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National standard for human syphilitic serum and the use of international unit in the routine work of syphilis serology
,~nwnal of Biological
Standardization
1973 1, 81-85
National standard for human syphilitic serum and the use of international unit in the routine work of syphilis serology* Margaret Surjhn~ and G. Fiistt
Following the large-scaleproduction and biological standardization of cardiolipin antigens a National Standard for syphilitic serum as well as some working standards have been prepared and a simple calculating method has been developedto facilitate the use of the International Unit in the daily routine work of syphilis serology.
INTRODUCTION
In recent years our department has made several efforts to standardize the syphilis serology used routinely throughout the whole country. Following the large-scale production of cardiolipin and lecithin preparations and the use of both the 50% haemolysis method and the direct probability sequential analysis (Pangborn, Almeida, Maltaner, Silverstein & Thompson, 1955), for the state control of these preparations (Surjarr & Fiist, 1968), we have produced Kolmer, VDRL and Kline antigens. For the screening test we have used the VDRL antigen as in the method of Portnoy (Portnoy & Garson, 1960). For each lot of Kolmer, VDRL and Portnoy antigens the specificity was controlled by the use of several hundreds of non-reactive sera and the sensitivity determined by the use of 22 reactive sera. These results have been evaluated by analysis for variance (Krag & Weis Bentzon, 1956). In order to simplify and shorten the technique of complement fixation we have introduced two modifications : (1) a rapid micro method using a microtitration system (Surj&n, F&t & Balogh, 1971); and (2) the immunoglobulins of the syphilitic sera have been studied with 2-mercaptoethanol and thereafter the temperature dependence of the complement fixation reactivity of the different types of immunoglobulins of the sera was * Receivedfor publication 17 April 1972. 1 Department of Immunology, National Institute of Public Health, Budapest,Hungary. 81
MARGARET
SURJAN
AND
G. FUST
determined. It was shown that the cold fixation method, taking a long time, was not justified because 1 h incubation at 37°C did not give lower titres (Surjan et al., 1971). In order to standardize the syphilis serology a National Standard Serum was prepared and a simplified method of calculation of the titres of reactive sera in terms of the International Unit/ml was devised. It is suggested that the general use of international units (i.u.) could provide an opportunity to achieve greater uniformity of results of serologic tests for syphilis than is obtained at present. However, the extensive use of i.u. in everyday routine is hindered by the present complicated and time-consuming method of conversion of the titres of reactive syphilitic sera into i.u./ml. In order to simplify this calculation we have worked out a method which requires only one addition and one subtraction and thereafter the i.u./ml content of the serum may be read from a table. In the present paper we have described the preparation of the National Standard Serum and the mathematical principle as well as the method of calculation of titres of reactive sera in terms of i.u./ml according to our new simplified method.
MATERIALS
AND
METHODS
Serological tests A quantitative microtechnique complement fixation test, as previously described (Surj&r et al., 1971) has been used throughout the study whereas the VDRL tests were performed according to the Tests for Syphilis described in the PHS Manual (PHS Publ. 411, 1969). Sera The blood used in the preparation of the national working standard was collected mainly from patients in the primary stage of syphilis as well as from some human beings in the secondary stage of the disease. Blood samples (100-120 ml, each) of appropriate titres were withdrawn under sterile conditions and, until freeze-drying, the sera were stored in the deep freeze at -20°C. The national working standard was a pool of sera taken from 12 syphilitic patients for which 580 ml of serum was used. This pooled serum was distributed in amounts of O-6 ml in ampoules which, after freeze-drying, were sealed under vacuum. Calibration of National Working Standard The calibration of the national working standard was carried out as proposed by the WHO (Weis Bentzon & Krag, 1961).
