British journal of Haernatology. 1991, 77, 535-538
ADONIS
000710489100086V
Variable inactivation of human factor VIII from different sources by human factor VIII inhibitors D. LITTLEWOOD,’” SALLY A . BEVAN,’ GEOFFREYKEMBALL-COOK,’ RICHARDJ. EVANS’ TREVORW. BARROWCLIFFE’ ‘University of Cambridge, Department of Clinical Veterinary Medicine, Madingley Road, Cambridge, ’Animal Health Trust, Newmarket, Sufolk, and ’National lnstitute of Biological Standards and Control South Mymms, Potters Bar, Herts. JANET
AND
Received 3 Septamber 1990; accepted for publication 11 December 1990
Summary. The source of human factor VIIII (FVIII) had a marked effect on the inhibitory activity of a panel of eight human FVllI inhibitors. Use of conventional FVIII concentrates gave lower inhibitor titres whereas a monoclonal antibody purified FVIII concentrate gave titres similar to or
greater than those with plasma. Addition of phospholipid (PL) protected highly purified FVIII against inhibition. The content of PL-bound FVIII in concentrates may account for the observed differences.
Factor VIII (FVIII) inhibitors occur in 12-20% of patients with severe haemophilia A (Shapiro& Hultin, 1975: Shapiro, 1979: Bloom, 1981: Koberts. 1981: Kizza. 1984). These inhibitors are specific anti-FVIII antibodies which may render replacement therapy with FVlII ineffective (Koberts. 1981 ), and their occurrence represents one of the most important complications of haemophilia. Biggs & Bidwell (1959) described a method of measuring inhibitors by assaying the amount of residual FVIII after incubation of dilutions of inhibitor with a known amount of FVIII of animal origin, after incubation at 37°C for 1 h. This was superceded by the New Oxford Method (Kizza& Biggs. 1973: Austen & Rhymes. 1975). This method uses FVIII concentrate as the source of FVIII with which the inhibitor, appropriately diluted, is incubated for 4 h and the residual FVIII measured allowing potency to be ascribed to the inhibitor where one antibody unit destroys 0 . 5 units of FVIII. Most laboratories now use the Bethesda assay (Kasper et a/, 1975).which uses plasma as the source ofFVIII. The residual FVlII remaining in the mixture after a 2 h incubation period is measured and the result converted to antibody units, where one Bethesda unit (BU) is defined as destroying 50% of FVIII activity. The Bethesda and New Oxford Methods of FVIII antibody assays have been compared (Austen et a/. 1982). This study demonstrated excessive disagreement between laboratories in antibody measurement, but. on average, the result for a given sample in Bethesda units was 1.21 x the result in New Oxford units. Two additional sources of variability in inhibitor assays could be the type of concentrate and the nature of deficient
plasma used for assay of FVIII. High purity concentrates, prepared by monoclonal antibody technology, are now available and, unlike conventional concentrates, contain very little von Willebrand factor (vWF). Also, many laboratories now use immunodepleted plasmas instead of human severe haemophilic plasma (SHP) for one-stage FVIII assays and most of these immunodepleted plasmas are deficient in vWF as well as FVIII. The development of antibodies to FVIII in haemophilic dogs has been reported (Giles et a / , 1984: Pijnappels et a / . 1986: Littlewood & Barrowcliffe. 1987). The haemophilic dogs described by Littlewood & Barrowcliffe ( 1987) developed antibodies after infusions of heterologous FVIII concentrates. In the course of studying the kinetics of these antibodies it was observed that there was less inhibition of FVIII concentrate compared with the inhibition of FVIII in plasma. Bethesda assays were performed with various inhibitor samples, using both pooled normal plasma and FVIII concentrate diluted to 1 unit/ml, and the inhibitor titre was found to be some 2.8-9.6 times lower against FVIII in concentrate than in plasma. Following the results obtained with canine antibodies, studies were undertaken using a panel of human antibody samples and various sources of human FVIII. The results of these studies are reported in this paper.
MATERIALS AND METHODS Antibodies. The panel of eight human FVIII inhibitors from polytransfused haemophiliacs was kindly provided by the University Hospital of Wales, Cardiff, and the Royal Free Hospital, London. Prior to use in assays, inhibitor samples
Correspondence: Dr Janet D. Littlewood. Animal Health Trust, P.O. Box 5. Newmarket. SufTolk CB8 7DW.
