THE TECHNIC OF THE KOLMER COMPLEMENT FIXATION TESTS FOR SYPHILIS EMPLOYING ONE-FIFTH AMOUNTS OF REAGENTS JOHN A. KOLMER

THE TECHNIC OF THE KOLMER COMPLEMENT FIXATION TESTS FOR SYPHILIS EMPLOYING ONE-FIFTH AMOUNTS OF REAGENTS JOHN A. KOLMER WITH THE TECHNICAL ASSISTANCE OP ELSA R. LYNCH Department of Bacteriology and Immunology of Temple University School of Medicine and the Research Institute of Cutaneous Medicine, Philadelphia, Penna. It has been found possible and highly satisfactory to conduct the Kolmer quantitative and simplified complement fixation tests for syphilis with one-fifth amounts of serum or spinal fluid and other reagents. This has proven important from the standpoint of economy with special reference to complement. Thus 17 to 18 cc. of guinea pig serum complement is ordinarily required for the conduct of 100 regular quantitative tests but only 5 cc. for the one-fifth technic and whereas about 6 cc. is required for the conduct of 100 regular simplified tests, only 2 cc. is required by the one-fifth method. In other words, the amounts of complement required in the latter are about three times less in both procedures, which is an important item and especially in those laboratories required to conduct large numbers of tests. For example, 10 cc. of complement is ordinarily sufficient for the conduct of 500 simplified tests by the one-fifth technic in State Laboratories, the military and naval Services and other laboratories required to conduct large numbers of tests. Under the circumstances it is advisable in the interests of economy for laboratories to maintain colonies of selected, large, male guinea pigs, varying in number according to requirements for complement, for the purpose of removing 4 or 5 cc. of blood from the heart of individual animals under light ether anesthesia, at varying intervals, with a 5 cc. syringe fitted with a short needle, gage 20. This has been our custom during the past several years with very satisfactory results as only very occasionally is an animal lost by injury after some experience with the technic has been acquired. Furthermore, while 0.6 cc. of serum and [1.5 cc. of spinal fluid are required in the regular quantitative tests, only 0.2 cc. serum and 0.3 cc. of spinal fluid are required in the one-fifth method and while 0.4 cc. serum and 1.0 cc. of spinal fluid are required for the regular simplified tests, only 0.2 cc. of either is required in the latter. This facilitates the use of multiple serological tests when only small amounts of serum or spinal fluid are available. Likewise there is an important economical reduction in the amount of Kolmer antigen employed. For example, 1 cc. is required for the conduct of 240 regular quantitative or 1200 regular simplified tests when the optimum dose is 0.5 cc. of 1:600 dilution, whereas 1 cc. suffices for 1200 quantitative or 6000 simplified tests by the one-fifth technic. This economy also applies to antisheep hemolysin. 109

110 JOHN A. KOLMER For example, when the unit is 0.5 cc. of 1:5000 dilution (corresponding to 0.5 cc. of 1:2500 for two units) 2 cc. of a 50 per cent dilution of serum in glycerol is sufficient for about 800 regular quantitative and 2500 regular simplified tests but ordinarily sufficient for about 4000 quantitative and 12,500 simplified tests by the one-fifth method. Since the results of comparative quantitative and simplified tests with sera and spinal fluids by the one-fifth method herewith described have yielded practically identical results in regard to both sensitivity and specificity as the regular procedures, it is apparent that the economy effected insofar as the reagents are concerned greatly facilitates the routine use of a complement fixation test in the serological diagnosis of syphilis. There is, however, no reduction in the amount of time and skill required as otherwise the technic is exactly the same as that of the regular procedures. Indeed, even greater care is required as the amounts of reagents are reduced to volumes of 0.1 or 0.2 cc. whereas one of the important principles incorporated into the technic of the regular procedures has been the use of reagents diluted to volumes of 0.5 to 1.0 cc. in order to-reduce to a minimum errors in pipetting. Furthermore, since