The urine specimen is treated with 1 or 2 drops of 33 per cent acetic acid to bring the pH down to 4.8 to 5.0, centrifuged clear, and diluted 1:1 with water. The diluted urine is layered carefully over concentrated HCl in a test tube. If Bence-Jones protein is present, it will be precipitated by the HCl and will form a fine or heavy ring at the interface of urine and HCl. The absence of a ring definitely rules out the presence of Bence-Jones protein. A positive test should be confirmed by the heating test because occasionally albumins may also precipitate, if present in large quantity. Protein, as evidenced by a positive sulfosalicylic acid test, must be present. If protein is negative, any HCl interface ring observed in the Bradshaw test is not due to Bence-Jones protein, but probably has formed from the reaction of the HCl with some unknown materials in the urine. The Bradshaw test serves as a useful screening test to determine which specimens should be subjected to the confirmatory heating test.
Heating Test (Jacobson and Milner)
The urine specimen should be fresh or refrigerated under toluene. The pH is checked with pH paper, and adjusted to the pH range 4.8 to 5.0 with 33 per cent acetic acid. The urine is then centrifuged to remove any turbidity (filtration is avoided). If a previous qualitative test for protein was over 3+, the original urine specimen is diluted with normal urine (fresh, free of protein) until it tests 2+ or less. Five to seven ml. of the prepared urine are transferred to a test tube, which is immersed in a beaker of water, warming over a slow-heating hot plate or gas burner. A thermometer is immersed in the urine, and the beaker is heated slowly, with the temperature on the thermometer being watched closely. If Bence-Jones protein is present, turbidity will begin to form in the urine at about 42 degree to 47 degree of Celcius, indicating that coagulation (precipitation) is beginning. The volume of precipitate will increase until the temperature reaches 60 degree of Celcius. If no precipitate is evident at this point, no Bence-Jones protein is present. Any precipitate forming at temperatures above 57 to 60 degree of Celcius is derived from urinary albumins and globulins.
After the Bence-Jones precipitate has formed, the beaker is rapidly heated to 100 degree of Celcius. As the temperature of the urine approaches 100 degree of Celcius the precipitate should dissolve completely or show a diminution in volume. If the precipitate is still present at temperatures of 95 to 98 degree of Celcius, the tube contents should be filtered through a hot funnel. The funnel, filter paper, and receiving tube may be heated either in a drying oven or by passing boiling hot water through them several times. As the filtrate of dissolved Bence-Jones proteins cools to 55 to 60 degree of Celcius, the protein should begin to precipitate out again. If only Bence-Jones protein is present, the test is clear-cut. If albumins are also present, the results of the test may, at times, be equivocal. Low temperature coagulation of the protein is observed, but there is none or only little reprecipitation on cooling.
After the Bence-Jones precipitate has formed, the beaker is rapidly heated to 100 degree of Celcius. As the temperature of the urine approaches 100 degree of Celcius the precipitate should dissolve completely or show a diminution in volume. If the precipitate is still present at temperatures of 95 to 98 degree of Celcius, the tube contents should be filtered through a hot funnel. The funnel, filter paper, and receiving tube may be heated either in a drying oven or by passing boiling hot water through them several times. As the filtrate of dissolved Bence-Jones proteins cools to 55 to 60 degree of Celcius, the protein should begin to precipitate out again. If only Bence-Jones protein is present, the test is clear-cut. If albumins are also present, the results of the test may, at times, be equivocal. Low temperature coagulation of the protein is observed, but there is none or only little reprecipitation on cooling.
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