Chromogenic assay: Background and design

1
Chromogenic assay Background and
design Steffen Rosén Rossix
2
?!
FVIII
3
General method requirements
High accuracy
High precision
High specificity with minimal interferences
Stable reagents
4
First publication on a quantitative chromogenic
FVIII assaySeghatchian MJ, Miller-Andersson M.
A colorimetric evaluation of factor VIIIC
potency. Med Lab Sci 35, 347-354 (1978).
Method used diluted human serum used as source
of FIXa and FX, platelet substitute (Diagnostic
Reagents, UK) , calcium chloride and S-2222
(Kabi Diagnostica, Sweden)
5
Basic considerations in developing a chromogenic
FVIII kit in the early 1980s
Use of non-human coagulation factors to decrease
the risk of viral infections due to hepatitis B
and C and later to HIV. No use of FVIII
deficiency plasma for the same reason.
Use of a relatively high sample dilution to
minimize interferences.
Use of a method design which mimics the part of
coagulation where FVIII plays its role.
6
FXa-plateau microplate method of Coatest Factor
VIII
Carlebjörk et al. Thromb Res 47, 5-14 (1987)
7
FXa-generation vs time
8
Criterion Insensitivity to preactivation by
thrombin
One-stage
Coatest FVIII
Thromb Haemost 54, 818-823 (1985)
9
Thromb Haemost 54, 818-823 (1985)
10
Chromogenic Factor VIII kits
Coatest Factor VIII (Chromogenix) Mikaelsson M,
Oswaldsson U. Scand J Haematol 33 (Suppl 41),
79-86 (1984) Factor VIII Chromogenic (Dade
Behring) Tran TH et al. Poster, XIth ISTH
Congress (1987) Waagenvord RJ et al Haemostasis
19, 196-204 (1989) Immunochrome FVIIIC (Baxter
Immuno) Lang H et al. Poster, XIIIth ISTH
Congress (1991) De Maistre E et al. Thromb
Haemost 79, 237-238 (1998) Coamatic Factor VIII
(Chromogenix) Andersson M et al. Poster, XXth WFH
Congress, (1992) Kleinveld HA et al. Scand J Clin
Lab Invest 59, 335-342 (1999)
11
Chromogenic method recommended by SSC/ISTH
1993 and adopted as the Ph Eur reference method
1995 for assignment of FVIII potency in FVIII
concentrates. Important features to ensure
optimal assay conditions Predilution to 1 IU/mL
with FVIII deficiency plasma with normal vWF
content Final dilution in buffer with 1 BSA
New challenges rFVIII Full length
rFVIII Recombinate (Baxter), Advate (Baxter),
Kogenate (Bayer) Truncated rFVIII ReFacto
(Wyeth)
12
Are the species of FIXa and FX of importance in
determination of FVIII activity?
Examples of other Ph Eur reference
methods Antithrombin, anti-IIa bovine
thrombin Prothrombin Ecarin FVII bovine or
human FX FX RVV
13
Study purpose Compare FVIII potency
assignments of FVIII concentrates when
using combinations of bovine and human FIXa and
FX and thrombin or prothrombin FV.
14
Materials
Bovine and human FIXa, bovine and human FX
(Haematologic Inc.) Bovine FII and human FII
(Enzyme Research) Bovine FV (gift from B
Dahlbäck), human FV (Alexis) Human thrombin
(Biovitrum), bovine thrombin (Instrumentation
Laboratory) Phospholipid emulsion
(Instrumentation Laboratory) S-2765
(Chromogenix) I-2581 (Chromogenix) Octonativ-M
(Octapharma) Recombinate (Baxter) ReFacto
(Wyeth) 6th WHO FVIII Concentrate Standard, Lot
97/616 (NIBSC)
15
Conc. during FX activationCa2 6.25
mmol/LPhospholipid (PC, PS, SM) 15
mmol/LThrombin 0.2 U/mL (ca 2
nmol/L)orexcess of FII and FV relative to
sample contributionFIXa 0.04 U/mL (4
nmol/L)FX 0 0.35 mmol/LFVIII 0.6 pmol/L
(presence of FII, FV) and 2 pmol/L (presence of
thrombin)
Fixed parameters in Km determinations
Medium for Km determinations 0.05 mol/L Tris-HCl
pH 7.3, I 0.15, 1 BSA
16
Manual microplate assay FIXa PL FIIa
/ FII FV 50 mL FX 50 mL Octonativ-M 50
mL Ca2 50 mL Activation 2 or 5 min,
37oC S-2765/I-2581 50 mL Hydrolysis x min,
37oC HAc, 20 50 mL
17
FVIII Octonativ-M
18
FVIII Octonativ-M
19
Apparent Km values and relative FXa activities
( r H-Wmean 0.9986 )
B. Thrombin (Coamatic FVIII, Immunochrome
FVIIIC, Factor VIII Chromogenic)
( r H-Wmean 0.9992 )
Porcine PL Km 3 nmol/L (all bovine)
20
FVIII potency assignments vs 6th IS with all
bovine and all human species
of FIXa and FX and of thrombin or prothrombin FV
Sample Bovine, FII FV Human, FII FV Bovine,
FIIa Human, FIIa IU/mL CV, IU/mL CV,
IU/mL CV, IU/mL CV,
Octonativ-M 41 4.6 40 8.1 41 4.6 42
8.8 Recombinate 86 8.1 88 4.3 87
4.3 92 6.7 ReFacto 83 8.0 81 7.7
87 1.1 87 5.2
rmean 0.998 (range 0.991-1.00) n 58
21
Recent results with Coatest Factor VIII using
activation times of 3.5 and 5 min (n 3) on
analysis of ReFacto vs 6th IS
Activation time 3.5
min 5 min Potency, IU/mL 80.0 81.8 CV,
4.3 3.1 r ReFacto 0.999 0.999 r 6th
IS 0.998 0.999
22
Typical dose-response curves obtained with 3.5
min FX activation time on analysis of ReFacto vs
the 6th WHO FVIII Conc. Std
6th WHO Std 97/616
ReFacto
23
Conclusions
Similar Km values, all in the nanomolar range,
were obtained with all species combinations of
FIXa and FX.
The only combination which results in inferior
FXa activity is bovine FIXa human FX.
No difference in the FVIII potency assignments
when using bovine species as compared to human
species of FIXa, FX and thrombin or prothrombin
FV under the conditions used.
Preliminary study with Coatest Factor VIII
indicates no difference in assigned potency of
ReFacto vs 6th IS when using a FX activation
time of 3.5 min as compared to 5 min
24
AcknowledgementsTo my dedicated and very
skilful FVIII coworker since 1978
Mrs Margareta Andersson
To Instrumentation Laboratory for kind support of
this study