the total volume of the one-fifth method is 0.7 cc. instead of 3 cc. as in the regular methods, the reading of positive reactions is not quite as easy as in the latter and especially the finer distinctions of positive reactions as differentiation between + and + + or between + + and + + + but, after all, this does not sufficiently interfere with or reduce the accuracy of readings from the practical standpoint. TECHNIC OF THE ONE-FIFTH METHODS As previously stated, the only changes involved are those involving the amounts of reagents employed. In other words, the methods for cleaning glassware, the preparation of saline solution, sheep corpuscles, antisheep hemolysin, complement, antigen, serums and spinal fluids are identical and exactly the same as in the regular quantitative and simplified tests 1 ' 8. The same applies to the manner of setting up the tests as well as to the primary and secondary incubations and the reading, recording and reporting of the reactions. While the changes involve the use of one-fifth amounts of serum and spinal fluids there is no need, however, for changing report blanks as the same may be employed as for the regular quantitative and simplified tests. (a) Test tubes and pipettes. Since the total volume in both the quantitative and simplified tests with sera and spinal fluids is 0.7 cc. instead of 3.0 cc, it is necessary to use smaller test tubes, those measuring 7.5 cm. in length and 1 cm. in inside diameter, as employed in the Kahn tests, being recommended. Accurate 1 cc. pipettes graduated in 0.01 cc. to the tips are employed and in the interests of accuracy, for the three required for measuring hemolysin, corpuscles and antigen, pipettes certified by the Bureau of Standards are recommended as well as an additional one (2 cc. graduated in 0.1 cc.) for measuring complement. It is also advisable to use wire racks adapted to the smaller test tubes employed although this is not absolutely essential. (b) Antigen. Plain Kolmer C.L. antigen or that re-enforced with acetone insoluble lipoids (3), which is preferred, is employed. It is diluted in exactly the same manner as for the regular quantitative or simplified test. The titration is also exactly the same as for the latter 1 ' 2 except that the dose is 0.1 cc. instead of 0.5 cc. of the optimum dilution. In other words, Kolmer antigen dispensed for use in dose of 0.5 cc. of a given dilution (usually 1:600 or higher) for the regular tests is used in the same dilution in dose of 0.1 cc. or onefifth the amount.

MICBO-KOLMEB TEST 111 (c) Sera and spinal fluids. Since the routine removal of natural antisheep hemolysin from sera by absorption with washed sheep corpuscles slightly increases the sensitivity of the quantitative reactions, this procedure is recommended when time and working conditions permit. However, it is not required for the conduct of the simplified tests and especially in State Laboratories where large numbers of tests are conducted. As in the regular quantitative test, sera are heated in a water bath at 55 C. for 15 to ZO minutes for the one-fifth quantitative test. This is also recommended for the one-fifth simplified test in order to reduce to a minimum the destruction of complement fixation antibody in the interests of sensitivity, but in those laboratories conducting the flocculation tests as well, the sera may be heated for 30 minutes at 55 to 56 C. in order to be prepared for both tests in a single operation. As is well known, sera are sometimes anticomplementary even after heating at 55 to 56 C. in a water bath for 30 minutes and especially if heavily contaminated with bacteria, chylous or heavily hemolysed. As stated later, the reactions in both the quantitative and simplified tests by the one-fifth methods can be frequently read and interpreted with safety when the sera are only slightly anticomplementary but otherwise they should be prepared by a modification of the Sachs method described later. Spinal fluids do not require heating unless kept for more than 3 days at room temperature (as during shipment) when heating at 55 to 56 C. for 10 minutes is advisable for the removal of thermolabile anticomplementary substances. (d) Hemolysin titration. This is conducted each time