Antigenic Differences among Bovine and Porcine Factor VIII Concentrates J. ROGER EDSON, M.D.,
AND CHERYL D. SWINEHART,
M.S.
From the Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455
ABSTRACT
that batches of Factor VIII from different purebred bovine strains yielded widely variable apparent antibody concentrations when reacted with the same amount of antiFactor-VIII antibody. 2 Incident to that study, we found that a 7-year-old batch of Maws commercial bovine Factor VIII concentrate gave a very low apparent antibody concentration. We questioned whether the low apparent antibody concentration was due to the age of the specimen, or had other causes. We have reacted batches of Maws commercial bovine and porcine W E PREVIOUSLY REPORTED
Received June 25, 1975; received revised manuscript September 18, 1975; accepted for publication September 18, 1975. Supported in part by the Gregory Bendt Memorial Fund for Hemophiliac Research. Address reprint requests to Dr. Edson.
Factor VIII concentrate manufactured over a 12-year period with three different hemophiliac antibodies (same amount of antibody with each Factor VIII source) and measured the apparent antibody concentrations. The results of this investigation and our interpretation of the data are reported herewith. Materials and Methods Bovine and Porcine AHF Concentrates Small samples of batches of bovine and porcine antihemophiliac factor (AHF) concentrates manufactured over a period of about 12 years were obtained through the courtesy of Mr. M. B. Walton, Maws Limited, Aldersgate House, Cromer Road, New Barnet, Hertfordshire, England EN5
957
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Edson, J. Roger, and Swinehart, Cheryl D.: Antigenic differences among bovine and porcine factor VIII concentrates. Am J Clin Pathol 65: 9 5 7 - 9 6 3 , 1976. Three anti-Factor-VIII antibodies from hemophiliacs were reacted with samples of batches of Maws commercial bovine and porcine Factor VIII concentrates manufactured over a 12-year period. The apparent antibody concentrations varied widely with the different batches of concentrates. The variations are probably due to intrinsic differences in the antigenic nature of the Factor VIII in the different preparations. With the older porcine concentrates, the low apparent concentrations may be in part due to the reaction of antibody with inactive Factor VIII. When animal Factor VIII concentrates are used in treating hemophiliac patients who have antiFactor-VIII antibodies, the least reactive batch should be chosen. Random batches of animal concentrates are not suited as a standard for measuring anti-Factor-VIII antibody concentration. (Key words: Factor VIII antigenic differences; Bovine Factor VIII; Porcine Factor VIII; Anti-Factor-VIII antibodies.)
958
A.J.C.P.—Vol.
EDSON AND SWINEHART
65
Table 1. Batches of AHF Studied
Bovine AHF concentrates
Factor VIII Range
Number of Assays*
Time to Dissolve (Min.)
Dilution for Use in Antibody Tests
Lot 32902 51283 51627 55239 59040 59796 8065 7492 9815 11785
1-25-60 4-15-64 10-26-64 6-17-75 5- 6-66 4-12-67 6-17-68 9-24-69 9- 7-70 5-18-71
580 890 180 160 185 150 93 97 94 98
570-586 810-980 140-206 140-190 150-225 130-172 85-100 84-104 78-109 50-150
3 3 3 6 3 5 4 3 4 7
45 90t 90t 45 90t 25 60 60 20
3.3 6.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
36276 36550 51756 55173 59085 59645 7469 9430 11775 31271
5-30-60 9- 6-60 12- 8-64 5-14-65 5-20-65 1-31-67 9-17-69 6- 3-70 5-18-71 11-25-71
490 940 840 190 215 260 1,040 395 285 730
450-530 910-1,000 770-920 150-215 206-226 205-310 1,000-1,060 265-485 205-335 610-810
3 3 3 3 3 3 3 3 3 3
60t 60 90* 75t 60t 75t 90* 60t 75t 60t
3.5 5.0 4.5 1.0 1.4 2.0 5.0 1.7 1.4 4.5
(%)
* Number of assays includes controls on antibody studies corrected for the dilution at which they were tested. t A small amount of powder did not dissolve. $ Dissolved very poorly.