RESULTS Production and control of National Working Standard The National Working Standard was compared with the International Standard Serum obtained from the Statens Seruminstitut, Copenhagen, using one ampoule of the International Standard reconstituted with 6.1 ml of 0.6% saline and giving a solution containing 8 i.u./ml. The National Working Standard was reconstituted by adding 0.6 ml distilled water and the two standard sera were compared by means of the quantitative Kolmer and the VDRL tests. The titration of both reconstituted sera was performed on each of six working days in three parallel tests. From each titration the 82
USE
OF
INTERNATIONAL
UNIT
IN
SYPHILIS
SEROLOGY
log,, titre (50%) was estimated by the KIrber method (KHrber, 1931), from which the average values for both sera and both test methods were calculated. The i.u. content of the National Working Standard was determined on the basis of the average titres obtained in the Kolmer reaction; the average titre (log,,) of the International Standard was l-3829, whereas that of the National Working Standard was 1.6839. The difference between these two values is 0.3010, the antilog of which is two. Accordingly, the reconstituted working standard contained 8 x 2 = 16 i.u./ml. Thus, a solution of the national working standard containing 8 i.u./ml could be obtained by adding to one ampoule of the dry serum reconstituted with 0.6 distilled water an additional 0.6 ml of 0.9% saline. In the VDRL reaction, however, the corresponding average titres were International Standard: l-1610, National Working Standard: 14084. Thus by the VDRL test the National Working Standard contained 14.16 i.u./ml. We have measured the amount of dry serum of the freeze-dried working standard that contained 1 i.u. Since the average amount of dry serum per ampoule was 67.5 + 1.83 mg [that is, the mean + the Standard Deviation (s.D.)], then by the Kolmer reaction 1 i.u. is contained in 5.989 mg, whereas by the VDRL reaction 1 i.u. is contained in 6.768 mg of the dry serum. Finally we examined the changes in titres of the sera stored, after reconstitution, at 46°C and it was found that reconstituted serum can be stored in a refrigerator for two weeks without any decrease in titre. General use of National
Working Standard for the standardization
of syphilis serology
Since our control serum is distributed and sent to 22 laboratories throughout the country, we are constantly kept informed of the daily results of the standard serum titrations. For each titration the logn, - titre is estimated by the KPrber method and the average values for each laboratory and for each test are calculated and compared to our results. In this manner we are able to control syphilis serology in the whole country. Conversion of the titres into i.u./ml of reactive sera
The titration of the National Working Standard, or that of the working standards calibrated against the National Working Standard, is included each day. If the log,,- titre of the standard serum containing 8 i.u./ml is designated x,s and that of the serum examined zcoT,the reactivity expressed in i.u./ml of the serum examined can be assessedaccording to the method of Bentzon & Krag (1961) using the following formula. The reactivity of serum in i.u./ml = 8. antilog (x~~-x,,~). In reactions when the reactivity is decreasing with the increasing dilution this value can be calculated by the Klrber method (Kgirber, 1931), according to the formula in the Appendix of WHO/VDT/SER0/89 (Weiss Bentzon & Krag, 1959) as follows. The reactivity of serum in i.u./ml= 8.antilog (o/R,,,) (x R,- C R,), where R max = maximal reactivity, w = the logarithm to the ratio between the subsequent doses, CR, = sums of scores (using the notation + = 0.5, + = 1, . . .. + + + + = 4) obtained in the serum examined, x R, = sum of scores obtained in standard serum. If the factor of dilution is two as was the casein most of the routine reactions of syphilis serology (Kolmer, VDRL, Kline, etc.), it is possible to set up the following specific formula. 83