the tests are employed as follows: (1) In a series of 10 small test tubes place 0.1 cc. amounts of the same ten dilutions of antisheep hemolysin (1:1000 to 1:16,000) as employed in the regular tests. (2) To each tube add 0.1 cc. of a 1:50 dilution of complement instead of 0.3 cc. of a 1:30 dilution as employed in the regular tests. The 1:50 dilution may be prepared by diluting 0.1 cc. of complement serum with 4.9 cc. of saline solution which gives sufficient for this titration as well as for the complement titration. (3) To each tube add 0.1 cc. of a 2 per cent suspension {thoroughly mixed) of washed sheep corpuscles. (4) To each tube add 0.4 cc. of saline solution to give a total volume of 0.7cc. (5) Place in a water bath at 37 C. for one hour and read the unit which is 0.1 cc. of the highest dilution that gives complete hemolysis. (6) Two units are used in the complement titration and in the complement fixation tests. For example, if the unit equals 0.1 cc. of 1:6000, two units equal 0.1 cc. of 1:3000. As in the regular tests, only high titer hemolysin should be employed and in our laboratory no difficulty is experienced in preparing hemolysin by the immunization of rabbits giving a unit of 0.1 cc. of 1:4000 or higher. Keep the hemolysin and corpuscles in suspension in the refrigerator when not in use. (e) Complement titration. This is also conducted each time the tests are employed as follows: (1) In a series of five small test tubes place 0.1, 0.2, 0.3, 0.4 and 0.5 cc. of the same 1:50 dilution of complement serum as employed in the hemolysin titration. (2) To each tube add 0.1 cc. of the proper dilution of antigen (optimum dose) instead of 0.5 cc. as in the regular tests. (3) Add saline solution to bring the total volume in each tube to approximately 0.5 cc. (0.3, 0.2 and 0.1 cc. in the first three tubes respectively). (4) Mix and place the tubes in a water bath at 37 C. for one hour. (5) Add 0.1 cc. hemolysin (2 units) and 0.1 cc. of a 2 per cent suspension (thoroughly mixed) of washed sheep corpuscles to each tube. (6) Mix, place in a water bath at 37 C. for one hour and read the exact unit which is the smallest amount just giving complete sparkling hemolysis (usually 0.1 to 0.3 cc. of 1:50 dilution). (7) For conducting the complement fixation tests two exact units are employed instead of two full units as in the regular tests. This dose of two exact units should be contained in 0.2 cc. of a proper dilution prepared as follows: Exact unit 0.1 cc. 0.2 cc. 0.3 cc. 0.4 cc. 0.5 cc. Two exact units 0.2 cc. 0.4 cc. 0.6 cc. 0.8 cc. 1.0 cc. Dose of two exact units 0.2 cc. of 1:50 dilution 0.2 cc. of 1:25 dilution 0.2 cc. of 1:17 dilution 0.2 cc. of 1:12$ dilution 0.2 cc. of 1:10 dilution

112 JOHN A. KOLMER In other words, the total amount of complement required for the complement fixation tests should be calculated and sufficient dilution prepared so that each dose of 0.2 cc. carries two exact units. It is always advisable to dilute complement serum with cold saline or egg albumin saline solution; undiluted and especially diluted complement should always be kept in a refrigerator when not in use. Egg albumin. As shown by Boerner and Lukens 4, and confirmed by Kolmer and Lynch 5, it is advisable to add egg albumin to all tests employing spinal fluid for the prevention of nonspecific prezone reactions. This is also advisable if and when such reactions are being observed in quantitative serum tests although not required in simplified tests. The egg albumin is prepared as follows: (a) Break a fresh egg and separate the white from the yolk. (6) Pick out heavy particles or filter through one layer of gauze, (c) Measure and beat briefly before adding 10 cc. to 90 cc. of sterile saline (10 per cent solution). This solution may be kept in a refrigerator for a week without the addition of a preservative, (d) For use the dose is 0.2 cc. 