mixed by vigorously shaking the test tube racks every 15 min. At the end of 45 min at 37°C, reaction tubes are removed from the water bath and 0.9 mL of PAG buffer is added to each tube.

11. The tubes are then immediately centrifuged for 2 min at 1000g. Alternatively, the tubes can be centrifuged at 150g for 10 min.

12. The cell-free supernatants are decanted into 2 mL auto analyzer cups and stored at 4°C in plastic racks (Elkay Products Inc., Shrewsbury, MA). To prevent evaporation, samples should be frozen at -20°C if histamine cannot be analyzed within 2 wk. Alternatively, histamine can be measured using a histamine immunoassay.

5.2. Histamine Autoanalyzer Method (11)

The fluorometric technique remains the most popular method of assessing histamine release and is capable of consistently producing accurate and sensitive measurements of this histamine. Automation of this technique by Siraganian allowed analysis of up to 30 samples per hour (11); with more modern machines, as many as 45-60 samples can be analyzed per hour. The procedure is based on the extraction of histamine and its coupling with o-phthalaldehyde at a highly alkaline pH to form a fluorescent product. Samples tested must be relatively free of protein and other interfering compounds such as histidine. This is achieved by first extracting histamine into n-butanol from a salt-saturated, alkalinized solution before the condensation step with OPT, histamine is back-extracted into an aqueous solution of dilute HCl by adding heptane. The histamine-ophthaldehyde complex is stable at an acid pH, which increases the fluorescent intensity of the compound. The automated technique requires a sample volume of 0.6-1.0 mL and is capable of detecting histamine levels in the range of 0.5 to >100 ng/mL. Although the automated methodology for histamine measurement is preferable, similar results can be obtained manually with minor loss of sensitivity and precision. The assay is linear from 0.5 to 1000 ng/mL. The advantage of automated fluorimetry over other systems is its ability to rapidly process a large number of samples. However, this is the method of choice only when samples are obtained from studies using low-protein buffer systems such as the in vitro release of histamine from basophile or mast cell cultures.

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