MARGARET
SURJAN
AND
G. FtfST
The reactivity of serum in i.u./ml = 8 .antilog O-075 (C RT-z R,). Table 1 was prepared on the basis of this formula from which the reactivity of the sera examined may be obtained in i.u./ml in the following way. TABLE
1. Conversion of the titres of reactive sera into i.u./ml (applicable where International Standard Serum has a potency of 8 i.u./ml) Potency of serum (i.u./ml)
zR,-zR, -24.0 -23.5 -23.0 -22.5 -22.0 -21.5 -21.0 -20.5 -20.0 - 19.5 - 19.0 -18.5 -18.0 -17.5 -17.0 -16.5 -16.0 -15.5 -15.0 -14.5 -14.0 -13.5 -13.0 -12.5 -12.0 -11.5 -11.0 -10.5 -10-o -9-s -9.0 -8.5
2
RT - 2 Rs
0.13 0.14 0.15 0.16 0.18 0.20 0.21 0.23 0.25 0.28 0.30 0.33 0.36 0.39 0.43 0.46 0.50 0.55 0.60 0.66 0.72 0.78 0.85 o-92 1.00 I.10 1.19 I.31 1.42 1.55 l-69 1.83
Potency of serum (i.u./ml)
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 +0*5 +1*0 +1*5 +2-o +2.5 +3*0 +3*5 +4.0 +4.5 +5-o +5*5 +6*0 +6-S +7*0 +7*5
Z&-Z&
Potency of serum (i.u./d)
+8.0 +8.5 + 9-o +9.5 + 10.0 + 10.5 +11*0 +11*5 + 12.0 + 12.5 +13.0 +13-s +14*0 +14-s +15-o fl5.5 +16.0 +16.5 +17*0 +17*5 +18-O +18*5 + 19.0 + 19.5 +20-o +20*5 +21*0 +21-S +22*0 +22-s +23.0 +23-S +24-O
2.00 2.19 2.39 2.61 2.84 3.13 3.40 3.67 4.00 4.37 4.76 5.18 5.67 6.15 6.72 7.34 8.00 8.72 9.52 10.32 11.28 12.32 13.64 14.64 16.00 17.26 18.96 20.72 22.56 24.56 26.80 29.20
32.00 34.72 37.84 41.28 45.04 49.04 5344 58.32 64.00 69.28 75.52 82.32 89.60 9840 103.20 116.00 128.00 13840 148.80 164.00 179.20 195.20 212.80 232.00 256.00 276.00 300.80 328.00 357.60 389.60 424.80 463.20 512.00
Results of the various dilutions of the standard serum are added and designated C The same procedure should be followed for each reactive serum and designated I; For example, the positive control serum may give the following result: ++++
++++
++++
++
If the result of the serum examined ++++
++++
++++
++++
f
-,
thus
x
R,= 4+4+4+2+0*5+0=
14.5.
was: +++
+, then
Then in the case of every positive serum the value of x 84
R,. R,.
z
R,= 4+4+4+4+3+1= 20. R,-x R, should be calculated.
USE
OF INTERNATIONAL
UNIT
IN SYPHILIS
SEROLOGY
If our example 20-14.5 = 5.5 ; and the titre of the serum examined can be read off in i.u./ml from Table 1, which in our example = 20.72 i.u./ml. If C R, is smaller than CR, then the difference will be negative and these values may also be read off from Table 1.
REFERENCES Kiirber, G. (1931). Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archiv fiir experimentelle Pathologie und Pharmakologie 162, 480. Cited by Gaddum, J. H. in Special Report Series Medical Research Council London, (1933), No. 183. Krag, P. & Weis Bentzon, M. (1956). Comparison between old and new provisional international reference preparations of cardiolipin and lecithins. Unpublished working document WHO/ VDT/SERO/64. Manual of Serologic Testsfor Syphilis (1969). Revision, PHS Publication No. 411. Washington, D.C.: U.S. Government Printing Office. Pangborn, M. C., Almeida, J. O., Maltaner, F., Silverstein, A. M. & Thompson, W. R. (1955). Cardiolipin Antigens, 2nd ed. World Health Organization Monograph Series No. 6. Geneva: WHO. Portnoy, J. & Garson, W. (1960). New and improved antigen suspension for rapid reagin tests for syphilis. Public Health Report, Washington 75, 985-988. SurjPn, M. & Fiist, G. (1968). The control of cardiolipin and lecithin products with the 50% hemolysis method, Zeitschrzft fiir Immunitcitsforschung und experimentelle Therapie 136, 303-310. SurjBn, M., Fiist, G. & Balogh, I. (1971. Sulphydryl sensitivity of syphilitic antibodies and temperature dependence in complement fixation. British Journal of Venereal Diseases 47,87-90. Weis Bentzon, M. & Krag, P. (1959). Report on the preparation and on the collaborative assay of the third international reference preparation of egg lecithin. Unpublished working document WHO/VDT/SERO/89. Weis Bentzon, M. & Krag, P. (1961). The International Standard for human syphilitic serum. Bulletin of the World Health Organization 24, 257-264.
