STANDARDIZATION OF ASSAYS OF FACTOR VIII AND FACTOR IX ° TREVOR W. ]~ARROWCLIFFE National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire
Although the assay methods for factor VIII and factor IX, which are still in use today, were developed over forty years ago, it is only in the last 15-20 years that there have been concerted efforts towards the standardization and quality control of these clinically important assays. After briefly describing the development of international and national concentrate and plasma standards, and the extent of agreement between laboratories in collaborative studies, this review will focus on recent developments which have impact o n the standardization of these assays in clinical laboratories. The introduction of plasmas depleted in factors VIII or IX by immunological methods as substrates for one-stage assays, and of the chromogenic method for factor VIII, are two important methodological changes, and comparisons with results from traditional methodology are of particular interest. Finally, some problems in the assay of factor VIII in concentrates of very high purity, prep~ared by monoclonal antibody and recombinant DNA technology, will be discussed. International standards f o r factors VIII a n d I X
The original definition of the unit of biological activity for both factors VIII and IX was the activity present in 1 ml o f fresh normal plasma. This proved inadequate in practice to effect standardization between laboratories because of the large variation of factor VIII and factor IX in the normal population. Table 1 shows the range of values of these factors in normal pools of at least 15 donors. The development of factor VIII concentrates as articles of commerce in the late 1960's accelerated the need for a stable reference preparation against which these products could be compared, and the 1st international standard for factor VIII concentrate was established in 19711,28. Since then the standard has been replaced at regular intervals, and all manufacturers of factor VIII concentrates around the world have used these standards directly or indirectly to calibrate their materials. Key-words: Assays; Factor VIII; FactorIX; Standards.
• Presented at the '2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implicationsfor the Clinical Laboratory', Rome, September 28-29, 1989. Res. Clin. Lab. 20, 155-165, 1990. 155
FACTOR VIII AND FACTOR IX ASSAYS
n-* o f l a b o r a t o r i e s
r a n g e o f values (IU/ml)
factor VIII
21
0.63-1.35
f a c t o r IX
23
0.82-1.37
Tab. 1 - F a c t o r V I I I a n d f a c t o r I X c o n t e n t o f n o r m a l p o o l s . (Data from international collaborative studies4.12. Pools in each laboratory are from at least 15 donors).
Similarly, a factor IX concentrate international standard was established in 197615.~9, a n d was replaced by the 2nd IS in 19874,3°. For assays o f clinical plasma samples laboratories use either nadonal plasma standards where available, commercial plasma standards, or local plasma pools. Calibration o f these plasma standards against the IS for factor VIII concentrate was found to give large discrepancies between laboratories and between assay methods n,19. T h e r e f o r e , it was decided to establish a plasma standard for factor VIII to coexist with the concentrate standard. This reference plasma was established in 1982, a n d calibrated for the o t h e r factor VIIIrelated activities as well as factor VIII:C, i.e. factor VIII:Ag, and von Willeb r a n d factor activity and andgen; it has recently b e e n replaced by the 2nd IS for factor VIII and von Willebrand factor in plasma. Similar considerations led to the establishment in 1987 o f a plasma IS for factor IX; this plasma was also calibrated for the other prothrombin complex factors II, VII, and X. Table 2 summarizes the various plasma a n d concentrate international standards which have b e e n established for factors VIII a n d IX.
Precision of factor VIII and factor IX assays In the calibration o f international standards, laboratories are asked to perform repeat assays on the same samples, a n d so information can be obtained on the within-laboratory variability between assays. Table 3 summarizes the average geometric coefficients o f variation (GCV's) within laboratories in five international collaborative studies. It can be seen that there has b e e n a general improvement in precision since 1971, and both the major methods can now be regarded as extremely precise, especially on assays o f concentrates. T h e r e is less information about the precision o f factor IX assays, but in the recent international collaborative study to establish plasma and concentrate standards for factor I X 4, the average GCV was 20.4%.
factorVIII
factorIX
concentrates
plasmas
concentrates
1st IS 1971
1st IRP 1983
1st IS 1974
2nd IS 1978
2nd IS 1989
2rid IS 1987
3rd IS 1983 4th IS 1989
Tab. 2 - International standards for factors VIII and IX. 156
plasmas 1st IS 1987
T. W. BA.RROWCLIFFE
avemge GCV% date of study
samples assayed
1971 1976 1981 1982 1983
Tab.
3 -
trate; P
C vs C vs P vs C vs C vs
one-stage
two-stage
45 16 12.4 9.9 6.2
12 10 13.3 8.9 3.9
P P P C C
Within-laboratory precision of factor VIII assays in collaborative studies. (c ~- concen-
= plasma).
O f course, these average GCV's conceal considerable variation b e t w e e n laboratories - in the study o f 1983, f o r instance, the within-laboratory GCV's r a n g e d f r o m 5 to 23% i2. Nonetheless, these figures do give an indication o f what is achievable. Between-laboratory
variability
T h e i m p r o v e d intralaboratory precision in r e c e n t studies is also reflected in i m p r o v e d a g r e e m e n t between laboratories, as can b e seen in tab. 4. T h e GCV's b e t w e e n laboratories in the most r e c e n t study o f factor VIII are similar to the within-laboratory precision, indicating that a g r e e m e n t b e t w e e n laboratories is a b o u t as g o o d as can be expected. A g r e e m e n t b e t w e e n laboratories in factor IX assays d u r i n g the r e c e n t international collaborative study was not as g o o d as for factor VIII. GCV's are s u m m a r i z e d in tab. 5, and the distribution o f potencies is illustrated in fig. 1. It must b e stressed that the degree o f variability between laboratories dep e n d s very m u c h o n the type o f sample being assayed a n d its similarity to the standard b e i n g used. Thus, for concentrates it has b e e n established in m a n y studies that variability is less w h e n c o m p a r e d to a concentrate standard than w h e n a plasma standard is used 4,5,~° - this is illustrated for factor IX by the results in tab. 5. T h e development o f new purification methods for concentrates, including m o n o c l o n a l antibody purification from b o t h plasma a n d rDNA sources, has m e a n t that some concentrates may differ in composition from the concentrate standards used. In this case, interlaboratory variability may b e considerably greater t h a n that shown in tab. 4, as discussed in a subsequent section.
GCV% date of study
samples assayed one-stage
1971
{C
1983
{C
1989
vs C vs
P C
376 26
two-stage 68 13
C vs
P C
11.6 7.4
17.1 3.5
C
C
5.6
5.9
vs
vs
Tab. 4 - Between-laboratory precision of factor VIII assays in collaborative studies. 157
FACTOR VIII AND FACTOR IX ASSAYS
comparison
GCV%
concentrate vs plasma concentrate vs concentrate
22.7 8,9
Tab. 5 - Between-laboratory precision of factor IX assays in an international collaborative study~.
