326 DIFGO MANUAL 



be overcome by using clean, dry, sterile glassware in which to prepare and store 

 the emulsion and in which to perform the test. The addition of sodium ethyl 

 mercuri-thiosalicylate (Merthiolate), in a concentration of 1:10,000 to the test 

 antigen emulsion and to the saline used for diluting the serum, has given good 

 results. The antigen emulsion should be stored at refrigerator temperature, 

 2-6° C., when not in use. 



Performance of the Hanger Flocculation Test 



The Hanger flocculation test is performed by adding 1 ml. of the aqueous 

 lipoid emulsion to a test tube (preferably a centrifuge tube) containing 0.2 ml. 

 of the patient's serum diluted with 4 ml. of 0.85 per cent sodium chloride. After 

 thorough shaking and stoppering with cotton, the tube is allowed to stand undis- 

 turbed in the dark at room temperature ( 20-25 °C.) and a notation is made at 

 the end of 24 and 48 hours as to the amount of flocculation and precipitation 

 that has taken place. With normal human sera the emulsion remains as a stable 

 homogeneous suspension, but with sera from patients with diffuse hepatitis the 

 lipoid material tends to flocculate or precipitate to the bottom of the tube. 

 A + + + + reaction indicates a complete precipitation leaving the supernatant 

 liquid water-clear. Gradations of the reaction between negative and -f ++H- 

 are designated in terms of +, ++ and +++• No test should be regarded as 

 negative until 48 hours have elapsed without flocculation. 



Neefe and Reinhold^^ discovered that the cephalin cholesterol flocculation 

 reaction was influenced by light and that this photosensitivity gave rise to falsely 

 positive reactions with normal sera. Hanger^^ and Mateer^^-^^ confirmed Neefe's 

 and Reinhold's observations. Their findings suggested that the test antigen 

 emulsion and the serum should be protected from light and that the serum- 

 saline-antigen test mixture should be incubated in the dark at room temperature 

 ( 20-25 °C.). Higher or lower incubation temperatures are not indicated. The 

 patient's serum should not be diluted until the test is to be performed. 



A rapid Cephalin Cholesterol Flocculation Test using centrifugation has been 

 described by Moloney, Donovan and Whoriskey.-*^ While less sensitive than the 

 regular Hanger test, the centrifugation technique is rapid, permitting the reading 

 of the test within a few minutes. Saifer and Zymaris-^ have described an easily 

 performed accurate photometric microprocedure for the quantitative determina- 

 tion of electrophoretically pure gamma globulin (Cohn's Fraction H) in the 

 range of 100 to 1,000 micrograms of protein using Bacto-Cephalin Cholesterol 

 Antigen in the presence of Hayem's solution. 



Bacto-Cephalin Cholesterol Antigen is supplied in 5 ml. bottles in packages 

 of 6. Each bottle or unit is sufficient for the preparation of 5 ml. of stock ether 

 antigen and 150 ml. of the final test antigen. 



1 Trans. Assoc. Am. Physicians, 53:148:1938. ^Science, 99:20:1944. 



3 J. Clin. Invest., 18:261:1939. i3 j. Qjn. Invest., 24:616:1945. 



3 Arch. Surgerv-, 43:231:1941. -* J. Clin. In\est., 24:296:1945. 



* Am. J. Digestive Diseases, 8:421:1941. " j. Am. Med. Assoc, 133:909: i947> 



^ J. Clin. Invest., 20:241:1941. i^ Gastroenterology, 8:52:1947. 



^ Am. J. Digestive Diseases, 9:13:1942. "Science, 100:83:1944. 



^Surgery, 11:732:1942. 1* Personal Communication, 1944. 



8 New Orleans Med. Surgical J., 95:60:1942. ^^ Personal Communication, 1944. 



9 Am. J. Clin. Path., 13:383:1943- 20 Am. J. Clin. Path., Tech. Supp., 18:568: 

 1° J. Am Med. Assoc, 121:723:1943. 1948. 



^ Am. J. Med. Sci., 205:90:1943. 21 j, Clin. Investigation, 31:1:1952. 