85
Standardization
1973 1, 81-85
National standard for human syphilitic serum and the use of international unit in the routine work of syphilis serology* Margaret Surjhn~ and G. Fiistt
Following the large-scaleproduction and biological standardization of cardiolipin antigens a National Standard for syphilitic serum as well as some working standards have been prepared and a simple calculating method has been developedto facilitate the use of the International Unit in the daily routine work of syphilis serology.
INTRODUCTION
In recent years our department has made several efforts to standardize the syphilis serology used routinely throughout the whole country. Following the large-scale production of cardiolipin and lecithin preparations and the use of both the 50% haemolysis method and the direct probability sequential analysis (Pangborn, Almeida, Maltaner, Silverstein & Thompson, 1955), for the state control of these preparations (Surjarr & Fiist, 1968), we have produced Kolmer, VDRL and Kline antigens. For the screening test we have used the VDRL antigen as in the method of Portnoy (Portnoy & Garson, 1960). For each lot of Kolmer, VDRL and Portnoy antigens the specificity was controlled by the use of several hundreds of non-reactive sera and the sensitivity determined by the use of 22 reactive sera. These results have been evaluated by analysis for variance (Krag & Weis Bentzon, 1956). In order to simplify and shorten the technique of complement fixation we have introduced two modifications : (1) a rapid micro method using a microtitration system (Surj&n, F&t & Balogh, 1971); and (2) the immunoglobulins of the syphilitic sera have been studied with 2-mercaptoethanol and thereafter the temperature dependence of the complement fixation reactivity of the different types of immunoglobulins of the sera was * Receivedfor publication 17 April 1972. 1 Department of Immunology, National Institute of Public Health, Budapest,Hungary. 81
MARGARET
SURJAN
AND
G. FUST
determined. It was shown that the cold fixation method, taking a long time, was not justified because 1 h incubation at 37°C did not give lower titres (Surjan et al., 1971). In order to standardize the syphilis serology a National Standard Serum was prepared and a simplified method of calculation of the titres of reactive sera in terms of the International Unit/ml was devised. It is suggested that the general use of international units (i.u.) could provide an opportunity to achieve greater uniformity of results of serologic tests for syphilis than is obtained at present. However, the extensive use of i.u. in everyday routine is hindered by the present complicated and time-consuming method of conversion of the titres of reactive syphilitic sera into i.u./ml. In order to simplify this calculation we have worked out a method which requires only one addition and one subtraction and thereafter the i.u./ml content of the serum may be read from a table. In the present paper we have described the preparation of the National Standard Serum and the mathematical principle as well as the method of calculation of titres of reactive sera in terms of i.u./ml according to our new simplified method.
MATERIALS
AND
METHODS
Serological tests A quantitative microtechnique complement fixation test, as previously described (Surj&r et al., 1971) has been used throughout the study whereas the VDRL tests were performed according to the Tests for Syphilis described in the PHS Manual (PHS Publ. 411, 1969). Sera The blood used in the preparation of the national working standard was collected mainly from patients in the primary stage of syphilis as well as from some human beings in the secondary stage of the disease. Blood samples (100-120 ml, each) of appropriate titres were withdrawn under sterile conditions and, until freeze-drying, the sera were stored in the deep freeze at -20°C. The national working standard was a pool of sera taken from 12 syphilitic patients for which 580 ml of serum was used. This pooled serum was distributed in amounts of O-6 ml in ampoules which, after freeze-drying, were sealed under vacuum. Calibration of National Working Standard The calibration of the national working standard was carried out as proposed by the WHO (Weis Bentzon & Krag, 1961).
