Standardization of VIII:C assays 79

STANDARDIZATION OF VIII:C ASSAYS: A MANUFACTURER’S VIEW

Marianne Mikaelsson and Ulla Oswaldsson

Research and Development, Biochemical Department KabiVitrurn AB, Stockholm, Sweden

ABSTRACT: Despite the use of an International Standard to define the unitage of Factor Vlll coagulant activity (VIII:C) discrepancies still exist with respect to labelled potencies. In order to examine possible causes of this disagreement concentrates of low, intermediate and high purity, norm!al plasma (NP) and the 1st International Reference Plasma (IRP) were assayed against the 3rd International Standard (IS) using one-stage, two-stage and chromogenic subs- trate assays.

When different concentrates were estimated in relation to the 3rd IS, and NP in relation to the 1st IRP, there was good agreement between the potency estimates obtained by the different assay methods. Consistent results were obtained when VIII:C deficient plasma was used instead of buffer as prediluent. When NP and IRP were assayed against the 3rd IS marked differences between the assay methods wer9 observed when buffer was used as prediluent. This effect was reduced when the 3rd IS was prediluted with VIII:C deficient plasma.

Many factors influence the standardization of VIII:C assays, but good agreement between methods can be achieved provided that like preparations are compared under standardized mnditions.

KEYWORDS: Assay method, Factor VIII, standardization.

INTRODUCTION

In recent years the standardization of VIII:C (Factor Vlll coagulant activity) assays has attracted considerable attention. Despite the use of an International Standard for the calibration of working standards, discre- pancies still exist with respect to the unit- age of VIII:C in therapeutic concentrates (Austen et al., 1981; Kasper, 1981).

Several collaborative studies (Barrow- cliffe and Kirkwood, 1978; Kirkwood and Barrowcliffe, 1978; Barrowcliffe et al., 1983, a and b) organized by the National Institute for Biological Standards and Con- trol (NIBSC) in Great Britain, have demons-

trated that one-stage and two-stage methods do not give the same results when comparing dissimilar preparations (plasma and concentrates). If, however, the principles of biological assays are applied, i.e. comparison of like with like, good agreement between assay methods can be achieved. Several factors apart from the assay method may contribute to discre- pancies, for example: type of reagents, manner of predilution, purity of the con- centrates.

This paper presents the results of a recent calibration study. The primary aim was to calibrate a new house standard, but

80 Standardization of VIII:C assays

the study was designed to illustrate the influence of various parameters on the potency estimates.

MATERIALS AND METHODS Factor Vlll preparations The various preparations assayed in this study are listed in Table 1. The 3rd IS, the

Table 1 Factor !A// preparations

~~ ~

IS 3rd International Standard

IRP

BS 3rd British Working Standard

LPC

HPC

NP Normal Human Plasma

(801556 intermediate purity concentrate)

1 st International Reference Preparation (80/511, plasma)

(811537, intermediate purity concentate)

Low Purity Factor Vlll Concentrate (LsS9, fraction 1-0, KabiVitrum)

High Purity Factor Vlll Concentrate (LsSl 0, Octonativ, KabiVitrum)

(82 11 03 local reference plasma)

1 st IRP and the 3rd BS were obtained from the National Institute for Biological Stan- dards and Control, London, U.K. The nor- mal plasma and the house standards LPC and HPC (solutions) were kept frozen in liquid nitrogen (below - 150°C). Freshly reconstituted or unfrozen samples were used for each independent set of assays.

Assay methods (73). The two-stage assay was performed according to Denson (1967) using com- bined reagent and substrate plasma from Diagnostic Reagents Ltd (England).

(E). Nectra one-stage assay. Artifical Fac- tor Vlll deficient plasma “Plarum” was prepared according to Nyman (1970) from platelet-poor serum, bovine Factor V and human fibrinogen (KabiVitrum AB, Stock- holm, Sweden). Cephotest (Cephalin and ellagic acid) was obtained from Nyegaard and Co (Oslo, Norway). 0.1 ml “Plarum”

diluted l + l with 0.15 M NaCI, 0.1 ml Cephotest and 0.1 ml sample or standard dilution were preincubated for 10.5 minutes at + 37°C in an Electra 600 Auto- matic clotting time recorder (Medical Laboratory Inc, Mount Vernon, NY U.S.) modified by KEBO-GRAVE (Sphga, Sweden). After addition of 0.1 ml 0.015 M CaCI2 the clotting time was registered.

(0). The modified one-stage method was primarily developed for samples containing metal salts. 1 part “Plarum” diluted 1+1 with saline and 1 part Actin (Activated P lT reagent containing cephalin and ellagic acid from American Dade, Miami, FL, U.S.) were mixed and preincubated at + 37°C for at least 30 minutes and was then usable for 5 hours. 0.2 ml activated mixture, 0.1 ml sample or standard dilution and 0.1 ml 0.01 5 M CaCI, were mixed and the clotting times were recorded by a Lode Coagulato- meter (Svenska Labex, Helsingborg, Sweden).