5HU. T h e dried contents of each individual vial were reconstituted in 20 ml. of buffered saline solution. T h e different lot numbers took different times to go into solution completely. After reconstitution, the dissolved material was quick-frozen in 2- to 3-ml. amounts and stored at —45 C. prior to use. Samples of each lot number were then assayed for Factor VIII activity. Data for the different batches of bovine and porcine AHF are given in Table 1. Anti-Factor VIII Antibodies Plasmas containing human antihuman Factor VIII antibodies were obtained from three patients with classic hemophilia A who had developed this complication. One of the antibody-containing specimens was obtained during the course of therapeutic plasmapheresis, one during a therapeutic
exchange transfusion, and the third by plasmapheresis after the patient had recovered from some emergency dental work. Two plasmas were used undiluted as sources of antibody, while the third was diluted with buffered saline solution before use in the antibody studies, as indicated in Tables 2 and 3. Antibody Studies Bovine or porcine AHF concentrate was diluted as indicated in the last column of Table 1 to give a Factor V I I I activity of between 100 and 200%. A 0.9ml. amount of Factor VIII concentrate (or diluted Factor VIII concentrate) was mixed with 0.1 ml. of antibody solution (plasma or diluted plasma). This mixture was incubated in a tightly stoppered siliconized tube for 4 hours, after which the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Porcine AHF concentrates
Mean Factor VIII
Date of Manufacture
June 1976
ANTIGENIC DIFFERENCES IN ANIMAL AHF
959
Table 2. Maws Bovine Factor VIII Concentrates, Residual Factor VIII Activity after 4-hour Incubation with Three Anti-Factor-VIII Antibodies, and Apparent Antibody Concentrations* Apparent Concentration, Pool-Miller U. per ml.
Residual Factor VIII (%)
32902 5128.3 51627 55239 59040 59796 8065 7492 9815 11785
1-25-60 4-15-64 10-26-64 6-17-65 5- 6-66 4-12-67 6-17-68 9-24-69 9- 7-70 5-18-71
Antibody Controls
A
B
C
160, 160 158, 146 186, 126 136, 148 240, 160 140, 126 89, 80 94, 92 68, 98 100,110 74, 74
136 118 126 72 136 71 56 49 28 7 12
31 12 53 13 22 7 4 4 3.5 2.5 3
112 80 83 68 118 42 25 35 16 3 6
AVERAGE ' Antibody B was diluted with an equal volume of buffered saline solution for i
residual Factor VIII activity was determined in a one-stage assay as previously described. 3 Control specimens with 0.9 ml. of Factor VIII concentrate solution mixed with 0.1 ml. of buffered saline solution were done in duplicate for each batch of concentrate, and the Factor VIII activity
Antibody A
Antibody B
Antibod)' C
1.4 4.2 1.9 5.7 2.9 5.2 2.0 3.5 5.4 17.4 16.4 16.9
35 44 20.2 41.4 47 50 32 35.8 32 48 58 53
3.4 6.0 5.9 6.2 4.7 9.7 8.5 5.4 8.5 22.8 22.6 22.7
ubation mixtures; the others were used undiluted.
determined at the end of 4 hours of incubation. Using these values, the apparent antibody concentration for each batch of Factor VIII concentrate was calculated using the method of Pool and Miller.4 Preliminary studies indicated that the Factor VIII activity in the antibody—con-
Table 3. Maws Porcine Factor VIII Concentrates, Residual Factor VIII Activity after 4-hour Incubation with Three Anti-Factor-VIII Antibodies, and Apparent Antibody Concentrations Apparent concentration. Pool-Miller U. per ml.