Predilution of concentrates
A n o t h e r factor which may affect the variability b e t w e e n laboratories is the m e t h o d o f predilution. It was s h o w n by LEE et al. 21 that predilution o f intermediate purity factor VIII concentrates in h e m o p h i l i c plasma increased their potency by approximately 25%, c o m p a r e d to the same c o n c e n t r a t e p r e d i l u t e d in b u f f e r only. A similar p h e n o m e n o n was f o u n d for factor IX in the international collaborative study 4. T h e r e f o r e , if c o n c e n t r a t e s are assayed in clinical laboratories against plasma standards, it is essential to predilute c o n c e n t r a t e s in the appropriate deficient plasma in o r d e r to obtain the correct potency. Since b o t h plasma a n d c o n c e n t r a t e i n t e r n a t i o n a l standards are available for factors VIII a n d IX, it was n e c e s s a r y to standardize the m e t h o d o f predilu-
8~
11 13 17 14 18 15
42-
~ r 50
_~
21 19 22 23 16 10 I i
100 potency (%overallmean)
200
300
200
3&
3 7
110 13 ~: r
5tO
i11 14 119 16 15 23 2C 100 potency (% overall mean)
Fig. 1 - Distribution of potencies of factor IX concentrate Q (2nd IS) vs T (lst IS). Each square represents the average result of 4 assays from individual laboratories. Assays were carried out with both buffer (upper histogram) and deficient plasma (lower histogram) as prediluents. (Datafroma n international collaborative study). 158
T. W. BARROWCLIFFE
potency (IU/ampoule)
date of study
1979 1981 1982 1984 1988
one-stage
two-stage
0.57 0.60 0.70 0.73 0.60
0.57 0.62 0.69 0.74 0.66
Tab. 6 - Comparison of one-stage and two-stage factor VIII assays on plasma samples.
t_ion in order to get equivalence between plasma and concentrate units. When the 3rd IS for factor VIII concentrate was established, it was assayed against both the concentrate and plasma standards, using predilution in hemophilic plasma for comparison against the plasma standard. In one-stage assays there was no significant difference between these two values, though a 20% difference remained in two-stage assays 5. For factor IX the situation was somewhat more complicated, since calibration of the 2nd IS against the plasma standard, using factor IX-deficient plasma as prediluent, yielded a value some 20% higher than that against the 1st IS concentrate 4,~z. The higher potency was chosen for the 2nd IS, to achieve comparability between plasma and concentrate units, and consistency with the factor VIII situation, and this effectively meant a reevaluation of the unit o f factor IX. For routine assays of concentrates against a concentrate standard in our laboratory, we have not found it necessary to predilute in deficient plasma. However, this policy may have to be revised when new high-purity concentrates become available, as discussed in a subsequent section.
Comparison of methods Comparisons between one-stage and two-stage assays of factor VIII have been extensively reviewed elsewhere z~, and will only be mentioned briefly here. The two-stage method has given better precision in most studies than the one-stage method, both within- and between-laboratories, although in recent studies the differences have been small (tabs 3 and 4). In most collaborative studies normal plasmas have given identical results with the two methods, though somewhat surprisingly a significant difference was found in the most recent collaborative study, as shown in tab. 6. It is welbknown that concentrates when assayed against a plasma standard give higher potencies by two-stage than by one-stage assays 5,11. Most concentrates when assayed against a concentrate standard give similar potencies by the two methods, but some products have consistently given significant differences, with one-stage potencies usually higher than two-stageZL Reasons for the discrepancies include the possible presence of activated factor VIII in some concentrates 6,7, and influence of non-factor VIII contaminants, such as lipids, on the assays 2. The introduction of a chromogenic method for factor VIII, which uses purified clotting factors and an amidolytic substrate for factor Xa, could eventually lead to improved standardization. The method is the same in principle as that of the two-stage method, and in comparative assays in our laboratory 159
FACTOR VIII AND FACTOR IX ASSAYS
IP concentrate n-" of laboratories
VHP concentrate
method potency (IU/ml)
GCV%
potency (IU/ml)
GCV%
26
one-s rage
6.10
5.6
4.97
11
two-stage
6.36
5.9
4.88
13.8 7.6
15
chromogenic
6_63
9,2
4,89
14.3
28
over'all
6_30
7.7
4.93
12.7
Tab. 7 - Potencies of intermediate purity (IP) and very high purity (VHP) concentrates in 4th IS study.
the c h r o m o g e n i c a n d two-stage m e t h o d s gave very similar results o n most concentrates 18. A collaborative study involving f o u r clinical laboratories also f o u n d n o significant differences in assays o f clinical plasma samples by one-stage a n d c h r o m o g e n i c assays 26. I n the most r e c e n t i n t e r n a t i o n a l collaborative study o f concentrates, the c h r o m o g e n i c m e t h o d gave identical results to the one-stage a n d two-stage methods o n a m o n o c l o n a l antibody purified concentrate, but slightly h i g h e r resuits o n a n intermediate purity c o n c e n t r a t e s. T h e 8% d i f f e r e n c e b e t w e e n chrom o g e n i c a n d one-stage assays o n the latter c o n c e n t r a t e was statistically significant (tab. 7). Although i m p r o v e d precision has b e e n claimed for the chromo° genic m e t h o d a n d d e m o n s t r a t e d in some laboratories ~3,23,26, between-laboratory GCV's in the most r e c e n t i n t e r n a t i o n a l collaborative study were h i g h e r t h a n for one-stage a n d two-stage assays, as shown in tab. 7. Comparison of laboratories in N E Q A S surveys
Although international collaborative studies have indicated the h i g h deg r e e o f a g r e e m e n t which can b e achieved b e t w e e n laboratories, it must b e r e c o g n i z e d that laboratories which take part in these studies are large centres p e r f o r m i n g m a n y assays o n a r e g u l a r basis. A b e t t e r assessment o f assay p e r f o r m a n c e by routine clinical laboratories is given b y the results o f national external quality assurance s c h e m e (NEQAS) surveys, which are c a r r i e d out in m a n y countries, including the UK. Table 8 summarizes the results o f factor VIII assays in two r e c e n t UK surveys o f o v e r 200 laboratories, a n d the distribution o f results o n o n e o f these samples is s h o w n in fig. 2. It can b e s e e n that a g r e e m e n t b e t w e e n laboratories is m u c h worse t h a n in the i n t e r n a t i o n a l studies. Apart f r o m the fact that these surveys involve a larger n u m b e r o f laboratories, m a n y o f which may per-
between-laboratory GCV% potency (IU/dl)
36 8.1
one-stage
two-stage
36.6 41.4
46.1 72.9
Tab. 8 - Factor VIII assays in UK NEQAS. (Datareproducedwith kind permissionfrom Dr. P. B. A. Kernoff). 160
T. W. BARROWCLIFFE
manufacturer
n ~- of laboratories
A B C D E 10 others
62 45 22 21 I0 54
total
214
Tab. 9 - A P T T r e a g e n t s u s e d i n o n e - s t a g e f a c t o r V I I I a s s a y s i n UK. (Data reproduced from the UK
NEQAS survey with kind permission from Dr. P. B. A. Kernofjg.