0. One-stage assay according to Velt- kamp (7968). In this method severely haemophilic plasma was used as substrate and kaolin 5 mg/ml + Diagen platelet substitute (Diagnostic Reagents Ltd, Lon- don) as activator. The activation time was 18 minutes. Clotting times were recorded by the Lode Coagulatometer.

(S). Chromogenic substrate assay. The CoatesP VIII:C kit (KabiVitrum AB, Stock- holm, Sweden) contains Factor IXa, Factor X, Phospholipid, I 2581 (thrombin inhibitor) and S 2222 (chromogenic substrate). The amount of Xa, formed in the presence of VIII:C, is measured spectropho- tometrically.

The assay methods are summarized in Table 2. Michaelis buffer (0.026 M sodium acetate, 0.026 M sodium veronal, 0.11 M sodium schloride, pH 7.4) with 1% bovine serumalbumin (BSA) was used for all dilu- tions, unless otherwise stated. In each test

Standardization of VIII:C assays 81

HPC Table 2 Assay methods

Method Principle Reagents

TS Two-stage (Denson 1967) No adsorption, no subsampling

E One-stage Electra Automated

Q Modified one-stage No preincubation of sample

V One-stage (Veltkamp 1968)

S Chrornogenic substrate

Diagen combined reagent

Cephotest “Plarum”

Actin “Plarum”

Kaolin + Diagen platelet substitute Haemophilic plasma

Coatest (S 2222)

two replicate sets of three dilutions of each preparation or standard (after predilution to 1 IU/ml) were tested. The potencies were calculated by regression analysis accor- ding to the multiple parallel line bioassay principle relating log clotting time to log concentration (European Pharmacopoeia, 1971). A special computer programme for VIII:C assays which compensates for any drift of the system was used. All tests were checked with respect to statistical validity (linearity and parallelism).

RESULTS In previous calibration studies occasional lack of accuracy and precision had been observed with the two-stage method. A suspicion that the use of citrate-saline as diluent was the major source of this varia- tion prompted us to start a preliminary study to investigate the influence of various diluents on the stability of VIII:C. As shown in Fig. 1 the VIII:C activity declined rapidly when the samples were diluted in citrate-saline. No significant losses were observed for Michaelis buffer with 1% BSA. Michaelis buffer without BSA was equivalent to Tris buffer. Addition of BSA to citrated-saline resulted in a marked sta-

%

100-

50-

BS C

- 0 3 6

M + BSA

TRlS

CITRATE

I l l

0 3 6 INCUBATION (HOURS)

Figure 1 - Influence of various diluents on the stability of VIII:C. The sample dilutions were incu- bated for 0, 3 and 6 hours at room temperature. On each occasion withdrawn samples were assayed by the two-stage method against a freshly unfrozen and diluted house standard. M = Michaelis buffer.

bilization of VIII:C but there was still an initial loss of activity for the HPC. In view of these findings we decided to use Michaelis buffer with 1 YO BSA throughout the study.

When changing the diluent the optimal conditions for the clotting reaction have to be re-established. The optimal Ca2+ concentration, for instance, lies within a fairly narrow range (Fig. 2). Thus the Ca2+ concentration as well as the activation time required for optimal results were determined for each of the methods used in this study.

In the main study, 10 independent sets of assays were performed for each of the 5 methods listed in Table 2. The six prepara- tions listed in Table 1 were assayed in each run. The 3rd IS (3.9 IU per ampoule), which was used as the standard, was tested at the beginning and at the end of each run. For the two-stage and the Electra methods a further 10 sets of assays were performed using “Plarum” as prediluent instead of buffer.

82 Standardization of VIII:C assays

TS E Q V S

overall mean

Assigned potency

I I , I 1

10 20 30 40 50 20 30 40 50 60

HPC BS LPC IRP NP

4.29 3.40 1.20 1.06 1.32 4.09 3.40 1.12 0.79 1.06 3.90 3.46 1.17 0.78 1.03 4.05 3.37 0.94 0.55 0.67 4.39 3.27 1.07 0.65 0.84

4.14 3.38 1.10 0.77 0.98

(1 1 (2) (3) 4.29 3.5 1.16 0.73 0.95

Concentration of added CaC12 (mM) Figure 2 - Influence of the concentration of Ca’+ on the clotting times. LPC was diluted 1:40 in Michaelis buffer with 1 % BSA and assayed by the Electra and the Veltkamp methods.

Table 3 Mean potency estimates for various preparations against the 3rd IS

VIII:C IU/ml Assav method

(1) TS, this calibration study (2) TS, previous calibration study against 2nd IS (3) E, this calibration study, assayed against the 1st IRP.

