LYSINS 161 



It must be emphasized that precision of measurement is an absolute 

 requirement for success; the activating power of complement for 

 hemolysin does not follow the law of multiple proportions it is rather 

 an inverse ratio, as Noguchi 1 has pointed out. Relatively less com- 

 plement is required to induce complete hemolysis in a system contain- 

 ing four units than is required for a system containing but a single 

 hemolytic unit. 



The serum to be examined for specific antibodies by the method 

 of complement-fixation must be sterile and free from hemoglobin. 

 The products of bacterial growths in serum may be anticomplementary 

 and the presence of hemoglobin in serum also tends to inhibit hemolysis. 

 Blood, therefore, should be withdrawn with aseptic precautions from 

 the median basilic vein of the patient into sterile test-tubes, and either 

 centrifugalized at once and the serum removed from the clot, or 

 placed in an inclined position in a cool place until the serum has 

 separated. The serum must be clear 2 and free from erythrocytes or 

 hemoglobin. 3 It is inactivated at 54 to 55 C. for half an hour in a 

 water-bath. 4 



The Technic of the Test. It is essential that the hemolytic system 

 erythrocytes, hemolysin, complement be standardized daily. 

 Varying amounts of hemolysin are added to constant amounts of 

 erythrocyte suspension and complement, as outlined above. A 

 known positive syphilitic serum and a known negative syphilitic 

 serum, together with suitable controls, must be examined along with 

 the unknown serum to be tested. 



The following reagents are required : 



1. Sterile physiological salt solution. 



2. Fresh guinea-pig serum (complement) add 0.1 c.c. to each tube. 



3. Five per cent, suspension of washed sheep erythrocytes in normal 

 salt solution use 1 c.c. to each tube. 



4. Hemolysin (amboceptor) use twice the hemolytic unit (the unit 

 must be determined daily). 



5. Known syphilitic serum inactivate and use 0.2 c.c. 



6. Known normal (non-syphilitic) serum, inactivated use 0.2 c.c. 



7. The serum to be tested inactivate, use 0.2 c.c. 



1 Serum Diagnosis of Syphilis. 



2 Blood is best obtained early in the morning, before the patient has eaten; blood 

 obtained at the height of digestion frequently contains fats which make the serum 

 turbid. 



3 Small amounts of blood, yielding a few drops of serum, may be obtained from the 

 finger-tip or the lobe of the ear. Massage must not be practised, for there is danger of 

 damaging erythrocytes with the liberation of hemoglobin. 



4 Noguchi, Serum Diagnosis of Syphilis, states that inactivation at 54 C. should be 

 practised the higher temperature weakens the reactive substance somewhat. 



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