The technique, while exceedingly simple, offers many chances for errors and individual variations so that we have thought it well to give directions and cautions at some length.
Bruck’s[[154]] technique is described as follows: “The test is made with 0.5 cc. clear serum in a test tube, to which is added 2 cc. of distilled water, and the whole shaken. Then, with a precision pipette, 0.3 cc. of the ac. nitr. purum of the German pharmacopeia is added and the whole thoroughly shaken and then set aside at room temperature for ten minutes. Then 16 cc. of distilled water at room temperature is added, and closing the tube with the finger, it is shaken up and down three times carefully, not vigorously enough to make it foam. This is repeated ten minutes later, and the tube is then set aside for half an hour. By this time the precipitate is entirely dissolved in the tube with the normal serum, while the syphilitic serum shows a distinct, flocculent turbidity. In two or three hours, or better still, in twelve hours, the gelatinous and characteristic precipitate is piled up on the floor of the test tube.”
The acid is prepared by diluting the Acidum nitricum of the U. S. P. (Sp. gr. 1.403) with distilled water until the hydrometer shows the specific gravity 1.149, which corresponds to the nitric acid of the German pharmacopeia, but since this requires a special hydrometer, a simpler method is to make a 25 per cent solution of the Acidum nitricum, which will give about the proper specific gravity.
The serum is obtained by allowing 10 cc. of blood to stand at room temperature for an hour, and then centrifuging. Serum that has stood for some time may be used as well as the fresh, and even bloody serum does not seem to confuse the results to any great degree. The serum gives the same results with or without inactivation. Post mortem blood gave results as constant as that obtained during life, in the few cases that we had in this series. But the reaction may be influenced markedly by the size of the test tubes. We have found that the 13×1.9 cm. is the most favorable size.
When one first thinks of this test it appears very simple and probably somewhat crude as a chemical reaction, but there are certain precautions that must be observed, and several hundred normal and syphilitic sera should be tried before the investigator can feel that he has a refined routine technique. There is the personal equation which must be watched, for here is probably the greatest source of error, and readily explains why two different persons get widely varying results with the same sera if they have done only a few dozen tests. We must take it for granted that the reaction is a quantitative one, where some positive reactions may differ only slightly from the normal non-syphilitic, and, furthermore, any normal serum may be made to give a positive reaction, and almost any positive serum be made to give a negative by improper manipulation at some point in the test. There are as many places for error to creep in as there are steps in the process. Bruck has omitted many details in his publication, which allow personal variations, and so we have tried to develop a routine process that will eliminate as many of these as possible.
We shall here attempt to explain the methods which we have found most satisfactory and at the same time indicate the places where error is likely to occur. The 0.5 cc. of serum is added to 2 cc. of distilled water, and shaken thoroughly. Now add slowly exactly 0.3 cc. of acid from a precision pipette, care being taken it does not flow down the side of the tube. The tube should be shaken gently while the acid is being added, for this prevents the formation of a flocculent precipitate in normal serum which is difficult to dissolve later. After the acid is added shake each tube gently to make sure that these flakes do not persist. It is difficult to shake each tube in exactly the same manner, as must be done if we expect uniform results.
The first 250 tests of this series were made by allowing the tubes to stand for ten minutes as Bruck advocates. Then we found that practically all sera gave a positive reaction if allowed to stand 15–20 minutes, and so in the other tests of the series an attempt was made to make the reaction more sensitive by allowing the tubes to stand only 6–7 minutes. During this time the tubes should be shaken gently once or twice. The manner in which the 16 cc. of water is added also influences the reaction. If allowed to flow freely in upon the precipitate, the positive may be forced into solution as well as the negative. Both pipette and tube should be slanted and the water allowed to flow down the side of the tube without disturbing the precipitate. If all has gone well up to this point, we may see a marked difference between the normal and syphilitic precipitates, in that the normal will begin to go into solution at once, thus clouding the water, while a positive precipitate will be composed of large flakes which show little or no tendency to go into solution or cloud the water above. It must be remembered that the most flocculent positive precipitate will go into solution if the fluid is splashed or shaken too hard while the tube is being inverted. If any doubt as to the character of the precipitate now exists, it may be allowed to stand ten minutes longer, and again inverted as before, or even repeated several times during the next hour or two. We see no reason why the tubes should be left to stand over night, for during this time a precipitate usually settles in the normal tubes. This, however, differs from the syphilitic precipitate in that it is still finely granular and goes back into solution readily when the tubes are inverted.
In view of these possible grounds for error, it is only logical to run controls of known positive and known negative sera along with each group of unknown bloods, and even then certain tubes will seem doubtful, in which event the test should be repeated with added precaution to see if a definite positive or negative reaction may be obtained.
In the last tests of this series we seemed to aid the reaction by rendering the serum-water solution alkaline by one or two drops of 10 per cent potassium hydroxide before the acid was added. The positive sera have a larger precipitate, while the normal seem to dissolve more readily.
| Table I | |||
| Syphilis: nervous system involved. | |||
| General Paresis | Wassermann and Bruck agree | positively | 47 |
| Wassermann and Bruck agree | negatively | 7 | |
| Wassermann and Bruck at | variance | 10 | |
| Tabes Dorsalis | Wassermann and Bruck agree | positively | 3 |
| Cerebrospinal | Wassermann and Bruck agree | positively | 8 |
| Wassermann and Bruck agree | negatively | 3 | |
| Juvenile Paresis | Wassermann and Bruck agree | positively | 1 |
| Summary: | Wassermann and Bruck agree | positively | 59 |
| Wassermann and Bruck agree | negatively | 10 | |
| Wassermann and Bruck at | variance | 10 | |
| Table II | |||
| Syphilis: nervous system not involved. | |||
| Syphilis | Wassermann and Bruck agree | positively | 12 |
| Wassermann and Bruck at | variance | 5 | |
| Congenital Syph. | Wassermann and Bruck agree | positively | 3 |
| Wassermann and Bruck agree | negatively | 2 | |
| Summary: | Wassermann and Bruck agree | positively | 15 |
| Wassermann and Bruck agree | negatively | 2 | |
| Wassermann and Bruck at | variance | 5 | |
| Table III | |||
| Non-syphilitic: Wassermann reaction negative. | |||
| Doubtful or positive Bruck | 86 | ||
| Bruck test negative | 216 | ||
| Total for three groups: | |||
| Wassermann and Bruck agree | positively | 74 | |
| Wassermann and Bruck agree | negatively | 230 | |
| Wassermann and Bruck at | variance | 101 | |