The mean potency estimates for the various preparations relative to the 3rd IS are shown in Table 3. The precision of the various assay systems is given in Table 4. The lowest overall figure for the coefficient

Of variation, 3.5%, was obtained for the f-~ew chromogenic substrate assay.

Fig. 3 shows the potency estimates for the various preparations expressed as per- cent of their assigned potency. Very good

Standardization of VIII:C assays 83

HPC against IS %

I

NP against IRP lo

+

Q

-

....................

~

..... 100

50

LPC against IS Y O

BS against IS 06

100

50

100

50

*

TS

-

t

E

-

................ 100 .......

-

........

-

.i.. c

V

t

S

-

t

S I Q V

50

-

NP against IS IRP against IS

7 5- ..... t

E

* .....

Q

...................... 100 .......

-

..... ...... .....

-

T 1 t

V

-

50

- TS

-

E

- Q

- I Figure 3 - Potency estimates (mean f SD, n = 1 O), expressed as % of assigned potency, for various preparations assayed against the 3rd IS or the 1 st IRP. Abbreviations for preparations and methods are explained in Table 1 and Table 2, respectively.

a4

Assay method

TS E Q V S

Standardization of VIII:C assays

'Yo cv

Overall mean H PC BS LPC IRP NP

3.5 8.8 3.5 12.9 13.6 8.5 5.3 7.8 9.5 8.8 6.4 7.6 3.2 6.0 6.0 4.1 5.2 4.9 3.3 4.6 3.7 6.8 4.6 4.6 1.3 5.5 4.2 3.1 3.6 3.5

agreement between the 5 assay methods was observed when the normal plasma (NP) was assayed against the IRP (plasma) and also when the concentrates HPC and BS were assayed against the 3rd IS. Good agreement between methods was also noticed for the LPC relative to the 3rd IS, except for the Veltkamp assay. In contrast with these results significant differences were observed between the assay methods when the plasma samples were compared with the 3rd IS. Almost identical patterns were obtained for IRP and NP. Despite this discrepancy the overall mean potency estimates for IRP and NP (Table 3) against the 3rd IS correlated well with the assigned potencies.

Fig. 4 illustrates how the prediluent affects the results. The use of substrate plasma as opposed to buffer for the initial dilutions did not change the results obtained for the concentrates HPC and BS relative to the 3rd IS by the two-stage and the Electra methods. When the plasma samples NP and IRP were assayed against the 3rd IS the potencies were overesti- mated when buffer was used as prediluent, especially for the two-stage assay. This difference was reduced when the 3rd IS was prediluted in substrate plasma.

Many laboratories have used haemophi- lic plasma as prediluent when assaying concentrates despite the fact that the pre- vious International Standard (2nd IS) con- tained only 1.1 IU per ampoule. Predilution

- BUFFER

assigned _._._ SUBSTRATE PLASMA 46 of

mtency

50

HPC BS IRP NP

.......

- Figure 4 - Influence of predilution procedure. Con- centrate and plasma samples were assayed by the two-stage and the Electra methods against the 3rd IS. The concentrates and the 3rd IS were prediluted in Michaelis buffer with 1% BSA or substrate plasma. Potencies are expressed as 'YO of the assigned potency.

of house standards in substrate plasma when calibrating against the 2nd IS means comparison of unlike material and may lead to overestimation (Lee et al., 1983). This statement was confirmed in a sepa- rate series of experiments. Two different concentrates were assayed by the Electra method against an undiluted standard (LPC 1.16 IU/ml). Predilution of the sam- ples in substrate plasma instead of buffer

Standardization of VIII:C assays 85

resulted in higher potency estimates as shown in Fig. 5. Concentrate A is identical with the HPC used in the main study. As shown in Figure 4 the potencies obtained for HPC against the 3rd IS were not affected by the prediluent when the sample and the standard were prediluted in the same way. Concentrate B is a US Factor Vlll concentrate. Using buffer as prediluent only 80% of the labelled potency was detected. Predilution in substrate plasma resulted in a higher estimate, 102% of the labelled activity.

of labelled potency

BUFFER SUBSTRATE PLASMA

100 ..........

5~

,. .... ................. 9

I A B

* .....

- A

,.. .. t

- B

...........

Figure 5 - Potency estimates for two high purity Factor Vlll concentrates A (=HPC) and B, expressed as % of labelled potency, assayed by the Electra method against an undiluted House Standard (LPC) after predilution in Michaelis buffer with 1% BSA or substrate plasma.

