Use of INR calibrator plasmas in the routine coagulation laboratory: a study of two thrombolastin reagents.

INR values may be either calculated with the ISI values supplied by thromboplastin manufacturers or are directly extrapolated from certified INR calibrator plasmas. We tested the principle of local INR calibration using INR calibrator plasmas (PT-Multi Calibrator, Siemens), two thromboplastin reagents (Neoplastin Plus, rabbit brain, Stago, coagulometer-specific ISI 1.31, and Innovin, recombinant human tissue factor, Siemens) and the same coagulometer (STA-R, Stago) in 100 patients on warfarin. Using a ISI value of 0.77 with Tomenson correction for Innovin (correction factor=1.09), INR values of patients were similar with the two reagents, with a bias of 0.03 INR units and no significant regression of the difference over the average INR by method comparison analysis. With the INR calibrator plasmas, INR values with Neoplastin Plus were lower than Innovin values with an average bias of 0.39 INR units and a significant regression of the difference over the average INR (r=-0.91). Significant bias (0.16 INR units, p<0.00001) and regression (r=-0.77) was also observed by comparison of Neoplastin Plus INRs with Innovin calibrated INRs. Based on a therapeutic INR interval of 2.0 to 3.5, discordance in warfarin dosing was approximately 3 times higher with INR calibration (27% vs 11%). Because of non commutability with fresh plasma samples, local INR calibration with lyophilized calibrator plasmas may not be valid for some reagent-instrument combinations.

Moderate hyperhomocysteinemia and early-onset central retinal vein occlusion.

PURPOSE: To evaluate the prevalence of moderate fasting hyperhomocysteinemia (HHcy) and postmethionine load (PML) HHcy among patients with early-onset central retinal vein occlusion (CRVO). METHODS: The prevalence of fasting HHcy and that of PML HHcy were evaluated in a consecutive series of 58 patients with CRVO who were younger than 56 years of age (mean age, 40.3 years) and in 103 controls (mean age, 39.6 years). Plasma folate, vitamin B12, and pyridoxal-5'-phosphate (PLP) levels were measured in 42 patients and 67 controls. RESULTS: Mantel-Haenszel odds ratios for CRVO patients were 3.00 (95% confidence interval [CI], 0.83-10.8) for fasting HHcy, 3.50 (95% CI, 1.07-11.4) for PML HHcy, and 3.00 (1.18-7.6) for fasting HHcy and PML HHcy in subjects with normal fasting total homocysteine (tHcy) levels. Moderate HHcy was associated with reduced plasma levels of folate and PLP (P < or = 0.04). There was no significant dependence of fasting and PML tHcy levels on any traditional risk factor evaluated. CONCLUSIONS: Moderate HHcy is an independent risk factor for early-onset CRVO.

Insulin action on protein metabolism in acromegalic patients.

Insulin resistance in acromegaly causes glucose intolerance and diabetes, but it is unknown whether it involves protein metabolism, since both insulin and growth hormone promote protein accretion. The effects of acromegaly and of its surgical cure on the insulin sensitivity of glucose and amino acid/protein metabolism were evaluated by infusing [6,6-(2)H(2)]glucose, [1-(13)C]leucine, and [2-(15)N]glutamine during a euglycemic insulin (1 mU x kg(-1) x min(-1)) clamp in 12 acromegalic patients, six studied again 6 mo after successful adenomectomy, and eight healthy controls. Acromegalic patients, compared with postsurgical and control subjects, had higher postabsorptive glucose concentration (5.5 +/- 0.3 vs. 4.9 +/- 0.2 micromol/l, P < 0.05, and 5.1 +/- 0.1 micromol/l) and flux (2.7 +/- 0.1 vs. 2.0 +/- 0.2 micromol x kg(-1) x min(-1), P < 0.01, and 2.2 +/- 0.1 micromol x kg(-1) x min(-1), P < 0.05) and reduced insulin-stimulated glucose disposal (+15 +/- 9 vs. +151 +/- 18%, P < 0.01, and 219 +/- 58%, P < 0.001 from basal). Postabsorptive leucine metabolism was similar among groups. In acromegalic and postsurgical subjects, insulin suppressed less than in controls the endogenous leucine flux (-9 +/- 1 and -12 +/- 2 vs. -18 +/- 2%, P < 0.001 and P < 0.05), the nonoxidative leucine disposal (-4 +/- 3 and -1 +/- 3 vs. -18 +/- 2%, P < 0.01 and P < 0.05), respectively, indexes of proteolysis and protein synthesis, and leucine oxidation (-17 +/- 6% in postsurgical patients vs. -26 +/- 6% in controls, P < 0.05). Within 6 mo, surgery reverses insulin resistance for glucose but not for protein metabolism. After adenomectomy, more leucine is oxidized during hyperinsulinemia.