535
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lanet D. Littlewood et a1
were heated a t 56°C for 30 min after thawing or reconstitution. Samples were then adsorbed with aluminium hydroxide suspension for 3 min prior to centrifugation at 1 2 000 g for 5 min. The precipitate was discarded and the supernatant stored in aliquots at -40°C. Factor VZZZ sources. Fresh frozen normal human plasma. pooled, from six donors, was obtained from the North London Blood Transfusion Service, Edgware. Human FVIII concentrates which were used were: 4th British Standard Concentrate (NIBSC 8 5/650): a commercial intermediate purity concentrate (IPC): a commercial monoclonal antibody-purified concentrate (MAPC). Inhibitor assays. The Bethesda assay (Kasper et al. 1975), using pooled normal human plasma, was used to measure FVIII inhibitor levels in each of the antibody samples. FVIII concentrates were diluted to 1 iu/ml in glyoxaline buffer, pH 7.4, prior to incubation with dilutions of inhibitor samples. After incubation for 2 h residual FVIII was measured by one-stage FVIII assay using FVIII incubated in buffer for 2 h as standard. For plasma and IPC inhibitor assays, the one-stage assay substrate was a biodepleted FVIII deficient plasma (Diagen or Organon Teknika), but in light of the studies of Dawson et a1 (1989) congenital SHP (Immuno or George King Biomedical) was used for the assays with MAPC. Antibody titre was calculated as for the Bethesda method. Dilutions of inhibitor giving values of residual FVIII between 30% and 75% were selected. The value closest to 50% was used in the calculation, or the dilution at 50% residual FVIII estimated graphically when two dilutions gave residual FVIII values equidistant from 50%. A minimum of two assays were performed with each concentrate. The reproducibility of the assay method was checked by performing the assay with one inhibitor (C1) at two dilutions, four times. against standard concentrate. The influence of von Willebrand factor (vWF) and other plasma proteins on the inhibition of concentrates was studied by performing assays using IPC and MAPC prediluted in congenital SHP (Immuno, George King Biomedical or Sigma) compared to concentrates diluted in glyoxaline buffer. Inhibitor assays were also performed with one antibody after adding purified vWF (kindly provided by Mr N. Dawson of NIBSC) to MAPC to give a final concentration of one unit of vWF per unit of FVIII. The proportion of FVIII antigen (FVIII :Ag) bound to phospholipd (PL) in plasma and in concentrates was estimated by FVII1:Ag assays using PL-site and non PL-site antibodies as described by Kemball-Cook & Barrowcliffe ( 1 986). The influence of phospholipid (PL) on the inactivation of highly purified FVIII by four inhibitor plasma was studied. MAPC diluted to 1 iu/ml was incubated with a n equal volume of PL (NIBSC reagent 83/555) at 30 pg/ml in 0.1 mM butylated hydroxyanisole. an anti-oxidant, in 0.15 M sodium chloride (BHAsaline), or BHA saline alone as control, for 10 min at 3 7OC. These mixtures were then incubated with inhibitor samples as for the Bethesda assay and assayed for residual FVIII after incubation for 2 h. The different concentrations of PL in incubation mixtures were corrected for by adding appropriate amounts of PL to the glyoxaline buffer used for assay dilutions in the one-stage FVIII assays.
n
250[ 200
Plasma
Std conc
IPC
MAPC
Factor YUI source
Fig 1. Inhibitory activity of human antibody C 1 against different sources of human factor VIII. Std conc.=4th British Standard Concentrate. 8 5 / 6 5 0 :IPC= intermediate purity concentrate:MAPC=monoclonal antibody purified concentrate.
Albumin (BPL, Elstree) was also added to the glyoxaline buffer to a final concentration of 1%. RESULTS Reproducility of inhibitor assays The geometric coefficient of variation of the inhibitor assays was found to be 9.95% and 8.27% respectively with the two dilutions of the antibody C1 at which the assays were performed. Comparison of factor VZZZ sources The Bethesda titres of the panel of human antibodies against plasma ranged from 9 . 3 4 to 9 2 4 BU/ml. The inhibition of the four sources of FVIII by human antibody C1 is shown in Fig 1. The inhibitory effect of each of the antibodies against plasma and the various FVIII concentrates is shown in Table I. expressed as Bethesda titres and in Table 11. as a percentage of the value against plasma. Inhibitor titres against the standard concentrate and IPC were approximately half and onefifth of those against plasma, respectively, the differences for these two concentrates being reasonably consistent among the eight antibodies tested (Table 11). For MAPC. however, the titres of the different antibodies relative to those against plasma were more variable, but in virtually all cases were equal to or higher than the plasma titres, the average being approximately 50% higher. The influence of prediluting IPC and MAPC in SHP on activation of FVIII in these concentrates was studied with seven antibodies. The ratio of the titres obtained with predilution in SHP compared to predilution in glyoxaline buffer for each antibody is shown in Table 111. The titre against IPC prediluted in SHP was generally the same or greater than that obtained with buffer predilution, the ratios ranging from 0.85 to 2.33. For MAPC the effect ofpredilution in SHP was more variable, with titres being similar for most antibodies. but much lower for antibodies Z and C1, these values being much closer to the titres against plasma FVIII.
Factor VZZZ Znactivation by Inhibitors Table I. Titres of human FVIII inhibitors against plasma and three concentrates Inhibitor titres (BU/ml) FVIII source
Plasma
Std conc.