DISCUSSION

Many different assay methods are used for the measurement of VIII:C activity. Despite the introduction of the International Unit- age, systematic discrepancies have been recognized, especially between one-stage and two-stage methods when comparing unlike material, i.e. plasma and concen-

trates. For manufacturers of Factor Vlll concentrates this anomaly may lead to great confusion when evaluating data on production yields as well as in vivo recov- ery. With the present knowledge of Factor Vlll regarding structure and function, there is no way to define a universal assay for an accurate analysis of VIII:C. However, the establishment of the new International Reference Plasma as a complement to the International Standard for concentrates has made comparisons between plasma and concentrate units possible.

In this study 5 different assay systems representing wide variations with respect to parameters such as methods, reagents and procedures were evaluated. Despite the marked methodological differences very good agreement was achieved when comparing like preparations, i.e. plasma against the 1st IRP and various concen- trates against the 3rd IS. Furthermore there were no significant differences between the values obtained for the LPC House Standard by the Electra and the two-stage methods in this calibration against the 3rd IS compared with the previous calibration against the 2nd IS. These results confirm the indications of continuity of the International Unitage as well as the suitability of the 3rd IS and the 1st IRP for the calibration of working standards, reported by Barrowcliffe et al. (1983, a and b).

Another valuable finding from the pre- sent investigation is, that the new chro- mogenic substrate assay seems to fulfil all requirements for accurate and precise measurement of V1II:C. As to precision, the chromogenic assay was superior to all the clotting methods, when examined under comparable conditions regarding general design and statistical analysis.

When assaying plasma against concen- trates, one-stage methods in general yield higher potencies than two-stage methods, according to the collaborative studies

86 Standardization of VIII:C assays

organized by NIBSC. In contrast with these reports and our previous experience, higher values were obtained for the two- stage method in this study. A possible explanation is the omission of the AI(OH)3 adsorption step especially in combination with the buffer exchange and stabilization with albumin. An interesting finding is that the anomalous results observed for the two-stage assay in comparing plasma and concentrates were normalized by predilu- tion of the concentrate standard in sub- strate plasma.

Finally, buffer is preferable as prediluent for working standards when calibrating against standards like the 2nd IS, which do not require predilution. The use of sub- strate plasma under these circumstances is not in agreement with the principles of biological assays and may contribute to the discrepancies that have been reported concerning labelled potencies. The estab- lishment of the 3rd IS with a higher potency has enabled standardization of the initial dilution step. The present results indicate that the relative potencies for concentrates remain the same, whether buffer or sub- strate plasma is chosen as prediluent pro- viding the predilution manner is uniform.

Reliable assays of V1II:C are of the greatest importance from both the clinical and the economical point of view. Opti- misation and standardization of the analyti- cal procedures prepare the ground for precise determinations. Accuracy, defined by the International Unitage for plasma as

well as concentrates, may be achieved by strict application of the general principles of biological assays.

REFERENCES

1. Austen, D.E.G., Rhymes, I.R. and Riua, C.R. (1981): Factor Vlll concentrates: What the label says. Lancet /I, 1167.

2. Barrowcliffe, T.W. and Kirkwood, T.B.L. (1978): An international collaborative assay of Factor Vlll clotting activity. Thromb. Haemost. 40, 260-270.

3. Barrowcliffe, T.W., Tydeman, M.S., Kirkwood, T.B.L. and Thomas, D.P. (1 983a): Standardization of Factor Vlll-Ill. Establishment of a stable refer- ence plasma for Factor Vlll related activities. Thromb. Haemost. 50, 690-696.

4. Barrowcliffe, T.W., Curtis, A.D. and Thomas, D.P. (1983b): Standardization of Factor VIII-IV. Establishment of the 3rd International Standard for Factor VIII:C concentrate. Thromb. Haemost.

5. Denson, K.W.E. (1967): The simplified two-stage assay for Factor Vlll using a combined reagent. Thromb. Diath. Haemorrh., Suppl. 26, 419-422.

6. Kasper, C.K. (1981): Decreased potency of Fac- tor Vlll concentrates. Thromb. Haemost. 46, 21 7.

7. Kirkwood, T.B.L. and Barrowcliffe, T.W. (1978): Discrepancy between one-stage and two-stage assay of Factor VIII:C. Br. J. Haematol. 40, 333- 338.

8. Lee, M.L., Magalang, E.A. and Kingdon, H.S. (1983): An effect of predilution on potency assays of Factor Vlll Concentrates. Thromb. Res. 30,

9. Nyman, D. (1970): The preparation of an artificial reagent for the one-stage Factor Vlll assay. Thromb. Diath. Haemorrh. 23, 306-31 1.

10. Veltkamp, J.J., Drion, E.F. and Loeliger, E.A. (1968): Detection of the carrier state in hereditary coagulation disorders. I . Thromb. Diath. Haemorrh. 19, 279-303.

50, 697-702.

51 1-519.

Correspondence: Marianne Mikaelsson - KabiVitrum AB - Research and Development, Biochemistry - S-112 87 Stockholm, Sweden.