Coagulation testing with the Du Pont aca discrete clinical analyzer.

Methods for the measurement of the functional concentration of AT III, PLG, and FBG have been developed for the Du Pont aca discrete clinical analyzer. These methods are fully automated, provide excellent reproducibility, and compare favorably with existing methodologies. The availability of these and future coagulation assays on this automated analyzer will provide the hospital laboratory with a reliable means for evaluating hemostatic function. In addition, excellent method reproducibility and use of defined reference standards should significantly contribute to the standardization of coagulation methods.

Development and analytical performance of automated tests for antithrombin III and plasminogen on the Du Pont aca analyzer.

We describe assays for functional antithrombin III (AT III) and plasminogen in plasma with the Du Pont aca discrete clinical analyzer. Both are two-stage kinetic assays, based on synthetic substrate methodologies, and require 20-microL sample volumes. In the AT III assay the sample is incubated with excess thrombin and heparin to form the functionally inactive AT III-thrombin complex. Residual thrombin is measured through its rate of hydrolysis of a lysine thioester and is inversely related to analyte concentration. In the plasminogen assay excess streptokinase is reacted with the sample to form an enzymatically active complex. The substrate hydrolysis rate of this complex is measured, which is linearly related to the concentration of plasminogen in the sample. Reaction conditions for both assays were optimized by univariate and response surface techniques. The assay for AT III has a range of 0 to 150% of the value for normal human plasma (% NHP) with a CV of 3% at 80% NHP. The plasminogen assay is linear from 25 to 200% NHP with a CV of less than 2% at 80% NHP. No significant interferences with either method by common blood components or drugs were found.

Optimization of enzyme-based assays in coagulation testing.

Optimized assays for antithrombin III and plasminogen have been developed based on a study of the kinetic parameters Km and Kcat for four commercially available substrates: the p-nitroanilide derivatives of D-Phe-pipecolyl-Arg (S-2238), and toluenesulfonyl-Gly-Pro-Arg (Chromozym TH), which are thrombin substrates; D-Val-Leu-Lys (S-2251), a plasminogen/streptokinase substrate; and alpha-N-carbobenzoxy-L-lysine thiobenzyl ester, a substrate for both enzymes. We used a centrifugal analyzer system for rapid data acquisition and interactive analysis. Optimized conditions for assay of a particular enzyme are not constant for different substrates in the same buffering agent. For example, in 1,4-piperazine diethanesulfonic acid buffer at 37 degrees C, thrombin-catalyzed hydrolysis of Chromozym TH is optimal at 125 mmol/L buffer, 100 mmol/L NaCl, and pH 8.2, whereas substitution of S-2238, also a tripeptide p-nitroanilide, yields optimal hydrolysis at 85 mmol/L buffer, 300 mmol/L NaCl, and pH 7.2. We conclude that optimized assay conditions are best obtained by an extensive survey of available buffers and a detailed investigation of the effects of variation in pH and in the concentrations of the buffer and auxiliary reagents through use of both one-factor-at-a-time and multivariate response surface experimentation.

Development and analytical performance of a functional assay for fibrinogen on the Du Pont aca analyzer.

A method for determination of functional fibrinogen has been developed for the Du Pont aca discrete clinical analyzer. This fully automated assay is based on the rate of fibrin turbidity formation when thrombin is added to the test sample. The rate of this reaction is enhanced by added calcium chloride and dextran. The assay range is from 0.50 to 8.00 g/L. Within-run reproducibility studies at the medical decision level of 1.0 g/L showed a CV of 2.5%. There was no interference by heparin up to 10 USP units per milliliter of plasma.

Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.

The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.