Residual Factor VIII (%)
Lot
Date of Manufacture
36276 36550 51756 55173 59085 59645 7469 9430 11775 31271
5-30-60 9- 6-60 12- 8-64 5-14-65 5-20-65 1-31-67 9-17-69 6- 3-70 5-18-71 11-25-71
Antibod) Controls 126, 164, 184, 192, 138, 120, 192, 256, 204, 162,
116 164 164 192* 146 93 192 232 228* 122
A
B
C
Antibody A
100 136 65 40 67 16 59 100 63 108
59 83 22 4.5 30 4.5 26 44 5.5 35
116 136 69 45 77 9.5 79 90 72 130
1.2 1.6 10.4 18.4 6.4 12.1 13.6 13.2 15.9 2.3
Antibod v B
Antibody C
15.6 19.8 66 88 40.2 62 71 102 97 28.2
0.2 1.6 10.3 17.1 5.1 15.5 10.2 14.6 14.5 5.7
* lii these im uhaiious. 0.1 ml. of a 1/2 dilution of antibodv B were used. In all others. 0.1 nil. of ,\ 1/3 dilution were used.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Lot
Date of Manufacture
960
A.J.C.P. —Vol. 65
EDSON AND SWINEHART
satisfactory in this test system, particularly with bovine Factor VIII concentrate. 2 Results T h e Factor VIII activities at the end of the incubation periods, and the calculated apparent antibody concentrations in PoolMiller units per ml., are given in Table 2 for bovine concentrates, and in Table 3 for porcine concentrates. With different lots of bovine Factor VIII concentrates, the apparent concentrations of antibody A ranged from 1.4 to 17.4 U. per ml. (12fold variation); those of antibody B, from 20 to 53 U. per ml. (2.7-fold variation);
Lot number and date of *ani
1960
1961
1962
1963
T—'—r1964
1965
1966
r 1967
1968
1969
1970
1971
Tear of aanufactura, MWS bovine AHG concentrate, teated in 1973 FIG. 1. Variations in apparent concentrations of three anti-Factor-VIII antibodies from hemophiliacs with different batches of Maws bovine AHG concentrates.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
centrate mixture did not plateau within 2 hours, but did within 4 hours; hence, the 4-hour incubation was chosen. In additional preliminary studies, it was found that aluminum hydroxide absorption of the incubation mixtures before assaying Factor VIII resulted in very erratic Factor VIII activity values, in some cases much higher, and in others much lower, than without absorption. Because of the number of preliminary studies and the small amount of material available to us, it was not possible to do replicate determinations on the antibody studies. Data previously reported by us, however, indicated that replication was
June 1976
1961 1962 1963 1964 196S 1966 1967 1968 Tear of manufacture, MAWS porcine AHG concentrate, tested In 1973
1969
1970
1971
FIG. 2. Variations in apparent concentrations of three anti-Factor-VIII antibodies from hemophiliacs with different batches of Maws porcine AHG concentrates.
those of antibody C, from 3.4 to 22.7 U. per ml. (6.7-fold variation). With different lots of porcine Factor VIII concentrate, the apparent concentrations of antibody A ranged from 1.2 to 18.4 U. per ml. (15fold variation); those of antibody B, from 16 to 102 U. per ml. (6.4-fold variation); those of antibody C from essentially zero to 17 U. per ml. T h e apparent concentrations of each antibody with the different batches of concentrate according to their dates of manufacture are shown graphically in Figures 1 and 2.
trations of a given anti-Factor-VIII antibody varied strikingly. 2 In the same study, Maws bovine Factor VIII concentrate lot #51283 was found to give a low apparent antibody concentration with the antibodies tested. We wondered whether the apparent low antibody concentration was due to the age of the concentrate (7 years at that time) or to some other factor. Figure 1 shows graphically the apparent concentrations of three hemophiliac antiFactor-VIII antibodies with different lots of Maws bovine Factor VIII concentrate manufactured over a 12-year period when Discussion tested in 1973. It can be seen readily We have previously shown that when re- that the apparent concentrations of antiacted with plasma Factor VIII from dif- bodies A and C parallel each other, and that ferent bovine strains the apparent concen- the lowest values were obtained with the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
1960
961
ANTIGENIC DIFFERENCES IN ANIMAL AHF
962
EDSON AND SWINEHART
A.J.C.P.—Vol.