RESULTS Production and control of National Working Standard The National Working Standard was compared with the International Standard Serum obtained from the Statens Seruminstitut, Copenhagen, using one ampoule of the International Standard reconstituted with 6.1 ml of 0.6% saline and giving a solution containing 8 i.u./ml. The National Working Standard was reconstituted by adding 0.6 ml distilled water and the two standard sera were compared by means of the quantitative Kolmer and the VDRL tests. The titration of both reconstituted sera was performed on each of six working days in three parallel tests. From each titration the 82
USE
OF
INTERNATIONAL
UNIT
IN
SYPHILIS
SEROLOGY
log,, titre (50%) was estimated by the KIrber method (KHrber, 1931), from which the average values for both sera and both test methods were calculated. The i.u. content of the National Working Standard was determined on the basis of the average titres obtained in the Kolmer reaction; the average titre (log,,) of the International Standard was l-3829, whereas that of the National Working Standard was 1.6839. The difference between these two values is 0.3010, the antilog of which is two. Accordingly, the reconstituted working standard contained 8 x 2 = 16 i.u./ml. Thus, a solution of the national working standard containing 8 i.u./ml could be obtained by adding to one ampoule of the dry serum reconstituted with 0.6 distilled water an additional 0.6 ml of 0.9% saline. In the VDRL reaction, however, the corresponding average titres were International Standard: l-1610, National Working Standard: 14084. Thus by the VDRL test the National Working Standard contained 14.16 i.u./ml. We have measured the amount of dry serum of the freeze-dried working standard that contained 1 i.u. Since the average amount of dry serum per ampoule was 67.5 + 1.83 mg [that is, the mean + the Standard Deviation (s.D.)], then by the Kolmer reaction 1 i.u. is contained in 5.989 mg, whereas by the VDRL reaction 1 i.u. is contained in 6.768 mg of the dry serum. Finally we examined the changes in titres of the sera stored, after reconstitution, at 46°C and it was found that reconstituted serum can be stored in a refrigerator for two weeks without any decrease in titre. General use of National
Working Standard for the standardization
of syphilis serology
Since our control serum is distributed and sent to 22 laboratories throughout the country, we are constantly kept informed of the daily results of the standard serum titrations. For each titration the logn, - titre is estimated by the KPrber method and the average values for each laboratory and for each test are calculated and compared to our results. In this manner we are able to control syphilis serology in the whole country. Conversion of the titres into i.u./ml of reactive sera
The titration of the National Working Standard, or that of the working standards calibrated against the National Working Standard, is included each day. If the log,,- titre of the standard serum containing 8 i.u./ml is designated x,s and that of the serum examined zcoT,the reactivity expressed in i.u./ml of the serum examined can be assessedaccording to the method of Bentzon & Krag (1961) using the following formula. The reactivity of serum in i.u./ml = 8. antilog (x~~-x,,~). In reactions when the reactivity is decreasing with the increasing dilution this value can be calculated by the Klrber method (Kgirber, 1931), according to the formula in the Appendix of WHO/VDT/SER0/89 (Weiss Bentzon & Krag, 1959) as follows. The reactivity of serum in i.u./ml= 8.antilog (o/R,,,) (x R,- C R,), where R max = maximal reactivity, w = the logarithm to the ratio between the subsequent doses, CR, = sums of scores (using the notation + = 0.5, + = 1, . . .. + + + + = 4) obtained in the serum examined, x R, = sum of scores obtained in standard serum. If the factor of dilution is two as was the casein most of the routine reactions of syphilis serology (Kolmer, VDRL, Kline, etc.), it is possible to set up the following specific formula. 83