'The quantitative complement fixation test. 1. For each serum (a) Arrange six test tubes and place in them the following amounts of saline solution respectively: 0.8, 0.2, 0.2, 0.2, 0.8 and 0.1 cc. (6) To tube No. 1 add 0.2 cc. of inactivated serum. Mix by drawing up in the pipette several times and transfer 0.2 cc. to No. 2 and No. 6 (serum control); discard 0.4 cc. (c) Mix No. 2 and transfer 0.2 cc. to No. 3; mix No. 3 and transfer 0.2 cc. to No. 4; mix No. 4 and transfer 0.2 cc. to No. 5; mix No. 5 and discard 0.8 cc. This leaves 0.2 cc.'in each of the first 5 tubes carrying the following amounts of serum: 0.04, 0.02, 0.01, 0.005 and 0.001 cc; No. 6 (the serum control) carries 0.3 cc. or 0.04 cc. of serum since it receives no antigen, thereby making the total volume in all tubes the same (0.7 cc.) when the test is finished. The amounts of serum, therefore, are one-fifth those employed in the regular quantitative test. 2. For each spinal fluid: (a) Arrange six test tubes and place in the first five the following amounts of saline solution respectively: 0.3, 0.2, 0.2, 0.2 and 0.2 cc. (6) To tube No. 1 add 0.3 cc. of spinal fluid. Mix by drawing up in the pipette several times and transfer 0.2 cc. to No. 2 and No. 6 (the control). (c) Mix No. 2 and transfer 0.2 cc. to No. 3; mix No. 3 and transfer 0.2 cc. to No. 4; Mix No. 4 and transfer 0.2 cc. to No. 5; mix No. 5 and discard 0.2 cc. This leaves 0.2 cc. in each tube carrying the following amounts of spinal fluid: 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.1 cc. (control) corresponding to one-fifth the amounts employed in the regular quantitative test. 3. To the first five tubes of each set of serum or spinal fluid add 0.1 cc. of a diluted antigen carrying the proper dose. 4. Allow to stand for 10 to 30 minutes at room temperature. If a longer interval elapses place the racks in a refrigerator. 5. Add 0.2 cc. complement (2 exact units) to each tube. In case of spinal fluid tests also. add 0.2 cc. of a 10 per cent solution of egg albumin in sterile saline solution. The same applies to tests employing serums if and when nonspecific prezone reactions are being observed although this is not otherwise required. 6. Include the following controls: (a) Antigen control containing 0.1 cc. diluted antigen, 0.2 cc. saline solution and 0.2 cc. diluted complement (carrying 2 exact units). If spinal fluids are being tested set up a second control in the same manner with the addition of 0.2 cc. of 10 per cent solution of egg albumin; the same applies if egg albumin is being used in the serum tests. (b) Hemolytic system control containing 0.3 cc. saline solution and 0.2 cc. diluted complement (carrying 2 exact units). (c) Corpuscle control containing 0.6 cc. saline solution. (d) Controls with positive and negative sera are advisable. 7. Mix the contents of each tube by gentle shaking and place in the refrigerator at 6 to 8 C. for 15 to 18 hours.

MICRO-KOLMER TEST 113 8. Place the tubes in a water bath at 37 C. for 10 minutes (not longer). 9. To all tubes, except the corpuscle control, add 0.1 cc. of hemolysin (carrying 2 units) and to all tubes add 0.1 cc. of 2 per cent corpuscle suspension (shaken up). 10. Mix the contents of each tube by gentle shaking and place in a water bath at 37 C. Watch the serum, antigen and hemolytic system controls and 10 minutes after these show complete hemolysis (usually 25 to 30 minutes) remove the tests and make the readings. In case of those tests in which the controls are not completely hemolysed continue the water bath incubation for a total of one hour, which frequently permits the reading of tests with serums and spinal fluids which are slightly anticomplementary. 11. Read the degree of inhibition of hemolysis and record for each tube as: (complete hemolysis); + (25 per cent inhibition recorded as 1); + (60 per cent inhibition recorded as 2) + + + (75 per cent inhibition recorded as 3); ++++ (100 per cent inhibition recorded as 4). All serum, antigen and hemolytic system controls should show complete hemolysis. The corpuscle control should show no hemolysis. 