form assays infrequently, there are two main reasons why results in NEQAS schemes may be more variable. Firstly, in NEQAS schemes only a single assay is usually performed, and very often the test sample is assayed using only one or two dilutions 14. In international studies, results from each laboratory are means from at least 4, sometimes 6, assays, with test samples tested at at least 3 dilutions. Thus, interlaboratory agreement in NEQAS surveys could probably be improved b y more repeat testing on each sample. The second reason is the multiplicity of reagent systems used. Tables 9 and 10 show the various substrate plasmas and the activated partial thromboplastin time (APTT) reagents used for one-stage factor VIII assays in the UI~ A total of 14-15 manufacturers are represented, although 4-5 manufacturers account for the majority of the reagents used. Although the use of different reagents does not necessarily lead to different results, different types of substrate plasma have previously been implicated in variability o£ one-stage assays17,24, and the introduction of new types of substrate plasma, prepared by immunodepletion, is of particular interest.
50
40
30
20 10
0
n
15 10
Fig.
2
-
25 20
35 30
45
40
55 65 75 85 95 105 115 50 60 70 80 90 100 110 factor VtI[:C (IU/dt)
Distribution of potencies of factor VIII in a clinical plasma sample among 214
laboratories. (Data reproduced.from the UK NEQAS survey with kind permission from Dr. R B. A. KernofJ}.
161
FACTOR VIII AND FACTOR IX ASSAYS
Immunodepletedplasmas As indicated in tab. 10, a large n u m b e r of laboratories are now using immunodepleted substrate plasmas for one-stage assays because of difficulties with supply of congenitally deficient plasma since the advent of AIDS. Both factor VIII- and factor IX-deficient plasmas are available. In the UK, two of the manufacturers of immunodepleted factor IX-deficient plasmas each carfled out a comparative trial of their products against congenitally factor IXdeficient plasma in several laboratories with comparable results 22,25. Immunodepleted factor IX-deficient plasmas, both commercial and home-made, have b e e n in use in our laboratory for over one year in assays o f factor IX, and results have been satisfactory. Our initial studies of factor VIII immunodepleted plasmas, however, found differences in results from hemophilic plasmas in assays of some concentrates. Two immunodepleted plasmas gave lower potencies than hemophilic plasma on three concentrates, the discrepancies being most marked for the two very high purity concentrates 16. An important aspect of the preparation o f factor VIII-deficient plasmas is that most manufacturers use antibodies to yon Willebrand factor (vWF), so that most plasmas are deficient in vWF as well as VIII:C. This violates one of the basic principles of coagulation assays, i.e. that reagent systems should be deficient only in the factor being measured. Although vWF is not directly involved in coagulation, it helps to stabilize factor VIII and protects it from degradation by activated protein C 2°. Thus, it might be expected that absence of vWF might be responsible for the lower potencies obtained with vWF-deficient plasma on high purity concentrates which themselves have very little vWF. This was confirmed in our laboratory, when addition of purified vWF to a vWF-deficient plasma gave higher potencies for two monoclonal antibody purified concentrates 16. The influence of vWF o n potency of recombinant factor VIII concentrates was even larger, as indicated in tab. 11. These results indicate that vWF-deficient plasmas are unsuitable for assay of very high purity concentrates. Whilst their use for clinical samples may be satisfactory, there have b e e n no published studies in which results have b e e n compared with hemophilic plasma. Accordingly, a multicentre study has been
substrate plasma
n-*
hemophilic commercial local
92 5
immunodepleted manufacturer A manufacturer B manufacturer C 9 other manufacturers
49 27 12 19
total
204
Tab. 10 - S u b s t r a t e p l a s m a s u s e d in o n e - s t a g e f a c t o r V I I I a s s a y s in UK. (Data reproducedfrom the
UK NEQAS survey with kind permission from Dr. R B. A. KernofJ).
162
T. W . B A R R O W C L I F F E
concentrate
increase in potency with addition of vWF (%)
Hemofll M
18.0
Monoctate
10.7
Hyland Recombinant
52.8
Cutter Recombinant
95.4
Tab. 11 - Effect o f v o n W i l l e b r a n d f a c t o r (vWF) o n p o t e n c y o f V H P f a c t o r V I I I c o n c e n t r a t e s .
[Purified vWF was added to a vWF-depletedplasma (Diagnostic Reagents Ltd.) and potencies (vs 3rd IS) compared with those in absence of added vWI;].
initiated in the UK, in which several clinical plasma samples, as well as concentrates, are assayed with a variety of factor VIII-deficient plasmas, both congenital and immunodepleted. This study, which is u n d e r the auspices of the British Committee for Standards in Haematology, and is not yet completed, should provide useful comparative data on these reagents.
Very high purity concentrates of factor VIII The advent of monoclonal antibody technology has led to the preparation for clinical use of concentrates of very high purity (VHP) from both plasma and rDNA sources. These concentrates, though formulated with albumin as a stabilizer, are different in both protein composition and methods of production from the concentrate standards used up to now, and our results on two monoclonal plasma-derived products indicated that these concentrates might be subject to more assay variability than conventional products 16. A coUaborative study was therefore carried out to investigate the variability between laboratories and between assay methods, and to examine t h e need for a possible very high purity standard. Seven laboratories took part and five concentrates, i.e. two monoclonal, one recombinant and two intermediate purity (IP), were assayed against existing W H O and US concentrate standards. The results showed that for all three methods, variability between laboratories was much higher for VHP concentrates than for IP materials 9. O f the three methods the chromogenic gave the most variability and the two-stage the least. One aspect o f VHP concentrates is that, though formulated with albumin, they tend to be less stable than IP concentrates, particularly when diluted in the assays. Although 0.1% albumin was used in the assay buffers during the above study, this may not be adequate to stabilize dilutions of some VHP concentrates. Accordingly, in the recently completed large-scale international collaborative study, organized to replace the 3rd IS for factor VIII concentrate, 1% albumin was included in all assay buffers for both candidate preparations, an IP and a VHP (monoclonal plasma-derived) concentrate. This may be responsible for the fact that interlaboratory variability on the VHP concentrate, though greater than on the IP concentrate (tab. 7), was much less than in the previous study 8. The results of these two studies emphasize the need for attention to methodological details in assessment of potency of these VHP concentrates, but indicate that at least one VHP product can be assayed satisfactorily against an 163
F A C T O R V I I I A N D F A C T O R I X ASSAYS
I P s t a n d a r d . I n v i e w o f this, a n d t h e f a c t t h a t I P c o n c e n t r a t e s stiII f o r m t h e bulk of the therapeutic products used around the world, the IP concentrate w a s c h o s e n as t h e 4 t h IS f o r f a c t o r V I I I c o n c e n t r a t e . T h e p o s s i b l e n e e d f o r a V H P s t a n d a r d will b e c a r e f u l l y a s s e s s e d i n t h e f u t u r e .