IPC
53 7
Table 111. Hect of predilution of concentrates in human SHP: inhibition of factor VIII expressed as a ratio of titres against concentrate diluted in SHP and in buffer
MAPC Ratio of titres
Antibody W X Y
141 12.2 43.3 9.3 165 172 31.1 924
2
c1 c4 c7 C8
64.7 6.2 3 3.9 4.4 80.9 122 14.9 432
18.3 3.4 13.2 1.1 29.6 46.9 5.4 156
118 14.1 89.1 13.3 236 210 32.0 2924
Std conc. = 4th British Standard Concentrate, 8 5/650: IPC = intermediate purity concentrate: MAPC= monoclonal antiody purified concentrate. Table 11. Inhibition ofconcentrates by human FVIII inhibitors as a percentage of their titres against plasma
FVIII source
IPC
MAPC
cs
1.66 1.37 2.33 0.85 0.91 1.59 0.99
1.38 0.76 0.47 0.55 1.54 0.90 0.73
Mean SD
1.39 0.53
0.90 0.41
Antibody W
Y 2
c1 c4 c7
IPC =intermediate purity concentrate: MAPC= monoclonal antibody purified concentrate: SD =standard deviation.
% of plasma titre FVIII source
Std conc.
IPC
MAPC
c4 c7 C8
45.9 51.5 78.3 47.3 49.0 70.9 47.9 46.8
13.0 28.1 30.5 12.1 17.9 27.3 17.4 16.9
83.7 116 208 142 143 122 103 316
Mean SD
54.7 12.6
20.4 7.2
154 75.1
Antibody W X
Y
z
c1
Std conc. =4th British Standard Concentrate, 85/650: IPC=intermediate purity concentrate: MAPC = monoclonal antibody purified concentrate: SD= standard deviation.
Eflects of vWF In the assays where purified vWF was added to MAPC there was a marked increase in the FVIII activity of the buffer (noninhibitor) control with added vWF compared to the control incubated without vWF. as previously found by Dawson et a1 (1989).However, when the residual FVIII in tests with and without vWF was calculated against the respective controls, with and without vWF. there was no reduction in antibody titre on addition of vWF. The titre of antibody C7 against MAPC alone was 27.3 BU/ml (95% confidence limit, 21.133.6) and with vWF added was 34.6 BU/ml(95%confidence limit 33.4-35.9). Eflect of PL The proportion of FVIII :Ag bound to PL in plasma and the
Table IV. Proportion of factor VIII bound to phospholipid in plasma and concentrates PL-bound
FV1II:Ag
Plasma Std conc. IPC MAPC
0 2 5% 44% 0
PL =phospholipid: FVIII :Ag = factor VIII antigen: Std conc.= 4th British Standard Concentrate, 85/650: IPC= intermediate purity concentrate: MAPC= monoclonal antibody purified concentrate.
three concentrates is shown in Table IV. There was none bound to PL in plasma of MPAC, but significant amounts were bound in the standard concentrate and a larger proportion in the IPC. Addition of PL to MAPC resulted in much less inactivation of FVIII with three o u t of the four antibodies studied (Fig 2). The inhibitor titre was reduced three-fold for one and ninefold for two of the antibodies. DISCUSSION The difference in inhibitor activity of canine anti-FVIII antibodies with human FVIII in concentrate compared with plasma FVIII, as described previously, was also found with this panel of human inhibitors. All eight inhibitors gave less inhibition of FVIII in standard concentrate compared with plasma. The least inhibition of FVIII was seen with the IPC.
Janet D. Littlewood et a1
538
350r
particularly to the highly purified concentrates, may be beneficial.
~ M A P C
300 E
\
250
MAPC t PL
ACKNOWLEDGMENTS The commercial concentrates used in this study were obtained from Alpha Therapeutic U.K., Thetford, Norfolk. U.K., and from Baxter Hyland Division, Los Angeles, California, U.S.A. Thanks is due to Mrs Karen Negus for preparation of the manuscript. This work was funded by a grant from the Medical Research Council.
-
m 3
F
200 -
+
.D” e +
150-
loo50 -
CI
rm c7
Antibody
Fig 2. Efect of preincubating monoclonal antibody purified FVIII concentrate with phospholipid on the inhibitory effect of four human antibodies.MAPC = monoclonal antibody purified concentrate alone: MAPC + PL= monoclonal antibody purified concentrate preincubated with phospholipid.