65
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Table 4. Ratios of Apparent Antibody Con- batch to batch. If this were the only cause of the observed differences, one would excentrations with Different Batches of pect to see parallel changes in the apparent Animal Factor VIII Concentrate concentrations of different antibodies with Ratio of different batches of concentrate. T h e nonApparent parallelism of antibody B and the other Antibody two antibodies with the different batches ConcenDate of trations of bovine concentrate speaks against this. ManuIf the variations in apparent antibody conB/10A C/A facture Lot centrations with different lot numbers of bovine Factor VIII concentrate were due Bovine Factor VIII consolely to variations in the amounts of ac2.5 2.4 centrates 32902 1-25-60 tive Factor VIII from batch to batch, 4-15-64 1.4 1.0 51283 one would also expect that the ratio of the 10-26-64 1.1 51627 3.1 0.7 55239 1.1 6-17-75 apparent concentration of one antibody 5- 6-66 1.6 59040 1.6 to another would not change from batch 1.0 4-12-67 59796 1.9 to batch. There are, in fact, wide variations 1.6 8065 6-17-68 4.2 1.0 9-24-69 1.5 7492 in the ratios of apparent antibody concen0.6 9815 9- 7-70 1.6 trations, as can be seen from the first 0.3 11785 5-18-71 1.3 half of Table 4. One might further exPorcine Factor VIII conpect that there would be more inactive centrates 36276 0.2 1.3 5-30-60 Factor VIII in the older batches of concen36550 9- 6-60 1.0 1.2 trate. T h e random differences in the ap128-64 51756 1.0 0.6 55173 5-14-65 0.9 0.5 parent concentration of antibody B, and 59085 5-20-65 0.8 0.6 the random variation of antibody ratios 59645 1-31-67 0.5 1.3 with bovine concentrates, make this un9-17-69 7469 0.7 0.5 likely. We think it more probable that we 6- 3-70 9430 1.1 0.8 5-18-71 11775 0.9 0.6 are dealing with real differences in the 2.5 11-25-71 31271 1.2 antigenic nature of the Factor VIII in the different batches of bovine concentrate. These could be due to intrinsic differences oldest concentrate, the highest values with among the cattle used as source material, the newest. With antibody B, however, the to alterations in Factor VIII in manufacapparent concentrations varied among the ture, or to a combination of these. bovine concentrate batches independently of the other two antibodies. While the The apparent concentrations of the highest value was with the newest concen- three antibodies with different batches of trate, the second highest value was with Maws porcine Factor VIII concentrate the 1964 batch of material that had given varied in a different way from those with us low values in our earlier study. Thus, bovine concentrates, as shown in Figure 2. the apparent antibody concentration with With the different batches of porcine a given batch of bovine Factor VIII con- concentrates there was parallelism of the centrate is as unpredictable as for individ- apparent concentrations of all three antiual plasma bovine Factor VIII. bodies. There was a much more definite One could postulate that antibody would trend for the apparent antibody concentrareact with both active and inactive Factor tion to increase with newer batches of conVIII, and that the relative amounts of centrate. In the lower half of Table 4, active and inactive material varied from it can be seen that the ratios of antibody
June 1976
ANTIGENIC DIFFERENCES IN ANIMAL AHF
for Factor VIII activity.1 Our studies indicate that, in general, animal Factor VIII concentrates would not provide a suitable standard Factor VIII preparation for the quantitation of human antibodies. References 1. Biggs, R, EvelingJ, Richards G: T h e assay of a n t i h a e m o p h i l i c - g l o b u l i n activity. Br J Haematol 1:20-34, 1955 2. Edson JR, Svvineharl CD: Antigenic differences in functionally normal antihemophiliac globulin in different bovine strains. J Lab Clin Med 83:467-477, 1974 3. Edson JR, McArthur JR, Branda RG, et al: Successful management of a subdural hematoma in a hemophiliac with an anti-Factor VIII antibody. Blood 41:113-122, 1973 4. Pool JG, Miller RG: Assay of the immune inhibitor in classic hemophilia: Application of virus-antibody reaction kinetics. Br J Haematol 22:517-528, 1972
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
concentrations were relatively constant from one batch of porcine concentrate to another, with the exception of the first two and the last batches. Thus, it is possible that antibody reacting with both active and inactive Factor VIII is contributing to the differences in apparent antibody concentrations with the porcine Factor VIII concentrates, in addition to intrinsic variations in antigenic reactivity. When animal Factor VIII concentrates are being considered for treatment of a hemophiliac patient who has an antiFactor-VIII antibody, it is very important to estimate the apparent antibody concentrations with the available concentrates, in order to select the preparation that is least reactive. In the past, animal Factor VIII has been used as a reference standard
963
AND CHERYL D. SWINEHART,
M.S.
