IntroductionThe biological action of ultra high dilutions is controversial [1,2]. Inhibition of anti-IgE induced basophil degranulation by successive histamine dilutions is of interest, as it studies a chemically defined compound (histamine) which exerts a negative feed back effect via the histamine H 2 receptor. The biological activity is measured using the human basophil degranulation test, which is relatively simple to perform and does not require specialised equipment. Inhibition of basophil degranulation was observed with histamine dilutions ranging between the 15 th and 19 th centesimal dilutions. Since most data were originally obtained from only one laboratory, this study aimed to verify these results in a multi-centre trial.
Materials and methods
LaboratoriesFour independent laboratories agreed to participate in the trial. Prior to the start of the trial, participants underwent a training period and their results were verified by the French laboratory.
Study protocolThe study was co-ordinated in Brussels and all histamine dilutions were coded randomly by the coordinator, who did not perform any of the tests. The dilutions were prepared in 3 separate laboratories, which did not participate in the trial. The samples were then posted to the trial laboratories. All reagents, including antibodies, histamine, staining solutions, microtitre plates etc., were from the same source. Data were returned to the co-ordinator and analysed independently by a biostatistician, who was not involved in any other part of the trial.
Preparation of histamine dilutionsHistamine hydrochloride (50 mg, Sigma) was dissolved in distilled water (5 ml), diluted (1/10, v/v) in distilled water and vortexed for 15 s (full speed). To obtain the dilutions for the trial, this solution was serially diluted (1/100 v/v) up to 19 times, always with vortexing as described. The dilutions 15, 16, 17, 18 and 19 were coded by the coordinator. In parallel, dilutions of distilled water alone were prepared in an identical manner and coded (controls). On receipt of the dilutions, each participating laboratory stored them at 4°C. Prior to use, the solutions were made isotonic by dilution (1/10 v/v) in HEPES buffer (NaCl 127 mM, KCl 5 mM, HEPES 20 mM, pH 7.4).
Human basophil degranulation testThe methods for the selection of volunteers, preincubation of cellular suspensions with the test dilutions and anti-IgE induced basophil degranulation have been described previously [3]. Cell suspensions (250 ml) were mixed with the test dilution (250 ml) and incubated at room temperature for 30 min. After mixing (3 s, medium vortex speed), aliquots (20 ml) were placed in the wells of a microtitre plate and mixed with anti-IgE (polyclonal anti-IgE affinity purified ATAB, USA, 20 ml; 1, 0.2, 0.04 mg/ml). The plates were then covered with a sealer tape (Dynatech Laboratories) and incubated for 30 min at 37°C. Thereafter, alcian blue (100 ml) was added to each well. Stained basophils (not activated) were counted using a haemocytometer (Fuchs Rosenthal). Approximately 80...