However, whilst the inhibition of highly purified FVIII in MAPC was less than that of plasma with canine antibodies (Littlewood, 1988) the panel of human antibodies showed similar or higher levels of inhibition of this concentrate compared with plasma. A number of possible reasons were considered for the difference in titres against the various FVIII sources. The lower titres against IPC cannot be due simply to absence of plasma protein, since titres were similar in the presence and absence of FVIII-deficient plasma. The possibility that the initial absence of vWF in MAPC could explain the higher titre than against the other concentrates was also considered.The addition of purified vWF to MAPC did not reduce titres of one antibody. When MAPC was prediluted in SHP the effect on titres was not consistent (Table III), with some antibodies showing an increase in titre and others showing a reduction of up to two-fold. However, the titres againt MAPC diluted in SHP were still equal to or greater than those against plasma. This indicates that the higher titres with MAPC compared to other FVIII concentrates are not due to the absence of vWF. The most likely explanation for the differencesin titre is the different proportions of PL-bound FVIII in the different materials. It has already been shown that PL can consistently protect against FVIII inhibition by some antibodies (Barrowcliffe et al. 1983, 1984: Littlewood & Barrowchffe, 1987; Littlewood, 1988). and that a proportion of FVIII in concentrates appears to be bound to PL (Kemball-Cook & Barrowcliffe, 1986).In MAPC none of the FVIII appeared to be bound to PL. The hypothesis that addition of PL to MAPC would reduce inhibition was tested and found to be true for all but one of the human antibodies studied. Addition of PL to MAPC reduced the inhibition by antibodies W, C1 and C7 to the same order as that obtained with IPC. Indeed, the PL content of concentrate appears to correlate with the degree of reduction of inhibition of FVIII by the human antibodies, compared to plasma titres. These findings have implications for the selection of concentrates for the treatment of inhibitor patients and add further support to the suggestion that addition of PL.
REFERENCES Austen. D.E.G.. Lechner. K., Riza. C.R. & Rhymes, I.L. (1982) A comparison of the Bethesda and New Oxford methods of factor VIII antibody assay. Thrombosis and Haemostasis. 4 7 , 72-75. Austen D.E.G. & Rhymes, I.L. (1975) A Laboratory Manual of Blood Coagulation, pp. 68. Blackwell Scientific Publications, Oxford. Barrowcliffe, T.W., Kemball-Cook. G. & Gray, E. (1983) Binding to phospholipid protects factor VIII from inactivation by human antibodies. journal oflaboratory and Clinical Medicine, 101, 34-43. Barrowclie, T.W.. Kemball-Cook, G. & Gray, E. (1984) Efect of phospholipid on factor VIII inactivation. Factor VllZ Znhibitors (ed. by L. Hoyer). pp. 251-263. Alan R. Liss. New York. Biggs. R. & Bidwell, E. (1959) A method for the study of antihaemophilic globulin inhibitors with reference to six cases. British Journal of Haematology, 5, 379-395. Bloom. A.L. (1981) Factor VIII inhibitors revisited. British Journal of Haematology 49, 3 19-324. Dawson, N.J.. Kemball-Cook,G. & Barrowcliffe, T.W. (1989) Assay discrepancies with highly purified factor VIII concentrates. Haemostasis, 19, 131-1 37. Giles, A.R.. Tinlin. S.. Hoogendoorn. H., Greenwood, P. & Greenwood, R. (1984) Development of factor VII1:C antibodies in dogs with hemophilia A (factor VII1:C deficiency).Blood, 6 3 . 4 5 1-456. Kasper. C.K.. Aldedort. L.M.. Counts, R.B.. Edson. J.R.. Frantatoni, J., Green, D.. Hampton, J.W., Hilgartner. M.W.. Lazerson, J . , Levine. P.H.. McMillan, C.W., Pool, J.G., Shapiro, S.S., Shulrnan, N.R.& von Eys, J. (1975) A more uniform measurement of factor VIII inhibitors. Thrombosis et Diathesis Haemorrhagica, 34. 869-872. Kemball-Cook, G. & Barrowcliffe. T.W. (1986) Factor VIII concentrates contain factor VIII procoagulant antigen bound to phospholipid. British Journal of Haematology. 63, 425-434. Littlewood. J.D. (1988) Factor VIII-phospholipid mixtures and factor VIII inhibitors: studies in haemophilic dogs. Ph.D. thesis, University of Cambridge. Littlewood. J.D. & Barrowcliffe. T.W. (1987) The development and characterisation of antibodies to human factor VIII in haemophilic dogs. Thrombosis and Haemostasis, 57. 314-321. Pijnappels, M.I.M., Briet. E.. van der Zweet. G. Th., Huisden. R., van Tilburg. N.H. & Eulderink. F. (1986) Evaluation of the cuticle bleeding time in canine haemophilia A. Thrombosis and Haemostasis, 55, 70-73. Rizza, C.R. (1984) The management of haemophiliacs who have antibodies to factor VIII. Scandinavian Iournal of Haematology. 33. SUPPI.40.187-193. R h a . C.R. & Biggs. R. (1973) The treatment of patients who have factor VIII antibodies. British Journal of Haematology, 52, 13-24. Roberts, H.R. (1981) Hemophiliacs with inhibitors: therapeutic options. New England lournal of Medicine. 305, 75 7-758. Shapiro, S.S. (1979) Antibodies to blood coagulation factors. Clinics in Hematology. 8, 207-214. Shapiro, S.S. & Hultin, M. (1975) Acquired inhibitors to the blood coagulation factors. Seminars in Thrombosis and Hemostasis. 1, 336-385.