From the Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455
ABSTRACT
that batches of Factor VIII from different purebred bovine strains yielded widely variable apparent antibody concentrations when reacted with the same amount of antiFactor-VIII antibody. 2 Incident to that study, we found that a 7-year-old batch of Maws commercial bovine Factor VIII concentrate gave a very low apparent antibody concentration. We questioned whether the low apparent antibody concentration was due to the age of the specimen, or had other causes. We have reacted batches of Maws commercial bovine and porcine W E PREVIOUSLY REPORTED
Received June 25, 1975; received revised manuscript September 18, 1975; accepted for publication September 18, 1975. Supported in part by the Gregory Bendt Memorial Fund for Hemophiliac Research. Address reprint requests to Dr. Edson.
Factor VIII concentrate manufactured over a 12-year period with three different hemophiliac antibodies (same amount of antibody with each Factor VIII source) and measured the apparent antibody concentrations. The results of this investigation and our interpretation of the data are reported herewith. Materials and Methods Bovine and Porcine AHF Concentrates Small samples of batches of bovine and porcine antihemophiliac factor (AHF) concentrates manufactured over a period of about 12 years were obtained through the courtesy of Mr. M. B. Walton, Maws Limited, Aldersgate House, Cromer Road, New Barnet, Hertfordshire, England EN5
957
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Edson, J. Roger, and Swinehart, Cheryl D.: Antigenic differences among bovine and porcine factor VIII concentrates. Am J Clin Pathol 65: 9 5 7 - 9 6 3 , 1976. Three anti-Factor-VIII antibodies from hemophiliacs were reacted with samples of batches of Maws commercial bovine and porcine Factor VIII concentrates manufactured over a 12-year period. The apparent antibody concentrations varied widely with the different batches of concentrates. The variations are probably due to intrinsic differences in the antigenic nature of the Factor VIII in the different preparations. With the older porcine concentrates, the low apparent concentrations may be in part due to the reaction of antibody with inactive Factor VIII. When animal Factor VIII concentrates are used in treating hemophiliac patients who have antiFactor-VIII antibodies, the least reactive batch should be chosen. Random batches of animal concentrates are not suited as a standard for measuring anti-Factor-VIII antibody concentration. (Key words: Factor VIII antigenic differences; Bovine Factor VIII; Porcine Factor VIII; Anti-Factor-VIII antibodies.)
958
A.J.C.P.—Vol.
EDSON AND SWINEHART
65
Table 1. Batches of AHF Studied
Bovine AHF concentrates
Factor VIII Range
Number of Assays*
Time to Dissolve (Min.)
Dilution for Use in Antibody Tests
Lot 32902 51283 51627 55239 59040 59796 8065 7492 9815 11785
1-25-60 4-15-64 10-26-64 6-17-75 5- 6-66 4-12-67 6-17-68 9-24-69 9- 7-70 5-18-71
580 890 180 160 185 150 93 97 94 98
570-586 810-980 140-206 140-190 150-225 130-172 85-100 84-104 78-109 50-150
3 3 3 6 3 5 4 3 4 7
45 90t 90t 45 90t 25 60 60 20
3.3 6.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
36276 36550 51756 55173 59085 59645 7469 9430 11775 31271
5-30-60 9- 6-60 12- 8-64 5-14-65 5-20-65 1-31-67 9-17-69 6- 3-70 5-18-71 11-25-71
490 940 840 190 215 260 1,040 395 285 730
450-530 910-1,000 770-920 150-215 206-226 205-310 1,000-1,060 265-485 205-335 610-810
3 3 3 3 3 3 3 3 3 3
60t 60 90* 75t 60t 75t 90* 60t 75t 60t
3.5 5.0 4.5 1.0 1.4 2.0 5.0 1.7 1.4 4.5
(%)
* Number of assays includes controls on antibody studies corrected for the dilution at which they were tested. t A small amount of powder did not dissolve. $ Dissolved very poorly.