SUMMARY The development of international standards over the last 15-20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment o f potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no yon Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail. REFERENCES 1. BA.NGr~ D. R., BraGs R., BRozovm M., D~NSON K. W. E., SWGG J. L.: A biological standard for measurement of blood coagulation factor VIII activity - Bull. Wld Hlth Org. 45, 337, 1971. 2. BARROWCUFFET. W.: Comparisons of 1-stage and 2-stage assays of factor VIII:C - Scand. J. Haematol. 33, 39, 1984. 3. B ~ o w c t s F ~ T. W.: The 1-stage versus the 2-stage factor VIII assay. In: TRn't~TT D.A. (Ed.): Advances in Coagulation Testing: Interpretation and Application. College of American Pathologists Press, Skokie, IL, 1985; chapt. 5. 4. B~OWCtaFVE T. W., CURTISA. D.: An international collaborative study of factors II, VII, IX and X - NIBSC unpublished report, 1986. 5. BAmmWCUFr~ T. W., CtmTIs A. D., THOra_~SD. P.: Standardisation of factor VIII. IV. Establishment of the 3rd international standard for factor VIII:C concentrate - Thrombos. Haemostas. 50, 697, 1983. 6. BAmmW¢~F~ T. W., DAWSONN.J., I~MBat~-CooR G.: Activated factor VIII in concentrates prepared by monoclonal antibody and recombinant technology - Thrombos. Haemostas. 60, 198, 1989. 7. B~a~OWCtaFFE T. W., EDWARDSS.J., I&_.MBa.t~-CooKG., THOMASD. P.: Factor VIII degradation products in heated concentrates - Lancet i, 1448, 1986. (Letter). 8. B~OWCtIFFE T. W., HEATH A. B.: Establishment of the 4th international standard for factor VIII concentrate - NIBSC unpublished report, 1989. 9. BARROWCLIFFET. W., KEMBALL-CooKG., HEATH A. B.: A collaborative study of intermediate and very high purity factor VIII concentrates - NIBSC unpublished report, 1989. 10. BARROWCLIFFET. W., KIRKWOODT. B. L.: An international collaborative assay of factor VIII clotting activity - Thrombos. Haemostas. 40, 260, 1978. 11. BAm~OWCLIFFET. W., KIRKWOODT. B. L.: Standardisation of factor VIII. I. Calibration of British standards for factor VIII clotting activity - Brit. J. Haematol. 46, 471, 1980. 12. BAm~OWCLIFFET. W., TYDEMANM. S., KIRk'WOODT. B. L., THOMASD. P.: Standardization of factor VIII. III. Establishment of a stable reference plasma for factor VIII-related activities Thrombos. Haemostas. 50, 690, 1983. 164
T. W. BARROWCLIFFE
13. BLOMBACKM.: Synthetic chromogenic substrate assay for factor VIII. In: TRIPLETT D. A. (Ed.): Advances in Coagulation Testing: Interpretation a n d Application. College of American Pathologists Press, Skokie, IL, 1985; chapt. 3. 14. BOY:EL E. G., TRIPLETT D. A.: Review of the College o f American Pathologists proficiency survey results for factor VIII:C assays 1983 through 1984. In: TRIPLETT D. A. (Ed.): Advances in Coagulation Testing: Interpretation and Application. College of American Pathologists Press, Skokie, IL, 1985; chapt. 9. 15. BROZOVICM., K/RKWOOD T. B. L., ROBERTSON I.: Study of a proposed international standard for blood coagulation factor IX - Thrombos. Haemostas. 35, 222, t976. 16. DAWSON N.J., KEMBALL-CooK G., BARROWCLIFFE T. W.: Assay discrepancies with highly purified factor VIII concentrates - Haemostasis 19, 131, 1989. 17. HELDEBRANT C., KL:sz'msva R., SHORT M., ARONSON D., BROWN L.,JAcoBY G., LEE M., THOMAS D., BARROWCUFFE T., HOWELL I., JOHNSON R. C.: Assay of AHF concentrates a n d standards: failure to eliminate variability with a m o n o g r a p h e d assay - Thrombos. Res. 30, 337, 1983. 18. HUBBARD A. R., CURTIS A. D., BARROWCL1FFET. W., EDWARDS S. J., JENNINGS C. A., KEMBALLCOOK G.: Assay of factor VIII concentrates: comparison of chromogenic and 2-stage clotting assays - Thrombos. Res. 44, 887, 1986. 19. gaRKWOOD T. B. L., BARROWCLIFFE T. W_: Discrepancy between 1-stage and 2-stage assay of factor VIII:C - Brit. J. Haematol. 40, 333, 1978. 20. KOEDAMJ. A., HAMER R.J., BEESER-VISSERN. H.,JAP E., SAN T., SCHIPPERS K., SIY,.MAJ. j.: T h e interaction between factor VIII a n d von Witlebrand factor - Thrombos. Haemostas. 58, 538, 1987. (Abstract). 21. LEE M. L., MAGALANGE. A., gaNC,DON H. S.: An effect of predilution on potency assays of factor VIII concentrates - Thrombos. Res. 30, 511, 1983. 22. MIDDLETON S. A.: Evaluation of an immunodepleted factor IX-deficient plasma - Speywood Laboratories unpublished report, 1986. 23. MIr.AELSSON M., OSWALDSSON U.: Standardization of VIII:C assays: a manufacturer's view Scand. J. Haematol. 33, 79, 1984. 24. OVERJ.: Quality control of the 1-stage factor VIII (VIII:C) assay in the coagulation laboratory - Scand. J. Haematol. 33, 89, 1984. 25. PROWSE C.: Evaluation of an immunodepteted factor IX-deficient plasma - SNBTS unpublished report, 1987. 26. RosI~N S., ANDERSON M., BLOMB~CKM., HAGGLIYI',IDU., LARRIEU M.J., WOLF M., BOYER C., ROTSSCHILD C., NILSSOnJ. M., SJORIn E., VINAZZER H.: Clinical application of a chromogenic substrate m e t h o d for determination of factor VIII activity - Thrombos. Haemostas. 54, 8t8, 1985. 27. STANDARDIZATIONof factors II, VII, IX a n d X in plasma a n d concentrates - Report o f the
ICTH Subcommittee o n factors VIII a n d IX, Brussels, 1987 - Thrombos. Haemostas. 59, 334, 1988. 28. Wld Hlth Org. techn. Rep. Set. 463, 14, 1971. 29. WId Hlth Org. techn. Rep. Ser. 6t0, 13, 1977. 30. Wld Hlth Org. techn. Rep. Ser. 771, 23, 1988.