ADONIS
000710489100086V
Variable inactivation of human factor VIII from different sources by human factor VIII inhibitors D. LITTLEWOOD,’” SALLY A . BEVAN,’ GEOFFREYKEMBALL-COOK,’ RICHARDJ. EVANS’ TREVORW. BARROWCLIFFE’ ‘University of Cambridge, Department of Clinical Veterinary Medicine, Madingley Road, Cambridge, ’Animal Health Trust, Newmarket, Sufolk, and ’National lnstitute of Biological Standards and Control South Mymms, Potters Bar, Herts. JANET
AND
Received 3 Septamber 1990; accepted for publication 11 December 1990
Summary. The source of human factor VIIII (FVIII) had a marked effect on the inhibitory activity of a panel of eight human FVllI inhibitors. Use of conventional FVIII concentrates gave lower inhibitor titres whereas a monoclonal antibody purified FVIII concentrate gave titres similar to or
greater than those with plasma. Addition of phospholipid (PL) protected highly purified FVIII against inhibition. The content of PL-bound FVIII in concentrates may account for the observed differences.
Factor VIII (FVIII) inhibitors occur in 12-20% of patients with severe haemophilia A (Shapiro& Hultin, 1975: Shapiro, 1979: Bloom, 1981: Koberts. 1981: Kizza. 1984). These inhibitors are specific anti-FVIII antibodies which may render replacement therapy with FVlII ineffective (Koberts. 1981 ), and their occurrence represents one of the most important complications of haemophilia. Biggs & Bidwell (1959) described a method of measuring inhibitors by assaying the amount of residual FVIII after incubation of dilutions of inhibitor with a known amount of FVIII of animal origin, after incubation at 37°C for 1 h. This was superceded by the New Oxford Method (Kizza& Biggs. 1973: Austen & Rhymes. 1975). This method uses FVIII concentrate as the source of FVIII with which the inhibitor, appropriately diluted, is incubated for 4 h and the residual FVIII measured allowing potency to be ascribed to the inhibitor where one antibody unit destroys 0 . 5 units of FVIII. Most laboratories now use the Bethesda assay (Kasper et a/, 1975).which uses plasma as the source ofFVIII. The residual FVlII remaining in the mixture after a 2 h incubation period is measured and the result converted to antibody units, where one Bethesda unit (BU) is defined as destroying 50% of FVIII activity. The Bethesda and New Oxford Methods of FVIII antibody assays have been compared (Austen et a/. 1982). This study demonstrated excessive disagreement between laboratories in antibody measurement, but. on average, the result for a given sample in Bethesda units was 1.21 x the result in New Oxford units. Two additional sources of variability in inhibitor assays could be the type of concentrate and the nature of deficient
plasma used for assay of FVIII. High purity concentrates, prepared by monoclonal antibody technology, are now available and, unlike conventional concentrates, contain very little von Willebrand factor (vWF). Also, many laboratories now use immunodepleted plasmas instead of human severe haemophilic plasma (SHP) for one-stage FVIII assays and most of these immunodepleted plasmas are deficient in vWF as well as FVIII. The development of antibodies to FVIII in haemophilic dogs has been reported (Giles et a / , 1984: Pijnappels et a / . 1986: Littlewood & Barrowcliffe. 1987). The haemophilic dogs described by Littlewood & Barrowcliffe ( 1987) developed antibodies after infusions of heterologous FVIII concentrates. In the course of studying the kinetics of these antibodies it was observed that there was less inhibition of FVIII concentrate compared with the inhibition of FVIII in plasma. Bethesda assays were performed with various inhibitor samples, using both pooled normal plasma and FVIII concentrate diluted to 1 unit/ml, and the inhibitor titre was found to be some 2.8-9.6 times lower against FVIII in concentrate than in plasma. Following the results obtained with canine antibodies, studies were undertaken using a panel of human antibody samples and various sources of human FVIII. The results of these studies are reported in this paper.
MATERIALS AND METHODS Antibodies. The panel of eight human FVIII inhibitors from polytransfused haemophiliacs was kindly provided by the University Hospital of Wales, Cardiff, and the Royal Free Hospital, London. Prior to use in assays, inhibitor samples
Correspondence: Dr Janet D. Littlewood. Animal Health Trust, P.O. Box 5. Newmarket. SufTolk CB8 7DW.
