THROMBOSIS RESEARCH 66; 599-602,1992 0049464#92 $6.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
A MICROTITER PLATE READER ASSAY FOR FACTOR VIII P. Chang, David L. Aronson, George WashingtonUniversity Medical Center, Washington, D.C.20037
(Received 12.12.1991; accepted in revised form 3.2.1992 by Editor M.W. Mosesson) (Ftecehmd by Executke Editorial Offii 11.3.1992)
INTRODUCTION A variety of end points have been utilized to detect the clotting of fibrinogen as the endpoint in coagulation testing. These have included visual inspection for clot, mechanical endpoints, and opacity. Most instrumentation for the detection of these endpoints have only a few channels available for testing samples at the same time and the testing of large numbers of samples is cumbersome and time consuming. The instrumentation is expensive and solely designed for clotting measurements. Microtiter plate readers are accurate and precise detectors of a wide range of optical endpoints and have been found valuable for the measurement of clot lysis (1,2). The time of clot formation has been measured below on a microtiter plate reader utilizing the increase in opacity and has been validated with measurements of factor VIII. MATERIALS AND METHODS A Vmax kinetic plate reader (Molecular Devices, Palo Alto, CA) in conjunction with an IBM PS/2 computer programmed with softmax (Molecular Devices) was used for the measurement of clotting time. The plate reader was programmed to read at a wavelength of 405 nm every 8 seconds with periodic shaking. The plate reader was further programmed to analyze the data on the basis of time to the maximum velocity of the optical density change(time to Vmax). A.R.P.(Assayed Reference Plasma) was purchased from Helena Laboratories (Beaumont, TX), APTT reagent from Fisher Scientific Co.(Pittsburgh, PA), Factor VIII deficient plasma with Factor VIII activity (1 % from George King Bio-Medical, Inc.(Overland Park, Kansas), Microtiter plates were 599
600
MICROTITER PLATE READER ASSAY
Vol. 66, No. 5
from VWR Scientific (Bridgeport, NJ), and a sythetic hirudin derivative, hirulog was kindly supplied by Dr. John Maraganore of Biogen Research Corp (3). This peptide contains the exosite inhibitor of hirudin with a D-phe-pro-arg-pro active Site inhibiting sequence. The factor VIII preparations tested were plasma derived factor VIII from Cutter Laboratories Containing von Willebrand factor; recombinant factor VIII (Cutter Laboratories, Berkeley, CA) and immunopurified factor VIII (Armour, Kankakee,IL) containing little von Willebrand factor. The test was performed at ambient temperature. Fifty microliters of samples or standards diluted in 0.02 M Tris, 0.15 M NaCl, pH 7.2, were placed in microtiter plates. This was followed by the sequential addition of 40 UL of hemophilia A plasma in the presence of 0.1 ugm/ml of Hirulog, and 25 ul of APTT reagent diluted with an equal volume of Tris-NaCl buffer. Following 5 minutes of vigorous mixing on a plate shaker (Minishaker, Dynatech, Chantilly, VA), 50 UL of 0.05 M calcium chloride is added to each column with an 8 channel pipettor (Eppendorf, Fremont, CA) and reading immediately commenced on the microtiter plate reader. The program was set to calculate the maximum velocity of the increase of optical density based on five consecutive readings at eight second intervals. RESULTS Figure 1 shows the pattern of increased optical density as plasma clots after the addition of calcium and in the presence of APTT reagent. The duration of clot formation as seen by the increased optical density is seen to be extremely short. Replicate samples have the calculated time to Vmax within 8 seconds the majority of the time.
Time in Minutes
FIG. 1 The change with time of optical density during the clotting of plasma as measured on the microtiter plate reader at 405 nm. The straight line delineates the time of maximum velocity.
with the addition of increasing concentrations of normal plasma to the hemophiliac plasma, there is decreased time to Vmax. A l/5 dilution of plasma clots in 300 set, and the blanks in about 1000 sec. The correlation coefficient of the logarithim of the dose of factor VIII versus the clotting time is typically aO.99.
Vol. 66, No. 5
MICROTITER PLATE READER ASSAY
601
In the absence of the inhibitor, hirulog, the log dose-response curves deviate from linearity. A l/5 dilution of plasma clots in about 180 set and the buffer blank in 400 sec. The variance across rows with each well containing the same samples were tested in the presence and absence of hirulog. In the presence of hirulog the time to Vmax across the rows has a coefficient of variance of 2-3% and in the absence of hirulog the coefficent of variance is 10%. When different quantities of therapeutic factor VIII preparations are compared with dilutions of the standard plasma the slopes of the clottimg times are parallel. Figure 2 compares the slopes of different types of factor VIII preparations with that obtained with a standard plasma.The preparations studied include plasma derived factor VIII containing Von Willebrand factor (vWf), monoclonal purified plasma factor VIII with very small quantities of vWf and recombinant factor VIII containing no vWf. When six samples were tested in quintuplicate, the within assay coefficient of variance averaged 7% (range 4.3-9.7). The between day variance over 5 days of a sample with 40% of normal plasma factor VIII was 8.4%.