Requests for reprints should be addressed to: TREVOR W. BARROWCLIFFE National Institute for Biological Standards and Control Blanche Lane, South Mirnms Potters Bar, Hertfordshire EN6 3QG - Great Britain
165
Although the assay methods for factor VIII and factor IX, which are still in use today, were developed over forty years ago, it is only in the last 15-20 years that there have been concerted efforts towards the standardization and quality control of these clinically important assays. After briefly describing the development of international and national concentrate and plasma standards, and the extent of agreement between laboratories in collaborative studies, this review will focus on recent developments which have impact o n the standardization of these assays in clinical laboratories. The introduction of plasmas depleted in factors VIII or IX by immunological methods as substrates for one-stage assays, and of the chromogenic method for factor VIII, are two important methodological changes, and comparisons with results from traditional methodology are of particular interest. Finally, some problems in the assay of factor VIII in concentrates of very high purity, prep~ared by monoclonal antibody and recombinant DNA technology, will be discussed. International standards f o r factors VIII a n d I X
The original definition of the unit of biological activity for both factors VIII and IX was the activity present in 1 ml o f fresh normal plasma. This proved inadequate in practice to effect standardization between laboratories because of the large variation of factor VIII and factor IX in the normal population. Table 1 shows the range of values of these factors in normal pools of at least 15 donors. The development of factor VIII concentrates as articles of commerce in the late 1960's accelerated the need for a stable reference preparation against which these products could be compared, and the 1st international standard for factor VIII concentrate was established in 19711,28. Since then the standard has been replaced at regular intervals, and all manufacturers of factor VIII concentrates around the world have used these standards directly or indirectly to calibrate their materials. Key-words: Assays; Factor VIII; FactorIX; Standards.
• Presented at the '2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implicationsfor the Clinical Laboratory', Rome, September 28-29, 1989. Res. Clin. Lab. 20, 155-165, 1990. 155
FACTOR VIII AND FACTOR IX ASSAYS
n-* o f l a b o r a t o r i e s
r a n g e o f values (IU/ml)
factor VIII
21
0.63-1.35
f a c t o r IX
23
0.82-1.37
Tab. 1 - F a c t o r V I I I a n d f a c t o r I X c o n t e n t o f n o r m a l p o o l s . (Data from international collaborative studies4.12. Pools in each laboratory are from at least 15 donors).
Similarly, a factor IX concentrate international standard was established in 197615.~9, a n d was replaced by the 2nd IS in 19874,3°. For assays o f clinical plasma samples laboratories use either nadonal plasma standards where available, commercial plasma standards, or local plasma pools. Calibration o f these plasma standards against the IS for factor VIII concentrate was found to give large discrepancies between laboratories and between assay methods n,19. T h e r e f o r e , it was decided to establish a plasma standard for factor VIII to coexist with the concentrate standard. This reference plasma was established in 1982, a n d calibrated for the o t h e r factor VIIIrelated activities as well as factor VIII:C, i.e. factor VIII:Ag, and von Willeb r a n d factor activity and andgen; it has recently b e e n replaced by the 2nd IS for factor VIII and von Willebrand factor in plasma. Similar considerations led to the establishment in 1987 o f a plasma IS for factor IX; this plasma was also calibrated for the other prothrombin complex factors II, VII, and X. Table 2 summarizes the various plasma a n d concentrate international standards which have b e e n established for factors VIII a n d IX.
Precision of factor VIII and factor IX assays In the calibration o f international standards, laboratories are asked to perform repeat assays on the same samples, a n d so information can be obtained on the within-laboratory variability between assays. Table 3 summarizes the average geometric coefficients o f variation (GCV's) within laboratories in five international collaborative studies. It can be seen that there has b e e n a general improvement in precision since 1971, and both the major methods can now be regarded as extremely precise, especially on assays o f concentrates. T h e r e is less information about the precision o f factor IX assays, but in the recent international collaborative study to establish plasma and concentrate standards for factor I X 4, the average GCV was 20.4%.
factorVIII
factorIX
concentrates
plasmas
concentrates
1st IS 1971
1st IRP 1983
1st IS 1974
2nd IS 1978
2nd IS 1989
2rid IS 1987
3rd IS 1983 4th IS 1989
Tab. 2 - International standards for factors VIII and IX. 156
plasmas 1st IS 1987
T. W. BA.RROWCLIFFE
avemge GCV% date of study
samples assayed
1971 1976 1981 1982 1983
Tab.
3 -
trate; P
C vs C vs P vs C vs C vs
one-stage
two-stage
45 16 12.4 9.9 6.2
12 10 13.3 8.9 3.9
P P P C C
Within-laboratory precision of factor VIII assays in collaborative studies. (c ~- concen-
= plasma).
O f course, these average GCV's conceal considerable variation b e t w e e n laboratories - in the study o f 1983, f o r instance, the within-laboratory GCV's r a n g e d f r o m 5 to 23% i2. Nonetheless, these figures do give an indication o f what is achievable. Between-laboratory
variability
T h e i m p r o v e d intralaboratory precision in r e c e n t studies is also reflected in i m p r o v e d a g r e e m e n t between laboratories, as can b e seen in tab. 4. T h e GCV's b e t w e e n laboratories in the most r e c e n t study o f factor VIII are similar to the within-laboratory precision, indicating that a g r e e m e n t b e t w e e n laboratories is a b o u t as g o o d as can be expected. A g r e e m e n t b e t w e e n laboratories in factor IX assays d u r i n g the r e c e n t international collaborative study was not as g o o d as for factor VIII. GCV's are s u m m a r i z e d in tab. 5, and the distribution o f potencies is illustrated in fig. 1. It must b e stressed that the degree o f variability between laboratories dep e n d s very m u c h o n the type o f sample being assayed a n d its similarity to the standard b e i n g used. Thus, for concentrates it has b e e n established in m a n y studies that variability is less w h e n c o m p a r e d to a concentrate standard than w h e n a plasma standard is used 4,5,~° - this is illustrated for factor IX by the results in tab. 5. T h e development o f new purification methods for concentrates, including m o n o c l o n a l antibody purification from b o t h plasma a n d rDNA sources, has m e a n t that some concentrates may differ in composition from the concentrate standards used. In this case, interlaboratory variability may b e considerably greater t h a n that shown in tab. 4, as discussed in a subsequent section.
GCV% date of study
samples assayed one-stage
1971
{C
1983
{C
1989
vs C vs
P C
376 26
two-stage 68 13
C vs
P C
11.6 7.4
17.1 3.5
C
C
5.6
5.9
vs
vs
Tab. 4 - Between-laboratory precision of factor VIII assays in collaborative studies. 157
FACTOR VIII AND FACTOR IX ASSAYS
comparison
GCV%
concentrate vs plasma concentrate vs concentrate
22.7 8,9
Tab. 5 - Between-laboratory precision of factor IX assays in an international collaborative study~.