5HU. T h e dried contents of each individual vial were reconstituted in 20 ml. of buffered saline solution. T h e different lot numbers took different times to go into solution completely. After reconstitution, the dissolved material was quick-frozen in 2- to 3-ml. amounts and stored at —45 C. prior to use. Samples of each lot number were then assayed for Factor VIII activity. Data for the different batches of bovine and porcine AHF are given in Table 1. Anti-Factor VIII Antibodies Plasmas containing human antihuman Factor VIII antibodies were obtained from three patients with classic hemophilia A who had developed this complication. One of the antibody-containing specimens was obtained during the course of therapeutic plasmapheresis, one during a therapeutic
exchange transfusion, and the third by plasmapheresis after the patient had recovered from some emergency dental work. Two plasmas were used undiluted as sources of antibody, while the third was diluted with buffered saline solution before use in the antibody studies, as indicated in Tables 2 and 3. Antibody Studies Bovine or porcine AHF concentrate was diluted as indicated in the last column of Table 1 to give a Factor V I I I activity of between 100 and 200%. A 0.9ml. amount of Factor VIII concentrate (or diluted Factor VIII concentrate) was mixed with 0.1 ml. of antibody solution (plasma or diluted plasma). This mixture was incubated in a tightly stoppered siliconized tube for 4 hours, after which the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Porcine AHF concentrates
Mean Factor VIII
Date of Manufacture
June 1976
ANTIGENIC DIFFERENCES IN ANIMAL AHF
959
Table 2. Maws Bovine Factor VIII Concentrates, Residual Factor VIII Activity after 4-hour Incubation with Three Anti-Factor-VIII Antibodies, and Apparent Antibody Concentrations* Apparent Concentration, Pool-Miller U. per ml.
Residual Factor VIII (%)
32902 5128.3 51627 55239 59040 59796 8065 7492 9815 11785
1-25-60 4-15-64 10-26-64 6-17-65 5- 6-66 4-12-67 6-17-68 9-24-69 9- 7-70 5-18-71
Antibody Controls
A
B
C
160, 160 158, 146 186, 126 136, 148 240, 160 140, 126 89, 80 94, 92 68, 98 100,110 74, 74
136 118 126 72 136 71 56 49 28 7 12
31 12 53 13 22 7 4 4 3.5 2.5 3
112 80 83 68 118 42 25 35 16 3 6
AVERAGE ' Antibody B was diluted with an equal volume of buffered saline solution for i
residual Factor VIII activity was determined in a one-stage assay as previously described. 3 Control specimens with 0.9 ml. of Factor VIII concentrate solution mixed with 0.1 ml. of buffered saline solution were done in duplicate for each batch of concentrate, and the Factor VIII activity
Antibody A
Antibody B
Antibod)' C
1.4 4.2 1.9 5.7 2.9 5.2 2.0 3.5 5.4 17.4 16.4 16.9
35 44 20.2 41.4 47 50 32 35.8 32 48 58 53
3.4 6.0 5.9 6.2 4.7 9.7 8.5 5.4 8.5 22.8 22.6 22.7
ubation mixtures; the others were used undiluted.
determined at the end of 4 hours of incubation. Using these values, the apparent antibody concentration for each batch of Factor VIII concentrate was calculated using the method of Pool and Miller.4 Preliminary studies indicated that the Factor VIII activity in the antibody—con-
Table 3. Maws Porcine Factor VIII Concentrates, Residual Factor VIII Activity after 4-hour Incubation with Three Anti-Factor-VIII Antibodies, and Apparent Antibody Concentrations Apparent concentration. Pool-Miller U. per ml.