535
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lanet D. Littlewood et a1
were heated a t 56°C for 30 min after thawing or reconstitution. Samples were then adsorbed with aluminium hydroxide suspension for 3 min prior to centrifugation at 1 2 000 g for 5 min. The precipitate was discarded and the supernatant stored in aliquots at -40°C. Factor VZZZ sources. Fresh frozen normal human plasma. pooled, from six donors, was obtained from the North London Blood Transfusion Service, Edgware. Human FVIII concentrates which were used were: 4th British Standard Concentrate (NIBSC 8 5/650): a commercial intermediate purity concentrate (IPC): a commercial monoclonal antibody-purified concentrate (MAPC). Inhibitor assays. The Bethesda assay (Kasper et al. 1975), using pooled normal human plasma, was used to measure FVIII inhibitor levels in each of the antibody samples. FVIII concentrates were diluted to 1 iu/ml in glyoxaline buffer, pH 7.4, prior to incubation with dilutions of inhibitor samples. After incubation for 2 h residual FVIII was measured by one-stage FVIII assay using FVIII incubated in buffer for 2 h as standard. For plasma and IPC inhibitor assays, the one-stage assay substrate was a biodepleted FVIII deficient plasma (Diagen or Organon Teknika), but in light of the studies of Dawson et a1 (1989) congenital SHP (Immuno or George King Biomedical) was used for the assays with MAPC. Antibody titre was calculated as for the Bethesda method. Dilutions of inhibitor giving values of residual FVIII between 30% and 75% were selected. The value closest to 50% was used in the calculation, or the dilution at 50% residual FVIII estimated graphically when two dilutions gave residual FVIII values equidistant from 50%. A minimum of two assays were performed with each concentrate. The reproducibility of the assay method was checked by performing the assay with one inhibitor (C1) at two dilutions, four times. against standard concentrate. The influence of von Willebrand factor (vWF) and other plasma proteins on the inhibition of concentrates was studied by performing assays using IPC and MAPC prediluted in congenital SHP (Immuno, George King Biomedical or Sigma) compared to concentrates diluted in glyoxaline buffer. Inhibitor assays were also performed with one antibody after adding purified vWF (kindly provided by Mr N. Dawson of NIBSC) to MAPC to give a final concentration of one unit of vWF per unit of FVIII. The proportion of FVIII antigen (FVIII :Ag) bound to phospholipd (PL) in plasma and in concentrates was estimated by FVII1:Ag assays using PL-site and non PL-site antibodies as described by Kemball-Cook & Barrowcliffe ( 1 986). The influence of phospholipid (PL) on the inactivation of highly purified FVIII by four inhibitor plasma was studied. MAPC diluted to 1 iu/ml was incubated with a n equal volume of PL (NIBSC reagent 83/555) at 30 pg/ml in 0.1 mM butylated hydroxyanisole. an anti-oxidant, in 0.15 M sodium chloride (BHAsaline), or BHA saline alone as control, for 10 min at 3 7OC. These mixtures were then incubated with inhibitor samples as for the Bethesda assay and assayed for residual FVIII after incubation for 2 h. The different concentrations of PL in incubation mixtures were corrected for by adding appropriate amounts of PL to the glyoxaline buffer used for assay dilutions in the one-stage FVIII assays.
n
250[ 200
Plasma
Std conc
IPC
MAPC
Factor YUI source
Fig 1. Inhibitory activity of human antibody C 1 against different sources of human factor VIII. Std conc.=4th British Standard Concentrate. 8 5 / 6 5 0 :IPC= intermediate purity concentrate:MAPC=monoclonal antibody purified concentrate.
Albumin (BPL, Elstree) was also added to the glyoxaline buffer to a final concentration of 1%. RESULTS Reproducility of inhibitor assays The geometric coefficient of variation of the inhibitor assays was found to be 9.95% and 8.27% respectively with the two dilutions of the antibody C1 at which the assays were performed. Comparison of factor VZZZ sources The Bethesda titres of the panel of human antibodies against plasma ranged from 9 . 3 4 to 9 2 4 BU/ml. The inhibition of the four sources of FVIII by human antibody C1 is shown in Fig 1. The inhibitory effect of each of the antibodies against plasma and the various FVIII concentrates is shown in Table I. expressed as Bethesda titres and in Table 11. as a percentage of the value against plasma. Inhibitor titres against the standard concentrate and IPC were approximately half and onefifth of those against plasma, respectively, the differences for these two concentrates being reasonably consistent among the eight antibodies tested (Table 11). For MAPC. however, the titres of the different antibodies relative to those against plasma were more variable, but in virtually all cases were equal to or higher than the plasma titres, the average being approximately 50% higher. The influence of prediluting IPC and MAPC in SHP on activation of FVIII in these concentrates was studied with seven antibodies. The ratio of the titres obtained with predilution in SHP compared to predilution in glyoxaline buffer for each antibody is shown in Table 111. The titre against IPC prediluted in SHP was generally the same or greater than that obtained with buffer predilution, the ratios ranging from 0.85 to 2.33. For MAPC the effect ofpredilution in SHP was more variable, with titres being similar for most antibodies. but much lower for antibodies Z and C1, these values being much closer to the titres against plasma FVIII.
Factor VZZZ Znactivation by Inhibitors Table I. Titres of human FVIII inhibitors against plasma and three concentrates Inhibitor titres (BU/ml) FVIII source
Plasma
Std conc.