FIG. 2 Comparison of the dose-response of plasma and therapeutic factor VIII preparations. Monoclonal-immunopurified factor VIII containing very low vWf and no large multimers; rDNA factor VIII-devoid of vwf; Plasma factor VIII-contains equal unitage of factor VIII and vWf.
DISCUSSION The methodology above for the measurement of Factor VIII has been validated by comparison with known dilutions of normal plasma in hemophilic plasma and by comparison of the slopes of different doses of plasma derived Factor VIII, monoclonal Factor VIII and recombinant Factor VIII. The slopes of the different preparations are all parallel despite the quite different purities of the Factor VIII.
602
MICROTITER PLATE READER ASSAY
Vol. 66, No. 5
The addition of calcium across the plate to start the clotting reaction takes about 15 seconds using the multichannel pipettor. We have added hirulog to the Factor VIII deficient plasma so that the time difference is minimal compared to the total clotting time. The clotting time is also subtantially prolonged since the reaction is run at ambient temperature. The advantage of this method is the rapidity with which many assays can be accomplished in a short time. In typical assays for Factor VIII using a single channel detector and a l-stage assay, the clotting times average about 1 minute. During the testing of many samples there is time for both the samples and the reagents to decay and alter the results. In this microtiter plate assay 48 samples can be tested in duplicate in the course of less than a half hour. Further the standards and the unknowns are measured at the same time giving added reliability to the measurement. Smaller quantities of hemophilic plasma can be used as in the experiments above accomplished with 40~1 per well. It may be possible to reduce this even further, the limiting factor being sufficient fibrinogen to produce a significant signal. More experience is needed to validate the accuracy of this methodology. It is unlikely that the presence of the hirulog or testing at room temperature will impact on the accuracy. The ability to run the standards and the samples at the same time should increase the accuracy and the precision. The data presented above indicate that the precision is as good or better than other l-stage Factor VIII assays. The same instrumentation could be utilized for the measurement of the other intrinsic coagulation components such as Factor IX and Factor XI or perhaps as a useful and reliable clot detecting endpoint for the APTT. References 1. Beebe DP, Aronson DL. An automated fibrinolytic assay performed in microtiter plates. Thrombosis Res 47, 123-128,1987. 2. Jones AJS, Meunier AM. A precise and rapid microtitre plate clot lysis assay: Methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis. Thromb Haemost 64, 455-463,199O. 3. Bourdon P, Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin. Biochemistry 29, 7095-7101, 1990.
BRIEF
COMMUNICATION
A MICROTITER PLATE READER ASSAY FOR FACTOR VIII P. Chang, David L. Aronson, George WashingtonUniversity Medical Center, Washington, D.C.20037
(Received 12.12.1991; accepted in revised form 3.2.1992 by Editor M.W. Mosesson) (Ftecehmd by Executke Editorial Offii 11.3.1992)
INTRODUCTION A variety of end points have been utilized to detect the clotting of fibrinogen as the endpoint in coagulation testing. These have included visual inspection for clot, mechanical endpoints, and opacity. Most instrumentation for the detection of these endpoints have only a few channels available for testing samples at the same time and the testing of large numbers of samples is cumbersome and time consuming. The instrumentation is expensive and solely designed for clotting measurements. Microtiter plate readers are accurate and precise detectors of a wide range of optical endpoints and have been found valuable for the measurement of clot lysis (1,2). The time of clot formation has been measured below on a microtiter plate reader utilizing the increase in opacity and has been validated with measurements of factor VIII. MATERIALS AND METHODS A Vmax kinetic plate reader (Molecular Devices, Palo Alto, CA) in conjunction with an IBM PS/2 computer programmed with softmax (Molecular Devices) was used for the measurement of clotting time. The plate reader was programmed to read at a wavelength of 405 nm every 8 seconds with periodic shaking. The plate reader was further programmed to analyze the data on the basis of time to the maximum velocity of the optical density change(time to Vmax). A.R.P.(Assayed Reference Plasma) was purchased from Helena Laboratories (Beaumont, TX), APTT reagent from Fisher Scientific Co.(Pittsburgh, PA), Factor VIII deficient plasma with Factor VIII activity (1 % from George King Bio-Medical, Inc.(Overland Park, Kansas), Microtiter plates were 599
600
MICROTITER PLATE READER ASSAY
Vol. 66, No. 5
from VWR Scientific (Bridgeport, NJ), and a sythetic hirudin derivative, hirulog was kindly supplied by Dr. John Maraganore of Biogen Research Corp (3). This peptide contains the exosite inhibitor of hirudin with a D-phe-pro-arg-pro active Site inhibiting sequence. The factor VIII preparations tested were plasma derived factor VIII from Cutter Laboratories Containing von Willebrand factor; recombinant factor VIII (Cutter Laboratories, Berkeley, CA) and immunopurified factor VIII (Armour, Kankakee,IL) containing little von Willebrand factor. The test was performed at ambient temperature. Fifty microliters of samples or standards diluted in 0.02 M Tris, 0.15 M NaCl, pH 7.2, were placed in microtiter plates. This was followed by the sequential addition of 40 UL of hemophilia A plasma in the presence of 0.1 ugm/ml of Hirulog, and 25 ul of APTT reagent diluted with an equal volume of Tris-NaCl buffer. Following 5 minutes of vigorous mixing on a plate shaker (Minishaker, Dynatech, Chantilly, VA), 50 UL of 0.05 M calcium chloride is added to each column with an 8 channel pipettor (Eppendorf, Fremont, CA) and reading immediately commenced on the microtiter plate reader. The program was set to calculate the maximum velocity of the increase of optical density based on five consecutive readings at eight second intervals. RESULTS Figure 1 shows the pattern of increased optical density as plasma clots after the addition of calcium and in the presence of APTT reagent. The duration of clot formation as seen by the increased optical density is seen to be extremely short. Replicate samples have the calculated time to Vmax within 8 seconds the majority of the time.