Residual Factor VIII (%)
Lot
Date of Manufacture
36276 36550 51756 55173 59085 59645 7469 9430 11775 31271
5-30-60 9- 6-60 12- 8-64 5-14-65 5-20-65 1-31-67 9-17-69 6- 3-70 5-18-71 11-25-71
Antibod) Controls 126, 164, 184, 192, 138, 120, 192, 256, 204, 162,
116 164 164 192* 146 93 192 232 228* 122
A
B
C
Antibody A
100 136 65 40 67 16 59 100 63 108
59 83 22 4.5 30 4.5 26 44 5.5 35
116 136 69 45 77 9.5 79 90 72 130
1.2 1.6 10.4 18.4 6.4 12.1 13.6 13.2 15.9 2.3
Antibod v B
Antibody C
15.6 19.8 66 88 40.2 62 71 102 97 28.2
0.2 1.6 10.3 17.1 5.1 15.5 10.2 14.6 14.5 5.7
* lii these im uhaiious. 0.1 ml. of a 1/2 dilution of antibodv B were used. In all others. 0.1 nil. of ,\ 1/3 dilution were used.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Lot
Date of Manufacture
960
A.J.C.P. —Vol. 65
EDSON AND SWINEHART
satisfactory in this test system, particularly with bovine Factor VIII concentrate. 2 Results T h e Factor VIII activities at the end of the incubation periods, and the calculated apparent antibody concentrations in PoolMiller units per ml., are given in Table 2 for bovine concentrates, and in Table 3 for porcine concentrates. With different lots of bovine Factor VIII concentrates, the apparent concentrations of antibody A ranged from 1.4 to 17.4 U. per ml. (12fold variation); those of antibody B, from 20 to 53 U. per ml. (2.7-fold variation);
Lot number and date of *ani
1960
1961
1962
1963
T—'—r1964
1965
1966
r 1967
1968
1969
1970
1971
Tear of aanufactura, MWS bovine AHG concentrate, teated in 1973 FIG. 1. Variations in apparent concentrations of three anti-Factor-VIII antibodies from hemophiliacs with different batches of Maws bovine AHG concentrates.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
centrate mixture did not plateau within 2 hours, but did within 4 hours; hence, the 4-hour incubation was chosen. In additional preliminary studies, it was found that aluminum hydroxide absorption of the incubation mixtures before assaying Factor VIII resulted in very erratic Factor VIII activity values, in some cases much higher, and in others much lower, than without absorption. Because of the number of preliminary studies and the small amount of material available to us, it was not possible to do replicate determinations on the antibody studies. Data previously reported by us, however, indicated that replication was
June 1976
1961 1962 1963 1964 196S 1966 1967 1968 Tear of manufacture, MAWS porcine AHG concentrate, tested In 1973
1969
1970
1971
FIG. 2. Variations in apparent concentrations of three anti-Factor-VIII antibodies from hemophiliacs with different batches of Maws porcine AHG concentrates.
those of antibody C, from 3.4 to 22.7 U. per ml. (6.7-fold variation). With different lots of porcine Factor VIII concentrate, the apparent concentrations of antibody A ranged from 1.2 to 18.4 U. per ml. (15fold variation); those of antibody B, from 16 to 102 U. per ml. (6.4-fold variation); those of antibody C from essentially zero to 17 U. per ml. T h e apparent concentrations of each antibody with the different batches of concentrate according to their dates of manufacture are shown graphically in Figures 1 and 2.
trations of a given anti-Factor-VIII antibody varied strikingly. 2 In the same study, Maws bovine Factor VIII concentrate lot #51283 was found to give a low apparent antibody concentration with the antibodies tested. We wondered whether the apparent low antibody concentration was due to the age of the concentrate (7 years at that time) or to some other factor. Figure 1 shows graphically the apparent concentrations of three hemophiliac antiFactor-VIII antibodies with different lots of Maws bovine Factor VIII concentrate manufactured over a 12-year period when Discussion tested in 1973. It can be seen readily We have previously shown that when re- that the apparent concentrations of antiacted with plasma Factor VIII from dif- bodies A and C parallel each other, and that ferent bovine strains the apparent concen- the lowest values were obtained with the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
1960
961
ANTIGENIC DIFFERENCES IN ANIMAL AHF
962
EDSON AND SWINEHART
A.J.C.P.—Vol.