IPC
53 7
Table 111. Hect of predilution of concentrates in human SHP: inhibition of factor VIII expressed as a ratio of titres against concentrate diluted in SHP and in buffer
MAPC Ratio of titres
Antibody W X Y
141 12.2 43.3 9.3 165 172 31.1 924
2
c1 c4 c7 C8
64.7 6.2 3 3.9 4.4 80.9 122 14.9 432
18.3 3.4 13.2 1.1 29.6 46.9 5.4 156
118 14.1 89.1 13.3 236 210 32.0 2924
Std conc. = 4th British Standard Concentrate, 8 5/650: IPC = intermediate purity concentrate: MAPC= monoclonal antiody purified concentrate. Table 11. Inhibition ofconcentrates by human FVIII inhibitors as a percentage of their titres against plasma
FVIII source
IPC
MAPC
cs
1.66 1.37 2.33 0.85 0.91 1.59 0.99
1.38 0.76 0.47 0.55 1.54 0.90 0.73
Mean SD
1.39 0.53
0.90 0.41
Antibody W
Y 2
c1 c4 c7
IPC =intermediate purity concentrate: MAPC= monoclonal antibody purified concentrate: SD =standard deviation.
% of plasma titre FVIII source
Std conc.
IPC
MAPC
c4 c7 C8
45.9 51.5 78.3 47.3 49.0 70.9 47.9 46.8
13.0 28.1 30.5 12.1 17.9 27.3 17.4 16.9
83.7 116 208 142 143 122 103 316
Mean SD
54.7 12.6
20.4 7.2
154 75.1
Antibody W X
Y
z
c1
Std conc. =4th British Standard Concentrate, 85/650: IPC=intermediate purity concentrate: MAPC = monoclonal antibody purified concentrate: SD= standard deviation.
Eflects of vWF In the assays where purified vWF was added to MAPC there was a marked increase in the FVIII activity of the buffer (noninhibitor) control with added vWF compared to the control incubated without vWF. as previously found by Dawson et a1 (1989).However, when the residual FVIII in tests with and without vWF was calculated against the respective controls, with and without vWF. there was no reduction in antibody titre on addition of vWF. The titre of antibody C7 against MAPC alone was 27.3 BU/ml (95% confidence limit, 21.133.6) and with vWF added was 34.6 BU/ml(95%confidence limit 33.4-35.9). Eflect of PL The proportion of FVIII :Ag bound to PL in plasma and the
Table IV. Proportion of factor VIII bound to phospholipid in plasma and concentrates PL-bound
FV1II:Ag
Plasma Std conc. IPC MAPC
0 2 5% 44% 0
PL =phospholipid: FVIII :Ag = factor VIII antigen: Std conc.= 4th British Standard Concentrate, 85/650: IPC= intermediate purity concentrate: MAPC= monoclonal antibody purified concentrate.
three concentrates is shown in Table IV. There was none bound to PL in plasma of MPAC, but significant amounts were bound in the standard concentrate and a larger proportion in the IPC. Addition of PL to MAPC resulted in much less inactivation of FVIII with three o u t of the four antibodies studied (Fig 2). The inhibitor titre was reduced three-fold for one and ninefold for two of the antibodies. DISCUSSION The difference in inhibitor activity of canine anti-FVIII antibodies with human FVIII in concentrate compared with plasma FVIII, as described previously, was also found with this panel of human inhibitors. All eight inhibitors gave less inhibition of FVIII in standard concentrate compared with plasma. The least inhibition of FVIII was seen with the IPC.
Janet D. Littlewood et a1
538
350r
particularly to the highly purified concentrates, may be beneficial.
~ M A P C
300 E
\
250
MAPC t PL
ACKNOWLEDGMENTS The commercial concentrates used in this study were obtained from Alpha Therapeutic U.K., Thetford, Norfolk. U.K., and from Baxter Hyland Division, Los Angeles, California, U.S.A. Thanks is due to Mrs Karen Negus for preparation of the manuscript. This work was funded by a grant from the Medical Research Council.
-
m 3
F
200 -
+
.D” e +
150-
loo50 -
CI
rm c7
Antibody
Fig 2. Efect of preincubating monoclonal antibody purified FVIII concentrate with phospholipid on the inhibitory effect of four human antibodies.MAPC = monoclonal antibody purified concentrate alone: MAPC + PL= monoclonal antibody purified concentrate preincubated with phospholipid.