Discrepant sensitivity of thromboplastin reagents to clotting factor levels explored by the prothrombin time in patients on stable oral anticoagulant treatment: impact on the international normalized ratio system.

BACKGROUND AND OBJECTIVES: We tested the principle of local International Normalized Ratio (INR) calibration using INR calibrator plasmas (PT Calibration Plasma Kit, Behring), two thomboplastin reagents (Neoplastin plus, rabbit brain, Stago, and Recombiplastin, recombinant human tissue factor, Ortho Diagnostics) and the same coagulometer (STA, Stago) on 92 patients on stable oral anticoagulant treatment. DESIGN AND METHODS: A four-point calibration was obtained with each reagent by linear regression (sec/INR) on a log-log scale (r > or = 0.999). The bias between the two reagents (Recombiplastin - Neoplastin Plus) was reduced from 31.7% to 17.5% and 7.5% (p=0.001) when results were expressed, respectively, as PT ratio (using the mean normal prothrombin time as denominator term), INR (using instrument-specific ISI supplied by the manufacturers) and calibrated INR, but there was a consistently significant regression of the differences over the average values even after log transformation (r > or = 0.586). The bias between the reagents was reduced to 1% (p=ns) when assuming Recombiplastin as the reference thromboplastin and applying Tomenson's correction, but limits of agreements were as large as 20%. Factor VII, X, V and II activity was measured with the two thromboplastin reagents in all plasma samples using immunodepleted plasmas (Stago). RESULTS: Statistically significant biases were observed for all clotting factors with the two reagents (Recombiplastin Neoplastin Plus) and ranged from 3.5 % (FII) to 37.2% (FVII). In addition, for FVII and FV there was a significant regression of the difference over the average value (after log-transformation, r > or = 0.282). The patients were divided into 3 groups according to their degree of anticoagulation (INR <2.0; INR between 2.0 and 3.5; INR >3.5). Factor levels differed significantly with the two reagents throughout the 3 groups of patients. In addition, the relative distributions of the 3 vitamin K-dependent factors also differed in the 3 groups with the two thromboplastin reagents. INTERPRETATION AND CONCLUSIONS: The discrepant sensitivity to factor VII, X and V levels of the two thromboplastin reagents explored in this study prevents INR calibration with commercially available calibrator plasmas and is responsible for a significant variability in INR values even under optimal conditions of INR calibration.

Relationship between international normalized ratio values, vitamin K-dependent clotting factor levels and in vivo prothrombin activation during the early and steady phases of oral anticoagulant treatment.