MARGARET
SURJAN
AND
G. FtfST
The reactivity of serum in i.u./ml = 8 .antilog O-075 (C RT-z R,). Table 1 was prepared on the basis of this formula from which the reactivity of the sera examined may be obtained in i.u./ml in the following way. TABLE
1. Conversion of the titres of reactive sera into i.u./ml (applicable where International Standard Serum has a potency of 8 i.u./ml) Potency of serum (i.u./ml)
zR,-zR, -24.0 -23.5 -23.0 -22.5 -22.0 -21.5 -21.0 -20.5 -20.0 - 19.5 - 19.0 -18.5 -18.0 -17.5 -17.0 -16.5 -16.0 -15.5 -15.0 -14.5 -14.0 -13.5 -13.0 -12.5 -12.0 -11.5 -11.0 -10.5 -10-o -9-s -9.0 -8.5
2
RT - 2 Rs
0.13 0.14 0.15 0.16 0.18 0.20 0.21 0.23 0.25 0.28 0.30 0.33 0.36 0.39 0.43 0.46 0.50 0.55 0.60 0.66 0.72 0.78 0.85 o-92 1.00 I.10 1.19 I.31 1.42 1.55 l-69 1.83
Potency of serum (i.u./ml)
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 +0*5 +1*0 +1*5 +2-o +2.5 +3*0 +3*5 +4.0 +4.5 +5-o +5*5 +6*0 +6-S +7*0 +7*5
Z&-Z&
Potency of serum (i.u./d)
+8.0 +8.5 + 9-o +9.5 + 10.0 + 10.5 +11*0 +11*5 + 12.0 + 12.5 +13.0 +13-s +14*0 +14-s +15-o fl5.5 +16.0 +16.5 +17*0 +17*5 +18-O +18*5 + 19.0 + 19.5 +20-o +20*5 +21*0 +21-S +22*0 +22-s +23.0 +23-S +24-O
2.00 2.19 2.39 2.61 2.84 3.13 3.40 3.67 4.00 4.37 4.76 5.18 5.67 6.15 6.72 7.34 8.00 8.72 9.52 10.32 11.28 12.32 13.64 14.64 16.00 17.26 18.96 20.72 22.56 24.56 26.80 29.20
32.00 34.72 37.84 41.28 45.04 49.04 5344 58.32 64.00 69.28 75.52 82.32 89.60 9840 103.20 116.00 128.00 13840 148.80 164.00 179.20 195.20 212.80 232.00 256.00 276.00 300.80 328.00 357.60 389.60 424.80 463.20 512.00
Results of the various dilutions of the standard serum are added and designated C The same procedure should be followed for each reactive serum and designated I; For example, the positive control serum may give the following result: ++++
++++
++++
++
If the result of the serum examined ++++
++++
++++
++++
f
-,
thus
x
R,= 4+4+4+2+0*5+0=
14.5.
was: +++
+, then
Then in the case of every positive serum the value of x 84
R,. R,.
z
R,= 4+4+4+4+3+1= 20. R,-x R, should be calculated.
USE
OF INTERNATIONAL
UNIT
IN SYPHILIS
SEROLOGY
If our example 20-14.5 = 5.5 ; and the titre of the serum examined can be read off in i.u./ml from Table 1, which in our example = 20.72 i.u./ml. If C R, is smaller than CR, then the difference will be negative and these values may also be read off from Table 1.
REFERENCES Kiirber, G. (1931). Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archiv fiir experimentelle Pathologie und Pharmakologie 162, 480. Cited by Gaddum, J. H. in Special Report Series Medical Research Council London, (1933), No. 183. Krag, P. & Weis Bentzon, M. (1956). Comparison between old and new provisional international reference preparations of cardiolipin and lecithins. Unpublished working document WHO/ VDT/SERO/64. Manual of Serologic Testsfor Syphilis (1969). Revision, PHS Publication No. 411. Washington, D.C.: U.S. Government Printing Office. Pangborn, M. C., Almeida, J. O., Maltaner, F., Silverstein, A. M. & Thompson, W. R. (1955). Cardiolipin Antigens, 2nd ed. World Health Organization Monograph Series No. 6. Geneva: WHO. Portnoy, J. & Garson, W. (1960). New and improved antigen suspension for rapid reagin tests for syphilis. Public Health Report, Washington 75, 985-988. SurjPn, M. & Fiist, G. (1968). The control of cardiolipin and lecithin products with the 50% hemolysis method, Zeitschrzft fiir Immunitcitsforschung und experimentelle Therapie 136, 303-310. SurjBn, M., Fiist, G. & Balogh, I. (1971. Sulphydryl sensitivity of syphilitic antibodies and temperature dependence in complement fixation. British Journal of Venereal Diseases 47,87-90. Weis Bentzon, M. & Krag, P. (1959). Report on the preparation and on the collaborative assay of the third international reference preparation of egg lecithin. Unpublished working document WHO/VDT/SERO/89. Weis Bentzon, M. & Krag, P. (1961). The International Standard for human syphilitic serum. Bulletin of the World Health Organization 24, 257-264.
85