Observations on the sensitivity of the haemolytic system in complement-fixation tests.

The development, in recent years, of optimalproportions techniques has made possible a great increase in the sensitivity of complement-fixation tests, without any loss of specificity. This is particularly true of the Wassermann reaction. Since 1949 Price (1949a, 1949b, 1950a, 1950b) has shown that the performance of the test is greatly improved when the reagents are used in optimal amounts. His technique is applicable to any complement-fixation test, and gives highly reproducible results, unaffected by changes in the batches of reagents, since all antigens, for example, are titrated before issue for use at the optimal dilution. Standardization of results is therefore possible not merely from day to day in any one laboratory but between all departments using Price's (Whitechapel) technique. Quantitative tests have thus acquired more than a purely local significance, and are of much greater value in the control of therapy. Price (1949a) has stressed that optimal-proportions techniques can only be used when the complement is stable, and has paid tribute to Richardson's (1941) work on the preservation of complement. Price has dealt with the antigen (1950a), complement (1949b), and serum dilutions (1950b), and Price and Wilkinson (1947) with the strength of the red-cell suspension, in the Wassermann reaction. Other variables are the method of sensitization of the haemolytic system, and the time interval between sensitization and use. Some serologists insist on the agitation of the cells during sensitization, while others consider this unimportant. The haemolytic system may be used immediately after sensitization, after standing at room temperature or at 40 C. for several hours, or even after overnight storage in the refrigerator. Alternatively, cells and amboceptor may be mixed and then kept at 40 C. until half an hour before use sensitization is completed in the 370 C. water-bath. The American custom of adding cell suspension and haemolysin separately during the actual performance of the test, as in Kolmer's method, does not appear to obtain in this country. With relatively insensitive techniques, employing two or more M.H.D. of complement as the test dose, some latitude in the preparation and use of the haemolytic system is permissible. But with more critical techniques, such as Price's in which the complement dose (at 14 M.H.D.) is near the theoretical minimum, considerable differences may result from minor variations in technique, especially in quantitative tests. In most departments using the Whitechapel technique it is customary to make screen tests in the morning and quantitative tests, as indicated, the same afternoon. The haemolytic system is usually left at room temperature in the interval. Such was our practice initially. It was, however, soon observed that sera giving weakly positive results, i.e., showing partial haemolysis, in the morning were not infrequently found negative when the quantitative test was performed in the afternoon. Series of parallel quantitative tests were, therefore, made to study the effects of variations in the method of preparation, storage, and the time lag before use of the sensitized cell suspension, in both the Wassermann reaction and the gonococcal complement-fixation test.