BACKGROUND AND OBJECTIVES: In vitro studies have shown that the rate of prothrombin activation is linearly related to the concentration of factor II (FII) in the assay system, suggesting a key role of prothrombin levels in the expression of the antithrombotic activity of oral anticoagulant treatment (OAT). We investigated the in vivo relationship between prothrombin activation and vitamin K-dependent clotting factor levels during the early and steady phases of OAT in patients and in healthy volunteers. DESIGN AND METHODS: The changes in international normalizezd ratio (INR) and in the plasma levels of FVII, FX, FII, protein C (PC) and prothrombin fragment 1.2 (F1+2) induced by OAT were monitored over 9 days in 10 patients not on heparin starting warfarin after heart valve replacement (HVR) and in 9 healthy volunteers submitted to an 8-day course of warfarin treatment. FII and F1+2 plasma levels were also measured in 100 patients on stable oral anticoagulant treatment with INRs ranging from 1.2 to 6.84. RESULTS: Because HVR patients had subnormal FVII, FX and FII levels after surgery, INR values > 2.0 were attained already 24 hours after the first warfarin dose. In healthy volunteers, INR values greater than 2.0 were first observed after 72 hours. Nadir levels of FVII, PC, FX and FII were reached between 40 and 88 hours in HVR patients and between 72 and 192 hours in healthy volunteers. The FII apparent half-disappearance time (t/2) was 99 hours in HVR patients and 115 hours in healthy volunteers (p = ns). In HVR patients there was no normalization of initially elevated F1+2 levels until day 7 with an apparent t/2 of 132 hours. In healthy volunteers, a decrease to subnormal F1+2 levels was observed by day 8 of treatment (apparent t/2 = 107 hours). In both HVR patients and healthy volunteers, FII and PC levels were independent predictors of the changes in F1+2 levels (p = 0.0001). In patients on stable OAT, only FII levels were independent predictors of the variation in F1+2 levels (p = 0.0001). INTERPRETATION AND CONCLUSIONS: During the early phase of oral anticoagulant treatment in vivo prothrombin activation is a function of the balance between FII and PC levels and is not significantly prevented until nadir levels of FII are obtained. This provides an explanation for the requirement of overlapping heparin and oral anticoagulant treatment for at least 48 hours after the achievement of therapeutic INR values in patients with thromboembolic diseases. In addition, in vivo prothrombin activation is a function of FII levels rather than INR values also in patients on stable oral anticoagulant treatment.

Amino acid kinetics during the anhepatic phase of liver transplantation.

Alanine and glutamine are interorgan nitrogen/carbon carriers for ureagenesis and gluconeogenesis, which are mainly but not necessarily only hepatic. The liver is central to alanine and glutamine metabolism, but most organs can produce and use them. We studied amino acid kinetics after liver removal to depict initial events of liver failure and to provide a model to study extrahepatic gluconeogenesis and nitrogen disposal in humans. We measured amino acid kinetics with [5,5,5-(2)H(3)]leucine and [3-(13)C]alanine or [1,2-(13)C(2)]glutamine tracers in 21 subjects during and after the anhepatic phase of liver transplantation: 12 were at 7 months posttransplantation, and 7 were healthy control subjects. Anhepatic leucine kinetics, including proteolysis, was unchanged. Alanine plasma and whole-body contents increased 3x and 2x, with a halved metabolic clearance and a doubled production, 2% greater than disposal. Free whole-body glutamine decreased 25% but increased 50% in plasma. Glutamine clearance was halved, and the production decreased by 25%, still 2% greater than disposal. Liver replacement decreased alanine and glutamine concentrations, leaving leucine unchanged. Liver removal caused doubled alanine fluxes, minor changes in glutamine, and no changes in leucine. The initial events after liver removal are an accumulation of three-carbon compounds, an acceleration of alanine turnover, and limited nitrogen storage in alanine and glutamine.

Plasma levels of endothelial protein C receptor respond to anticoagulant treatment.

The endothelial protein C receptor (EPCR) facilitates protein C activation and plays a protective role in the response to Escherichia coli-mediated sepsis in primates. Previously, a soluble form of EPCR (sEPCR) in human plasma was characterized, and several studies indicated that generation of sEPCR is regulated by inflammatory mediators, including thrombin-mediated up-regulation of surface metalloproteolytic activity in vitro. This study addressed the question of whether plasma sEPCR levels reflect changes in thrombin generation in patients undergoing anticoagulant treatment. The sEPCR levels in patients treated with coumarin-type oral anticoagulants were significantly lower than those in healthy asymptomatic adult volunteers (105.3 +/- 70.8 ng/mL [n = 55] versus 165.8 +/- 115.8 ng/mL [n = 200]; P <.0001). A similar decline in plasma sEPCR levels was found in patients treated with unfractionated heparin. In healthy volunteers, sEPCR levels declined to about 100 ng/mL within 3 days after initiation of an 8-day period of warfarin administration and increased within 2 days after its cessation. Plasma sEPCR levels returned to pretreatment values within 1 week, and the changes in plasma sEPCR levels mirrored changes in values for international normalized ratios. A similar decline in sEPCR levels with time was observed in 7 patients beginning treatment with warfarin for a thrombotic disorder. Prothrombin fragment 1 + 2 levels also decreased in volunteers and patients given warfarin. These results show that plasma sEPCR levels decline in response to treatment with anticoagulants whose mechanism of action is known to decrease in vivo thrombin production.