However, whilst the inhibition of highly purified FVIII in MAPC was less than that of plasma with canine antibodies (Littlewood, 1988) the panel of human antibodies showed similar or higher levels of inhibition of this concentrate compared with plasma. A number of possible reasons were considered for the difference in titres against the various FVIII sources. The lower titres against IPC cannot be due simply to absence of plasma protein, since titres were similar in the presence and absence of FVIII-deficient plasma. The possibility that the initial absence of vWF in MAPC could explain the higher titre than against the other concentrates was also considered.The addition of purified vWF to MAPC did not reduce titres of one antibody. When MAPC was prediluted in SHP the effect on titres was not consistent (Table III), with some antibodies showing an increase in titre and others showing a reduction of up to two-fold. However, the titres againt MAPC diluted in SHP were still equal to or greater than those against plasma. This indicates that the higher titres with MAPC compared to other FVIII concentrates are not due to the absence of vWF. The most likely explanation for the differencesin titre is the different proportions of PL-bound FVIII in the different materials. It has already been shown that PL can consistently protect against FVIII inhibition by some antibodies (Barrowcliffe et al. 1983, 1984: Littlewood & Barrowchffe, 1987; Littlewood, 1988). and that a proportion of FVIII in concentrates appears to be bound to PL (Kemball-Cook & Barrowcliffe, 1986).In MAPC none of the FVIII appeared to be bound to PL. The hypothesis that addition of PL to MAPC would reduce inhibition was tested and found to be true for all but one of the human antibodies studied. Addition of PL to MAPC reduced the inhibition by antibodies W, C1 and C7 to the same order as that obtained with IPC. Indeed, the PL content of concentrate appears to correlate with the degree of reduction of inhibition of FVIII by the human antibodies, compared to plasma titres. These findings have implications for the selection of concentrates for the treatment of inhibitor patients and add further support to the suggestion that addition of PL.
REFERENCES Austen. D.E.G.. Lechner. K., Riza. C.R. & Rhymes, I.L. (1982) A comparison of the Bethesda and New Oxford methods of factor VIII antibody assay. Thrombosis and Haemostasis. 4 7 , 72-75. Austen D.E.G. & Rhymes, I.L. (1975) A Laboratory Manual of Blood Coagulation, pp. 68. Blackwell Scientific Publications, Oxford. Barrowcliffe, T.W., Kemball-Cook. G. & Gray, E. (1983) Binding to phospholipid protects factor VIII from inactivation by human antibodies. journal oflaboratory and Clinical Medicine, 101, 34-43. Barrowclie, T.W.. Kemball-Cook, G. & Gray, E. (1984) Efect of phospholipid on factor VIII inactivation. Factor VllZ Znhibitors (ed. by L. Hoyer). pp. 251-263. Alan R. Liss. New York. Biggs. R. & Bidwell, E. (1959) A method for the study of antihaemophilic globulin inhibitors with reference to six cases. British Journal of Haematology, 5, 379-395. Bloom. A.L. (1981) Factor VIII inhibitors revisited. British Journal of Haematology 49, 3 19-324. Dawson, N.J.. Kemball-Cook,G. & Barrowcliffe, T.W. (1989) Assay discrepancies with highly purified factor VIII concentrates. Haemostasis, 19, 131-1 37. Giles, A.R.. Tinlin. S.. Hoogendoorn. H., Greenwood, P. & Greenwood, R. (1984) Development of factor VII1:C antibodies in dogs with hemophilia A (factor VII1:C deficiency).Blood, 6 3 . 4 5 1-456. Kasper. C.K.. Aldedort. L.M.. Counts, R.B.. Edson. J.R.. Frantatoni, J., Green, D.. Hampton, J.W., Hilgartner. M.W.. Lazerson, J . , Levine. P.H.. McMillan, C.W., Pool, J.G., Shapiro, S.S., Shulrnan, N.R.& von Eys, J. (1975) A more uniform measurement of factor VIII inhibitors. Thrombosis et Diathesis Haemorrhagica, 34. 869-872. Kemball-Cook, G. & Barrowcliffe. T.W. (1986) Factor VIII concentrates contain factor VIII procoagulant antigen bound to phospholipid. British Journal of Haematology. 63, 425-434. Littlewood. J.D. (1988) Factor VIII-phospholipid mixtures and factor VIII inhibitors: studies in haemophilic dogs. Ph.D. thesis, University of Cambridge. Littlewood. J.D. & Barrowcliffe. T.W. (1987) The development and characterisation of antibodies to human factor VIII in haemophilic dogs. Thrombosis and Haemostasis, 57. 314-321. Pijnappels, M.I.M., Briet. E.. van der Zweet. G. Th., Huisden. R., van Tilburg. N.H. & Eulderink. F. (1986) Evaluation of the cuticle bleeding time in canine haemophilia A. Thrombosis and Haemostasis, 55, 70-73. Rizza, C.R. (1984) The management of haemophiliacs who have antibodies to factor VIII. Scandinavian Iournal of Haematology. 33. SUPPI.40.187-193. R h a . C.R. & Biggs. R. (1973) The treatment of patients who have factor VIII antibodies. British Journal of Haematology, 52, 13-24. Roberts, H.R. (1981) Hemophiliacs with inhibitors: therapeutic options. New England lournal of Medicine. 305, 75 7-758. Shapiro, S.S. (1979) Antibodies to blood coagulation factors. Clinics in Hematology. 8, 207-214. Shapiro, S.S. & Hultin, M. (1975) Acquired inhibitors to the blood coagulation factors. Seminars in Thrombosis and Hemostasis. 1, 336-385.