Predilution of concentrates
A n o t h e r factor which may affect the variability b e t w e e n laboratories is the m e t h o d o f predilution. It was s h o w n by LEE et al. 21 that predilution o f intermediate purity factor VIII concentrates in h e m o p h i l i c plasma increased their potency by approximately 25%, c o m p a r e d to the same c o n c e n t r a t e p r e d i l u t e d in b u f f e r only. A similar p h e n o m e n o n was f o u n d for factor IX in the international collaborative study 4. T h e r e f o r e , if c o n c e n t r a t e s are assayed in clinical laboratories against plasma standards, it is essential to predilute c o n c e n t r a t e s in the appropriate deficient plasma in o r d e r to obtain the correct potency. Since b o t h plasma a n d c o n c e n t r a t e i n t e r n a t i o n a l standards are available for factors VIII a n d IX, it was n e c e s s a r y to standardize the m e t h o d o f predilu-
8~
11 13 17 14 18 15
42-
~ r 50
_~
21 19 22 23 16 10 I i
100 potency (%overallmean)
200
300
200
3&
3 7
110 13 ~: r
5tO
i11 14 119 16 15 23 2C 100 potency (% overall mean)
Fig. 1 - Distribution of potencies of factor IX concentrate Q (2nd IS) vs T (lst IS). Each square represents the average result of 4 assays from individual laboratories. Assays were carried out with both buffer (upper histogram) and deficient plasma (lower histogram) as prediluents. (Datafroma n international collaborative study). 158
T. W. BARROWCLIFFE
potency (IU/ampoule)
date of study
1979 1981 1982 1984 1988
one-stage
two-stage
0.57 0.60 0.70 0.73 0.60
0.57 0.62 0.69 0.74 0.66
Tab. 6 - Comparison of one-stage and two-stage factor VIII assays on plasma samples.
t_ion in order to get equivalence between plasma and concentrate units. When the 3rd IS for factor VIII concentrate was established, it was assayed against both the concentrate and plasma standards, using predilution in hemophilic plasma for comparison against the plasma standard. In one-stage assays there was no significant difference between these two values, though a 20% difference remained in two-stage assays 5. For factor IX the situation was somewhat more complicated, since calibration of the 2nd IS against the plasma standard, using factor IX-deficient plasma as prediluent, yielded a value some 20% higher than that against the 1st IS concentrate 4,~z. The higher potency was chosen for the 2nd IS, to achieve comparability between plasma and concentrate units, and consistency with the factor VIII situation, and this effectively meant a reevaluation of the unit o f factor IX. For routine assays of concentrates against a concentrate standard in our laboratory, we have not found it necessary to predilute in deficient plasma. However, this policy may have to be revised when new high-purity concentrates become available, as discussed in a subsequent section.
Comparison of methods Comparisons between one-stage and two-stage assays of factor VIII have been extensively reviewed elsewhere z~, and will only be mentioned briefly here. The two-stage method has given better precision in most studies than the one-stage method, both within- and between-laboratories, although in recent studies the differences have been small (tabs 3 and 4). In most collaborative studies normal plasmas have given identical results with the two methods, though somewhat surprisingly a significant difference was found in the most recent collaborative study, as shown in tab. 6. It is welbknown that concentrates when assayed against a plasma standard give higher potencies by two-stage than by one-stage assays 5,11. Most concentrates when assayed against a concentrate standard give similar potencies by the two methods, but some products have consistently given significant differences, with one-stage potencies usually higher than two-stageZL Reasons for the discrepancies include the possible presence of activated factor VIII in some concentrates 6,7, and influence of non-factor VIII contaminants, such as lipids, on the assays 2. The introduction of a chromogenic method for factor VIII, which uses purified clotting factors and an amidolytic substrate for factor Xa, could eventually lead to improved standardization. The method is the same in principle as that of the two-stage method, and in comparative assays in our laboratory 159
FACTOR VIII AND FACTOR IX ASSAYS
IP concentrate n-" of laboratories
VHP concentrate
method potency (IU/ml)
GCV%
potency (IU/ml)
GCV%
26
one-s rage
6.10
5.6
4.97
11
two-stage
6.36
5.9
4.88
13.8 7.6
15
chromogenic
6_63
9,2
4,89
14.3
28
over'all
6_30
7.7
4.93
12.7
Tab. 7 - Potencies of intermediate purity (IP) and very high purity (VHP) concentrates in 4th IS study.
the c h r o m o g e n i c a n d two-stage m e t h o d s gave very similar results o n most concentrates 18. A collaborative study involving f o u r clinical laboratories also f o u n d n o significant differences in assays o f clinical plasma samples by one-stage a n d c h r o m o g e n i c assays 26. I n the most r e c e n t i n t e r n a t i o n a l collaborative study o f concentrates, the c h r o m o g e n i c m e t h o d gave identical results to the one-stage a n d two-stage methods o n a m o n o c l o n a l antibody purified concentrate, but slightly h i g h e r resuits o n a n intermediate purity c o n c e n t r a t e s. T h e 8% d i f f e r e n c e b e t w e e n chrom o g e n i c a n d one-stage assays o n the latter c o n c e n t r a t e was statistically significant (tab. 7). Although i m p r o v e d precision has b e e n claimed for the chromo° genic m e t h o d a n d d e m o n s t r a t e d in some laboratories ~3,23,26, between-laboratory GCV's in the most r e c e n t i n t e r n a t i o n a l collaborative study were h i g h e r t h a n for one-stage a n d two-stage assays, as shown in tab. 7. Comparison of laboratories in N E Q A S surveys
Although international collaborative studies have indicated the h i g h deg r e e o f a g r e e m e n t which can b e achieved b e t w e e n laboratories, it must b e r e c o g n i z e d that laboratories which take part in these studies are large centres p e r f o r m i n g m a n y assays o n a r e g u l a r basis. A b e t t e r assessment o f assay p e r f o r m a n c e by routine clinical laboratories is given b y the results o f national external quality assurance s c h e m e (NEQAS) surveys, which are c a r r i e d out in m a n y countries, including the UK. Table 8 summarizes the results o f factor VIII assays in two r e c e n t UK surveys o f o v e r 200 laboratories, a n d the distribution o f results o n o n e o f these samples is s h o w n in fig. 2. It can b e s e e n that a g r e e m e n t b e t w e e n laboratories is m u c h worse t h a n in the i n t e r n a t i o n a l studies. Apart f r o m the fact that these surveys involve a larger n u m b e r o f laboratories, m a n y o f which may per-
between-laboratory GCV% potency (IU/dl)
36 8.1
one-stage
two-stage
36.6 41.4
46.1 72.9
Tab. 8 - Factor VIII assays in UK NEQAS. (Datareproducedwith kind permissionfrom Dr. P. B. A. Kernoff). 160
T. W. BARROWCLIFFE
manufacturer
n ~- of laboratories
A B C D E 10 others
62 45 22 21 I0 54
total
214
Tab. 9 - A P T T r e a g e n t s u s e d i n o n e - s t a g e f a c t o r V I I I a s s a y s i n UK. (Data reproduced from the UK
NEQAS survey with kind permission from Dr. P. B. A. Kernofjg.