Time in Minutes
FIG. 1 The change with time of optical density during the clotting of plasma as measured on the microtiter plate reader at 405 nm. The straight line delineates the time of maximum velocity.
with the addition of increasing concentrations of normal plasma to the hemophiliac plasma, there is decreased time to Vmax. A l/5 dilution of plasma clots in 300 set, and the blanks in about 1000 sec. The correlation coefficient of the logarithim of the dose of factor VIII versus the clotting time is typically aO.99.
Vol. 66, No. 5
MICROTITER PLATE READER ASSAY
601
In the absence of the inhibitor, hirulog, the log dose-response curves deviate from linearity. A l/5 dilution of plasma clots in about 180 set and the buffer blank in 400 sec. The variance across rows with each well containing the same samples were tested in the presence and absence of hirulog. In the presence of hirulog the time to Vmax across the rows has a coefficient of variance of 2-3% and in the absence of hirulog the coefficent of variance is 10%. When different quantities of therapeutic factor VIII preparations are compared with dilutions of the standard plasma the slopes of the clottimg times are parallel. Figure 2 compares the slopes of different types of factor VIII preparations with that obtained with a standard plasma.The preparations studied include plasma derived factor VIII containing Von Willebrand factor (vWf), monoclonal purified plasma factor VIII with very small quantities of vWf and recombinant factor VIII containing no vWf. When six samples were tested in quintuplicate, the within assay coefficient of variance averaged 7% (range 4.3-9.7). The between day variance over 5 days of a sample with 40% of normal plasma factor VIII was 8.4%.
FIG. 2 Comparison of the dose-response of plasma and therapeutic factor VIII preparations. Monoclonal-immunopurified factor VIII containing very low vWf and no large multimers; rDNA factor VIII-devoid of vwf; Plasma factor VIII-contains equal unitage of factor VIII and vWf.
DISCUSSION The methodology above for the measurement of Factor VIII has been validated by comparison with known dilutions of normal plasma in hemophilic plasma and by comparison of the slopes of different doses of plasma derived Factor VIII, monoclonal Factor VIII and recombinant Factor VIII. The slopes of the different preparations are all parallel despite the quite different purities of the Factor VIII.
602
MICROTITER PLATE READER ASSAY
Vol. 66, No. 5
The addition of calcium across the plate to start the clotting reaction takes about 15 seconds using the multichannel pipettor. We have added hirulog to the Factor VIII deficient plasma so that the time difference is minimal compared to the total clotting time. The clotting time is also subtantially prolonged since the reaction is run at ambient temperature. The advantage of this method is the rapidity with which many assays can be accomplished in a short time. In typical assays for Factor VIII using a single channel detector and a l-stage assay, the clotting times average about 1 minute. During the testing of many samples there is time for both the samples and the reagents to decay and alter the results. In this microtiter plate assay 48 samples can be tested in duplicate in the course of less than a half hour. Further the standards and the unknowns are measured at the same time giving added reliability to the measurement. Smaller quantities of hemophilic plasma can be used as in the experiments above accomplished with 40~1 per well. It may be possible to reduce this even further, the limiting factor being sufficient fibrinogen to produce a significant signal. More experience is needed to validate the accuracy of this methodology. It is unlikely that the presence of the hirulog or testing at room temperature will impact on the accuracy. The ability to run the standards and the samples at the same time should increase the accuracy and the precision. The data presented above indicate that the precision is as good or better than other l-stage Factor VIII assays. The same instrumentation could be utilized for the measurement of the other intrinsic coagulation components such as Factor IX and Factor XI or perhaps as a useful and reliable clot detecting endpoint for the APTT. References 1. Beebe DP, Aronson DL. An automated fibrinolytic assay performed in microtiter plates. Thrombosis Res 47, 123-128,1987. 2. Jones AJS, Meunier AM. A precise and rapid microtitre plate clot lysis assay: Methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis. Thromb Haemost 64, 455-463,199O. 3. Bourdon P, Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin. Biochemistry 29, 7095-7101, 1990.