12. Reactions may be interpreted and reported as very strongly positive, strongly positive, moderately positive, weakly positive, doubtful or negative as in the regular quantitative test 1 ' 2 ; or they may be reported as positive, doubtful or negative as recommended by the American Committee on Evaluation of Serodiagnostic Tests for Syphilis of the United States Public Health Service cooperating with the American Society of Clinical Pathologists. Slightly anticomplementary reactions may be safely reported as follows: 441 2 = positive,441 1 = positive, 441 ± = positive, 32 ± = positive, 441 3 = doubtful, 32 1 = doubtful, 21 ± = doubtful, 3 ± = doubtful, 1 ± = negative, 1 1 = negative, 2 1 = negative, 2 2 = negative. However, it is always advisable to repeat the tests and especially in the case of doubtful reactions while with sera which are heavily contaminated with bacteria and those which are chylous or heavily tinged with hemoglobin from spontaneous hemolysis in which the presence of thermostabile anticomplementary substances is suspected, a modified Sachs method is recommended for their preparation as described later. The simplified complement fixation test. 1. For each serum: (a) Arrange two test tubes and place 0.8 cc. of saline in No. 1 and 0.1 cc. in No. 2 (may be omitted to save time). (b) Add 0.2 cc. of inactivated serum to No. 1. (c) Mix by drawing up in the pipette several times; transfer 0.2 cc. to No. 2 and discard 0.6 cc. This leaves 0.04 cc. of serum in each of the two tubes (No. 2 being the serum control) or one-fifth the amounts employed in the regular test. 2. For each spinal fluid: (a) Arrange two test tubes and place 0.1 cc. saline solution in No. 1 and 0.2 cc. in No. 2. (6) Add 0.1 cc. of spinal fluid to each. Each tube, therefore, carries 0.1 cc. of spinal fluid or one-fifth the amounts of the regular simplified test, tube No. 2 being the control. 3. To the first tubes add 0.1 cc. of diluted antigen carrying the proper dose and mix thoroughly. 4. Allow to stand at room temperature for 10 to 30 minutes. If a longer interval elapses place the racks in a refrigerator. 5. Add 0.2 cc. of complement (2 exact units) to all tubes. 6. In the case of spinal fluid tests add 0.2 cc. of a 10 per cent solution of egg albumin in saline to each tube. 7. Include the following controls: (a) Antigen control containing 0.1 cc. diluted antigen, 0.2 cc. saline solution and 0.2 cc. diluted complement (carrying 2 exact units). If spinal fluids are being tested set up a second control in the same manner with the addition of 0.2 cc. of 10 per cent solution of egg albumin. (6) Hemolytic system control containing 0.3 cc. saline solution and 0.2 cc. diluted complement (carrying 2 exact units), (c) Corpuscle control containing 0.6 cc. saline solution, (d) Controls of positive and negative serums are advisable. 8. Mix the contents of each tube by gentle shaking and place in the refrigerator at 6 to 8 C. for 15 to 18 hours.

114 JOHN A. KOLMER 9. Place the tubes in a water bath at 37 C. for 10minutes (not longer). 10. To all tubes, except the corpuscle control, add 0.1 cc. of hemolysin (carrying 2 units) and to all tubes add 0.1 cc. of 2 per cent corpuscle suspension (shaken up). 11. Mix the contents of each tube by gentle shaking and place in a water bath at 37 C. Watch the serum, antigen, and hemolytic system controls and 10 minutes after these show complete hemolysis (usually $5 to SO minutes) remove the tests and make the readings. In the case of those tests in which the controls are not completely hemolysed, continue the water bath incubation for a total of one hour which frequently permits the reading of tests with serums and spinal fluids which are slightly anticomplementary. 12. The reactions may be reported as positive, doubtful, or negative, as recommended by the American Committee on Evaluation of Serodiagnostic Tests for Syphilis of the United States Public Health Service cooperating with the American Society of Clinical Pathologists, as follows: Positive: ++++ (4), +++ (3), ++ (2) or + (1), in the first tube; doubtful if ± in the first tube, and negative. However, Kolmer (6) recommends reporting as strongly positive (++++ or +++), weakly positive (+-1- or +), doubtful (±) and negative ( ) in the first tube. Slightly anticomplementary reactions may be safely reported as follows: 4± = positive, 41 = positive, 42 => doubtful, 31 = doubtful, 3± = doubtful, 22 = negative, 11 = negative, 1± = negative, ±± = negative. But it is always advisable to repeat the tests and especially in the case of doubtful reactions, while serums which are heavily contaminated with bacteria and those which are chylous or heavily tinged with hemoglobin from spontaneous hemolysis in which the presence of thermostabile anticomplementary substances is suspected, should be prepared by the modified Sachs method. Modified Sachs method for the preparation and testing of anticomplementary sera 1. Heat 0.5 cc. of serum at 55 C. in a water bath for 15 minutes. If the serum has been previously heated and is being retested, heat for 10 minutes at 55-56 C. 2. Add 4.1 cc. of accurately titrated N/300 hydrochloric acid and mix. 3. After standing \ hour at room temperature, centrifuge thoroughly and discard the sediment. 