65
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
Table 4. Ratios of Apparent Antibody Con- batch to batch. If this were the only cause of the observed differences, one would excentrations with Different Batches of pect to see parallel changes in the apparent Animal Factor VIII Concentrate concentrations of different antibodies with Ratio of different batches of concentrate. T h e nonApparent parallelism of antibody B and the other Antibody two antibodies with the different batches ConcenDate of trations of bovine concentrate speaks against this. ManuIf the variations in apparent antibody conB/10A C/A facture Lot centrations with different lot numbers of bovine Factor VIII concentrate were due Bovine Factor VIII consolely to variations in the amounts of ac2.5 2.4 centrates 32902 1-25-60 tive Factor VIII from batch to batch, 4-15-64 1.4 1.0 51283 one would also expect that the ratio of the 10-26-64 1.1 51627 3.1 0.7 55239 1.1 6-17-75 apparent concentration of one antibody 5- 6-66 1.6 59040 1.6 to another would not change from batch 1.0 4-12-67 59796 1.9 to batch. There are, in fact, wide variations 1.6 8065 6-17-68 4.2 1.0 9-24-69 1.5 7492 in the ratios of apparent antibody concen0.6 9815 9- 7-70 1.6 trations, as can be seen from the first 0.3 11785 5-18-71 1.3 half of Table 4. One might further exPorcine Factor VIII conpect that there would be more inactive centrates 36276 0.2 1.3 5-30-60 Factor VIII in the older batches of concen36550 9- 6-60 1.0 1.2 trate. T h e random differences in the ap128-64 51756 1.0 0.6 55173 5-14-65 0.9 0.5 parent concentration of antibody B, and 59085 5-20-65 0.8 0.6 the random variation of antibody ratios 59645 1-31-67 0.5 1.3 with bovine concentrates, make this un9-17-69 7469 0.7 0.5 likely. We think it more probable that we 6- 3-70 9430 1.1 0.8 5-18-71 11775 0.9 0.6 are dealing with real differences in the 2.5 11-25-71 31271 1.2 antigenic nature of the Factor VIII in the different batches of bovine concentrate. These could be due to intrinsic differences oldest concentrate, the highest values with among the cattle used as source material, the newest. With antibody B, however, the to alterations in Factor VIII in manufacapparent concentrations varied among the ture, or to a combination of these. bovine concentrate batches independently of the other two antibodies. While the The apparent concentrations of the highest value was with the newest concen- three antibodies with different batches of trate, the second highest value was with Maws porcine Factor VIII concentrate the 1964 batch of material that had given varied in a different way from those with us low values in our earlier study. Thus, bovine concentrates, as shown in Figure 2. the apparent antibody concentration with With the different batches of porcine a given batch of bovine Factor VIII con- concentrates there was parallelism of the centrate is as unpredictable as for individ- apparent concentrations of all three antiual plasma bovine Factor VIII. bodies. There was a much more definite One could postulate that antibody would trend for the apparent antibody concentrareact with both active and inactive Factor tion to increase with newer batches of conVIII, and that the relative amounts of centrate. In the lower half of Table 4, active and inactive material varied from it can be seen that the ratios of antibody
June 1976
ANTIGENIC DIFFERENCES IN ANIMAL AHF
for Factor VIII activity.1 Our studies indicate that, in general, animal Factor VIII concentrates would not provide a suitable standard Factor VIII preparation for the quantitation of human antibodies. References 1. Biggs, R, EvelingJ, Richards G: T h e assay of a n t i h a e m o p h i l i c - g l o b u l i n activity. Br J Haematol 1:20-34, 1955 2. Edson JR, Svvineharl CD: Antigenic differences in functionally normal antihemophiliac globulin in different bovine strains. J Lab Clin Med 83:467-477, 1974 3. Edson JR, McArthur JR, Branda RG, et al: Successful management of a subdural hematoma in a hemophiliac with an anti-Factor VIII antibody. Blood 41:113-122, 1973 4. Pool JG, Miller RG: Assay of the immune inhibitor in classic hemophilia: Application of virus-antibody reaction kinetics. Br J Haematol 22:517-528, 1972
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
concentrations were relatively constant from one batch of porcine concentrate to another, with the exception of the first two and the last batches. Thus, it is possible that antibody reacting with both active and inactive Factor VIII is contributing to the differences in apparent antibody concentrations with the porcine Factor VIII concentrates, in addition to intrinsic variations in antigenic reactivity. When animal Factor VIII concentrates are being considered for treatment of a hemophiliac patient who has an antiFactor-VIII antibody, it is very important to estimate the apparent antibody concentrations with the available concentrates, in order to select the preparation that is least reactive. In the past, animal Factor VIII has been used as a reference standard
963
