Evaluation of chromogenic substrate assays for fibrinolytic analytes in dogs

Anne Lanevschi From the Department of Pathology and Microbiology, College of Veterinary Medicine, St-Hyacinthe, Quebec, Canada J2S 7C6 (Lanevschi), and Departments of Veterinary Clinical Sciences (Kramer, Greene) and Veterinary and Comparative Anatomy, Pharmacology, and Physiology (Meyers), College of Veterinary Medicine, Pullman, WA 99164.

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John W. Kramer From the Department of Pathology and Microbiology, College of Veterinary Medicine, St-Hyacinthe, Quebec, Canada J2S 7C6 (Lanevschi), and Departments of Veterinary Clinical Sciences (Kramer, Greene) and Veterinary and Comparative Anatomy, Pharmacology, and Physiology (Meyers), College of Veterinary Medicine, Pullman, WA 99164.

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Stephen A. Greene From the Department of Pathology and Microbiology, College of Veterinary Medicine, St-Hyacinthe, Quebec, Canada J2S 7C6 (Lanevschi), and Departments of Veterinary Clinical Sciences (Kramer, Greene) and Veterinary and Comparative Anatomy, Pharmacology, and Physiology (Meyers), College of Veterinary Medicine, Pullman, WA 99164.

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Kenneth M. Meyers From the Department of Pathology and Microbiology, College of Veterinary Medicine, St-Hyacinthe, Quebec, Canada J2S 7C6 (Lanevschi), and Departments of Veterinary Clinical Sciences (Kramer, Greene) and Veterinary and Comparative Anatomy, Pharmacology, and Physiology (Meyers), College of Veterinary Medicine, Pullman, WA 99164.

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Abstract

Objective

To evaluate the ability of commercial, chromogenic kits designed to measure human fibrinolytic pathway components to measure the canine plasma fibrinolytic pathway enzymes, tissue plasminogen activator (tPA) and plasminogen (PLG), and their respective inhibitors, plasminogen activator inhibitor 1 (PAI) and α2-anti-plasmim (AP).

Animals

20 healthy dogs of various ages and breeds.

Procedure

The commercial procedure was adapted to a microtitration plate. Standard curves were generated by use of a canine plasma pool.

Results

Modifications of the commercial kit consisted of change in incubation periods and the substitution of urokinase for the streptokinase. Plasminogen and AP procedures yielded intra- and interassay coefficients of variation (CV) ranging from 2 to 6.4%. The tPA activity gave an acceptable intra-assay CV of 4.2%, but an equivocal interassay CV of 18%. The PAI assay gave unacceptable intra-assay and interassay CV of 59 and 66%, respectively.

Conclusions

Modifications of the commercial PLG and AP procedures were appropriate for use with fresh and frozen canine plasma. However, equivocal results were obtained for canine plasma tPA. Although the PAI assay was able to detect the inhibitor, it gave unacceptable quantifiable results. Human and canine plasma contained similar amounts of PLG and AP, but 25% more tPA was found in canine plasma than human plasma.

Clinical Relevance

With modifications, the commercial human PLG and AP chromogenic kits may serve to elucidate such canine fibrinolytic disorders as disseminated coagulopathy. The high cost of the chromogenic substrate limits its application. (Am J Vet Res 1996;57: 1124-1130)

Abstract

Objective

To evaluate the ability of commercial, chromogenic kits designed to measure human fibrinolytic pathway components to measure the canine plasma fibrinolytic pathway enzymes, tissue plasminogen activator (tPA) and plasminogen (PLG), and their respective inhibitors, plasminogen activator inhibitor 1 (PAI) and α2-anti-plasmim (AP).

Animals

20 healthy dogs of various ages and breeds.

Procedure

The commercial procedure was adapted to a microtitration plate. Standard curves were generated by use of a canine plasma pool.

Results

Modifications of the commercial kit consisted of change in incubation periods and the substitution of urokinase for the streptokinase. Plasminogen and AP procedures yielded intra- and interassay coefficients of variation (CV) ranging from 2 to 6.4%. The tPA activity gave an acceptable intra-assay CV of 4.2%, but an equivocal interassay CV of 18%. The PAI assay gave unacceptable intra-assay and interassay CV of 59 and 66%, respectively.

Conclusions

Modifications of the commercial PLG and AP procedures were appropriate for use with fresh and frozen canine plasma. However, equivocal results were obtained for canine plasma tPA. Although the PAI assay was able to detect the inhibitor, it gave unacceptable quantifiable results. Human and canine plasma contained similar amounts of PLG and AP, but 25% more tPA was found in canine plasma than human plasma.

Clinical Relevance

With modifications, the commercial human PLG and AP chromogenic kits may serve to elucidate such canine fibrinolytic disorders as disseminated coagulopathy. The high cost of the chromogenic substrate limits its application. (Am J Vet Res 1996;57: 1124-1130)

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