form assays infrequently, there are two main reasons why results in NEQAS schemes may be more variable. Firstly, in NEQAS schemes only a single assay is usually performed, and very often the test sample is assayed using only one or two dilutions 14. In international studies, results from each laboratory are means from at least 4, sometimes 6, assays, with test samples tested at at least 3 dilutions. Thus, interlaboratory agreement in NEQAS surveys could probably be improved b y more repeat testing on each sample. The second reason is the multiplicity of reagent systems used. Tables 9 and 10 show the various substrate plasmas and the activated partial thromboplastin time (APTT) reagents used for one-stage factor VIII assays in the UI~ A total of 14-15 manufacturers are represented, although 4-5 manufacturers account for the majority of the reagents used. Although the use of different reagents does not necessarily lead to different results, different types of substrate plasma have previously been implicated in variability o£ one-stage assays17,24, and the introduction of new types of substrate plasma, prepared by immunodepletion, is of particular interest.
50
40
30
20 10
0
n
15 10
Fig.
2
-
25 20
35 30
45
40
55 65 75 85 95 105 115 50 60 70 80 90 100 110 factor VtI[:C (IU/dt)
Distribution of potencies of factor VIII in a clinical plasma sample among 214
laboratories. (Data reproduced.from the UK NEQAS survey with kind permission from Dr. R B. A. KernofJ}.
161
FACTOR VIII AND FACTOR IX ASSAYS
Immunodepletedplasmas As indicated in tab. 10, a large n u m b e r of laboratories are now using immunodepleted substrate plasmas for one-stage assays because of difficulties with supply of congenitally deficient plasma since the advent of AIDS. Both factor VIII- and factor IX-deficient plasmas are available. In the UK, two of the manufacturers of immunodepleted factor IX-deficient plasmas each carfled out a comparative trial of their products against congenitally factor IXdeficient plasma in several laboratories with comparable results 22,25. Immunodepleted factor IX-deficient plasmas, both commercial and home-made, have b e e n in use in our laboratory for over one year in assays o f factor IX, and results have been satisfactory. Our initial studies of factor VIII immunodepleted plasmas, however, found differences in results from hemophilic plasmas in assays of some concentrates. Two immunodepleted plasmas gave lower potencies than hemophilic plasma on three concentrates, the discrepancies being most marked for the two very high purity concentrates 16. An important aspect of the preparation o f factor VIII-deficient plasmas is that most manufacturers use antibodies to yon Willebrand factor (vWF), so that most plasmas are deficient in vWF as well as VIII:C. This violates one of the basic principles of coagulation assays, i.e. that reagent systems should be deficient only in the factor being measured. Although vWF is not directly involved in coagulation, it helps to stabilize factor VIII and protects it from degradation by activated protein C 2°. Thus, it might be expected that absence of vWF might be responsible for the lower potencies obtained with vWF-deficient plasma on high purity concentrates which themselves have very little vWF. This was confirmed in our laboratory, when addition of purified vWF to a vWF-deficient plasma gave higher potencies for two monoclonal antibody purified concentrates 16. The influence of vWF o n potency of recombinant factor VIII concentrates was even larger, as indicated in tab. 11. These results indicate that vWF-deficient plasmas are unsuitable for assay of very high purity concentrates. Whilst their use for clinical samples may be satisfactory, there have b e e n no published studies in which results have b e e n compared with hemophilic plasma. Accordingly, a multicentre study has been
substrate plasma
n-*
hemophilic commercial local
92 5
immunodepleted manufacturer A manufacturer B manufacturer C 9 other manufacturers
49 27 12 19
total
204
Tab. 10 - S u b s t r a t e p l a s m a s u s e d in o n e - s t a g e f a c t o r V I I I a s s a y s in UK. (Data reproducedfrom the
UK NEQAS survey with kind permission from Dr. R B. A. KernofJ).
162
T. W . B A R R O W C L I F F E
concentrate
increase in potency with addition of vWF (%)
Hemofll M
18.0
Monoctate
10.7
Hyland Recombinant
52.8
Cutter Recombinant
95.4
Tab. 11 - Effect o f v o n W i l l e b r a n d f a c t o r (vWF) o n p o t e n c y o f V H P f a c t o r V I I I c o n c e n t r a t e s .
[Purified vWF was added to a vWF-depletedplasma (Diagnostic Reagents Ltd.) and potencies (vs 3rd IS) compared with those in absence of added vWI;].
initiated in the UK, in which several clinical plasma samples, as well as concentrates, are assayed with a variety of factor VIII-deficient plasmas, both congenital and immunodepleted. This study, which is u n d e r the auspices of the British Committee for Standards in Haematology, and is not yet completed, should provide useful comparative data on these reagents.
Very high purity concentrates of factor VIII The advent of monoclonal antibody technology has led to the preparation for clinical use of concentrates of very high purity (VHP) from both plasma and rDNA sources. These concentrates, though formulated with albumin as a stabilizer, are different in both protein composition and methods of production from the concentrate standards used up to now, and our results on two monoclonal plasma-derived products indicated that these concentrates might be subject to more assay variability than conventional products 16. A coUaborative study was therefore carried out to investigate the variability between laboratories and between assay methods, and to examine t h e need for a possible very high purity standard. Seven laboratories took part and five concentrates, i.e. two monoclonal, one recombinant and two intermediate purity (IP), were assayed against existing W H O and US concentrate standards. The results showed that for all three methods, variability between laboratories was much higher for VHP concentrates than for IP materials 9. O f the three methods the chromogenic gave the most variability and the two-stage the least. One aspect o f VHP concentrates is that, though formulated with albumin, they tend to be less stable than IP concentrates, particularly when diluted in the assays. Although 0.1% albumin was used in the assay buffers during the above study, this may not be adequate to stabilize dilutions of some VHP concentrates. Accordingly, in the recently completed large-scale international collaborative study, organized to replace the 3rd IS for factor VIII concentrate, 1% albumin was included in all assay buffers for both candidate preparations, an IP and a VHP (monoclonal plasma-derived) concentrate. This may be responsible for the fact that interlaboratory variability on the VHP concentrate, though greater than on the IP concentrate (tab. 7), was much less than in the previous study 8. The results of these two studies emphasize the need for attention to methodological details in assessment of potency of these VHP concentrates, but indicate that at least one VHP product can be assayed satisfactorily against an 163
F A C T O R V I I I A N D F A C T O R I X ASSAYS
I P s t a n d a r d . I n v i e w o f this, a n d t h e f a c t t h a t I P c o n c e n t r a t e s stiII f o r m t h e bulk of the therapeutic products used around the world, the IP concentrate w a s c h o s e n as t h e 4 t h IS f o r f a c t o r V I I I c o n c e n t r a t e . T h e p o s s i b l e n e e d f o r a V H P s t a n d a r d will b e c a r e f u l l y a s s e s s e d i n t h e f u t u r e .