4. To the supernatant fluid add 0.4 cc. of 10 per cent sodium chloride solution. The acid is fixed by the precipitate of globulin; hence neutralization is unnecessary. 5. This gives a 1:10 dilution of original serum ready for testing as follows in both the quantitative and simplified tests: (1) Arrange two rows of 5 test tubes (the rear row are the serum controls). (2) Place 0.2 cc. of saline solution in Nos. 2, 3 and 4 of both rows and 0.8 cc. in No. 5 of each row. (3) Place 0.2 cc. of prepared serum (1:10) in Nos. 1 and 2 of the front row. Mix No. 2 and transfer 0.2 cc. to No. 3. Mix No. 3 and transfer 0.2 cc. to No. 4. Mix No. 4 and transfer 0.2 cc. to No. 5; mix No. 5 and discard 0.8 cc. (4) Repeat in the same manner with the rear row. (5) The five tubes of each row now carry 0.02, 0.01, 0.005,0.0025 and 0.0005 cc. of serum respectively. (6) Add antigen (0.1 cc. of proper dilution) to each tube of the front row. (7) Mix the contents of all tubes and allow to stand at room temperature for 10 to 30 minutes. If a longer time elapses place the racks in a refrigerator. (8) Add 0.2 cc. of complement (2 full units) and 0.2 cc. of 10 per cent egg albumin to each of the 5 tubes of both rows. Also add 0.2 cc. of 10 per cent egg albumin to the antigen control. Or dilute the complement with a 10 per cent solution of egg albumin instead of with plain saline solution (0.2 cc. to carry 2 units) and add 0.2 cc. to all tubes of both rows including the antigen control. Complete the test in the usual manner. Upon completion of the test all of the tubes in the rear row should show complete hemolysis. However, the first tubes carrying 0.02 cc. and sometimes the second tubes carrying

MICHO-KOLMER TEST 115 0.01 cc. of serum, of both rows, may not show complete hemolysis. With negative serums the corresponding front tubes show the same degree of inhibition of hemolysis, and if the degree of inhibition is slight a negative report may be rendered. With positive serums inhibition of hemolysis is much more marked in the tubes of the front row. It is advisable to report the reactions as positive, doubtful or negative. SUMMARY Methods are described for conducting the Kolmer quantitative and simplified complement fixation tests with sera and spinal fluids employing one-fifth the amounts of serum, spinal fluid and other reagents employed in the regular procedures. The reactions observed with the one-fifth methods are practically identical in sensitivity and specificity with those observed in the regular tests with a marked economical saving in all of the reagents employed, with special reference to the amount and cost of the complement required and thereby rendering available the Kolmer complement fixation tests on a large and routine basis. REFERENCES (1) KOLMER, J. A., AND BOERNER, F.: Approved Laboratory Technic, third edition. D. Appleton-Century Company, New York, 616-634, 1941. (2) Tech nics of serodiagnostic tests for syphilis. Supplement No. 11 to Ven. Dis. Information, 1940. (3) KOLMER, J. A., AND RICHTER, C. E.: A note on acetone-insoluble lipoids in relation to antigen for the Wassermann reaction. Amer. Jour. Clin. Path., 4: 235, 1934. (4) BOERNER, F., AND LUKENS, M.: The use of egg albumin as a protective protein in the spinal fluid Wassermann test. Amer. Jour. Clin. Path., 11: 71 (technical section), 1941. (5) KOLMER, J. A., AND LYNCH, E. R.: The prevention of non-specific and prezone reactions in the Wassermann test with sera and spinal fluids by the addition of egg albumin to the complement. Amer. Jour. Clin. Path., 11: 402, 1941. (6) KOLMER, J. A.: Concerning the method proposed for reporting the serological reactions for syphilis as positive, doubtful or negative. Amer. Jour. Clin. Path., 9: 121, 1939.