SUMMARY The development of international standards over the last 15-20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment o f potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no yon Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail. REFERENCES 1. BA.NGr~ D. R., BraGs R., BRozovm M., D~NSON K. W. E., SWGG J. L.: A biological standard for measurement of blood coagulation factor VIII activity - Bull. Wld Hlth Org. 45, 337, 1971. 2. BARROWCUFFET. W.: Comparisons of 1-stage and 2-stage assays of factor VIII:C - Scand. J. Haematol. 33, 39, 1984. 3. B ~ o w c t s F ~ T. W.: The 1-stage versus the 2-stage factor VIII assay. In: TRn't~TT D.A. (Ed.): Advances in Coagulation Testing: Interpretation and Application. College of American Pathologists Press, Skokie, IL, 1985; chapt. 5. 4. B~OWCtaFVE T. W., CURTISA. D.: An international collaborative study of factors II, VII, IX and X - NIBSC unpublished report, 1986. 5. BAmmWCUFr~ T. W., CtmTIs A. D., THOra_~SD. P.: Standardisation of factor VIII. IV. Establishment of the 3rd international standard for factor VIII:C concentrate - Thrombos. Haemostas. 50, 697, 1983. 6. BAmmW¢~F~ T. W., DAWSONN.J., I~MBat~-CooR G.: Activated factor VIII in concentrates prepared by monoclonal antibody and recombinant technology - Thrombos. Haemostas. 60, 198, 1989. 7. B~a~OWCtaFFE T. W., EDWARDSS.J., I&_.MBa.t~-CooKG., THOMASD. P.: Factor VIII degradation products in heated concentrates - Lancet i, 1448, 1986. (Letter). 8. B~OWCtIFFE T. W., HEATH A. B.: Establishment of the 4th international standard for factor VIII concentrate - NIBSC unpublished report, 1989. 9. BARROWCLIFFET. W., KEMBALL-CooKG., HEATH A. B.: A collaborative study of intermediate and very high purity factor VIII concentrates - NIBSC unpublished report, 1989. 10. BARROWCLIFFET. W., KIRKWOODT. B. L.: An international collaborative assay of factor VIII clotting activity - Thrombos. Haemostas. 40, 260, 1978. 11. BAm~OWCLIFFET. W., KIRKWOODT. B. L.: Standardisation of factor VIII. I. Calibration of British standards for factor VIII clotting activity - Brit. J. Haematol. 46, 471, 1980. 12. BAm~OWCLIFFET. W., TYDEMANM. S., KIRk'WOODT. B. L., THOMASD. P.: Standardization of factor VIII. III. Establishment of a stable reference plasma for factor VIII-related activities Thrombos. Haemostas. 50, 690, 1983. 164
T. W. BARROWCLIFFE
13. BLOMBACKM.: Synthetic chromogenic substrate assay for factor VIII. In: TRIPLETT D. A. (Ed.): Advances in Coagulation Testing: Interpretation a n d Application. College of American Pathologists Press, Skokie, IL, 1985; chapt. 3. 14. BOY:EL E. G., TRIPLETT D. A.: Review of the College o f American Pathologists proficiency survey results for factor VIII:C assays 1983 through 1984. In: TRIPLETT D. A. (Ed.): Advances in Coagulation Testing: Interpretation and Application. College of American Pathologists Press, Skokie, IL, 1985; chapt. 9. 15. BROZOVICM., K/RKWOOD T. B. L., ROBERTSON I.: Study of a proposed international standard for blood coagulation factor IX - Thrombos. Haemostas. 35, 222, t976. 16. DAWSON N.J., KEMBALL-CooK G., BARROWCLIFFE T. W.: Assay discrepancies with highly purified factor VIII concentrates - Haemostasis 19, 131, 1989. 17. HELDEBRANT C., KL:sz'msva R., SHORT M., ARONSON D., BROWN L.,JAcoBY G., LEE M., THOMAS D., BARROWCUFFE T., HOWELL I., JOHNSON R. C.: Assay of AHF concentrates a n d standards: failure to eliminate variability with a m o n o g r a p h e d assay - Thrombos. Res. 30, 337, 1983. 18. HUBBARD A. R., CURTIS A. D., BARROWCL1FFET. W., EDWARDS S. J., JENNINGS C. A., KEMBALLCOOK G.: Assay of factor VIII concentrates: comparison of chromogenic and 2-stage clotting assays - Thrombos. Res. 44, 887, 1986. 19. gaRKWOOD T. B. L., BARROWCLIFFE T. W_: Discrepancy between 1-stage and 2-stage assay of factor VIII:C - Brit. J. Haematol. 40, 333, 1978. 20. KOEDAMJ. A., HAMER R.J., BEESER-VISSERN. H.,JAP E., SAN T., SCHIPPERS K., SIY,.MAJ. j.: T h e interaction between factor VIII a n d von Witlebrand factor - Thrombos. Haemostas. 58, 538, 1987. (Abstract). 21. LEE M. L., MAGALANGE. A., gaNC,DON H. S.: An effect of predilution on potency assays of factor VIII concentrates - Thrombos. Res. 30, 511, 1983. 22. MIDDLETON S. A.: Evaluation of an immunodepleted factor IX-deficient plasma - Speywood Laboratories unpublished report, 1986. 23. MIr.AELSSON M., OSWALDSSON U.: Standardization of VIII:C assays: a manufacturer's view Scand. J. Haematol. 33, 79, 1984. 24. OVERJ.: Quality control of the 1-stage factor VIII (VIII:C) assay in the coagulation laboratory - Scand. J. Haematol. 33, 89, 1984. 25. PROWSE C.: Evaluation of an immunodepteted factor IX-deficient plasma - SNBTS unpublished report, 1987. 26. RosI~N S., ANDERSON M., BLOMB~CKM., HAGGLIYI',IDU., LARRIEU M.J., WOLF M., BOYER C., ROTSSCHILD C., NILSSOnJ. M., SJORIn E., VINAZZER H.: Clinical application of a chromogenic substrate m e t h o d for determination of factor VIII activity - Thrombos. Haemostas. 54, 8t8, 1985. 27. STANDARDIZATIONof factors II, VII, IX a n d X in plasma a n d concentrates - Report o f the
ICTH Subcommittee o n factors VIII a n d IX, Brussels, 1987 - Thrombos. Haemostas. 59, 334, 1988. 28. Wld Hlth Org. techn. Rep. Set. 463, 14, 1971. 29. WId Hlth Org. techn. Rep. Ser. 6t0, 13, 1977. 30. Wld Hlth Org. techn. Rep. Ser. 771, 23, 1988.
Requests for reprints should be addressed to: TREVOR W. BARROWCLIFFE National Institute for Biological Standards and Control Blanche Lane, South Mirnms Potters Bar, Hertfordshire EN6 3QG - Great Britain
165
