The importance of locally derived reference ranges and standardized calculation of dilute Russell's viper venom time results in screening for lupus anticoagulant.

Three commercial dilute Russell's viper venom time (DRVVT) kits were evaluated at four UK centres experienced at performing lupus anticoagulant (LA) tests. Each centre established a normal reference range for the DRVVT ratio calculated against local pooled normal plasma from 20 healthy normal subjects. Plasma from LA-positive patients and LA-negative thrombophilia patients was also tested. DRVVT ratios and the degree of correction were assessed in a variety of ways to reflect not only the UK national Guidelines, but also the manufacturers' recommendations. The reference range data showed a normal distribution in each case, but considerable variation in the mean and SD between the centres and reagents, with the mean +2SD value ranging from 1.06 to 1.19. The use of an arbitrary DRVVT ratio of < 1.1 as the cut-off value for normality, which is applied in many laboratories, is therefore inappropriate. Although no single kit had a clear overall advantage in terms of sensitivity and specificity, the way in which the screen and confirmation data were analysed had a major impact on the interpretation of the results. A data analysis method employing a mean plus two standard deviations (SDs) cut-off for normality, and judgement regarding confirmation of LA based on a percentage correction of DRVVT ratio, was the simplest and most consistent, with overall sensitivity and specificity values of 81% and 94%, respectively, for uncomplicated LA-positive and LA-negative thrombophilia samples. We conclude that the 1991 BSCH Guidelines are in need of revision, each laboratory should establish its own normal reference range for the DRVVT ratio and a common method should be used for calculating the degree of correction with confirmation reagents, so that LA results can be correctly interpreted between laboratories. Standardizing DRVVT interpretation in this way should improve the consistency of LA detection.

Calculation vs calibration curve for INR determination. Results of an interlaboratory proficiency scheme.

We surveyed 271 laboratories participating in a quality assessment program to ascertain whether the use of a calibration curve for determining the international normalized ratio (INR) would improve interlaboratory accuracy and precision. Lyophilized warfarinized samples with INR values assigned through manual calibration against internationally assigned rabbit reference thromboplasts were assayed for prothrombin time. Calibration analysis on the results was performed by linear regression. In all but 1 sample, the mean INR value computed by the calibration method was closer to the "true" value than the mean for the conventional calculation method using the International Sensitivity Index (ISI); the ISI calculation consistently overestimated the true value. Interlaboratory variation decreased using the calibration method. Variation from reagent to reagent was greater than from instrument to instrument, but was reduced by the calibration method. The specificity of the ISI for instrument type did not seem to alter the findings. Use of in-house calibrators to verify the ISI improved precision but not necessarily accuracy. The formation of a stable calibration line is consistent over time, but further studies are required to confirm whether such calibration improves the accuracy and precision of INR determination in practice.

The determination of INR in stored whole blood.

AIMS: To examine the reliability of international normalised ratio (INR) determination on samples stored as whole blood for up to two days at room temperature. METHODS: The INR of 40 patients receiving oral anticoagulants was determined on fresh blood and on samples stored for 24 and 48 hours, using five locally calibrated prothrombin time systems. These incorporated Manchester reagent, Recombiplastin, IL PT Fibrinogen HS Plus, Manchester combined capillary prothrombin time reagent, and a freeze dried in-house reference rabbit brain thromboplastin, RBT 1010. In addition, factors II, V, VII, and X were determined on samples obtained from 18 of these patients before and after incubation at room temperature. RESULTS: The INR of the samples changed by differing amounts during storage, depending on which system was employed. Although the mean change after 24 hours storage was relatively small, there were individual samples that changed by > 0.5 INR with all systems. These changes would lead to adjustment in dosage of certain patients. After 48 hours these effects were greater with all systems except that employing Recombiplastin. There were only small reduction in the measured factors by 48 hours. CONCLUSIONS: After storage of samples for only 24 hours, some patients' INR changed sufficiently to affect dosage. In view of these observations, the practice of storing whole blood samples for INR determination cannot be recommended.

Why do outpatients fail to keep their clinic appointments? Results from a survey and recommended remedial actions.

We undertook a survey of 2555 outpatients (both new and review) to look at the reasons for high non-attendance (DNA) rates. Completed questionnaires (n = 983, 38.5% response) indicated the main reasons: forgetting or not receiving the appointment because of illness, and less often because of feeling better; transport problems; and short notice. Our remedial actions included improving the clerical system, announcing DNA rates on a regular basis in GP surgeries and in the outpatient department, introducing a tear-off slip with the appointment letter for patients to return, installing a free answerphone, active GP/consultant involvement in DNA-related problems and local publicity campaigns. A new scheme in our trust--outpatient telephone follow-up--may also help in reducing DNA rates.

Local INR correction: justification for a simplified approach.

AIMS: Errors in reporting International Normalised Ratios (INR) may be corrected by assignment of a System International Sensitivity Index (System ISI). This 57 centre study tests the validity of several procedures for INR correction. METHODS: Prothrombin times of eight lyophilised coumarin calibrants, a lyophilised normal pool calibrant, and eight frozen coumarin plasmas were determined at each centre. The calibrants were calibrated using international reference preparations. The eight frozen coumarin plasmas were calibrated in a four centre international exercise. The relations tested were: (a) the logarithm of local prothrombin time against the logarithm of reference prothrombin time; (b) reference INR against local prothrombin time; and (c) logarithm of reference INR against logarithm of local prothrombin time. These methods were analysed by both linear and orthogonal regression. RESULTS: All system groups required correction, the mean percentage deviation of the uncorrected data from the calibrated values was 19.0%. There was also considerable variation in INR, with the coefficient of variance (CV) ranging from 11.30 to 17.29%. Correction of INR was possible with all methods (CV reduced to < 7%). However, only when a plot of the logarithm of local prothrombin time against the logarithm of reference prothrombin time was fitted by orthogonal regression, or a plot of logarithm of reference INR against logarithm of local prothrombin time was fitted by either type of regression analysis, did the best fit line through the calibrant plasmas also pass close to the local mean normal prothrombin time. CONCLUSIONS: While INR correction may be achieved by all the above methods, that relating log reference INR to log local prothrombin time by linear regression analysis is the simplest to perform.

Heat stability of two candidate international reference preparations for recombinant human tissue factor.

Adequate heat stability of international reference preparations (IRP) for thromboplastin (tissue factor) is an essential requirement. Accelerated degradation testing was performed by three laboratories on two candidate IRP for recombinant human tissue factor. Heat treatment of these candidates resulted in slight shortening of the PT, contrasting with heat-induced prolongation of the PT observed with a conventional human brain derived IRP. Heat stability of these candidates was improved when compared with the stability of previous recombinant human tissue factor preparations. The PT-ratio did not change significantly when the candidates were stored for 28 days at 44 degrees C. It can therefore be concluded that both candidates are acceptable with regard to stability.

System ISI calibration: a universally applicable scheme is possible only when coumarin plasma calibrants are used.

Coagulometer-induced errors in International Normalized Ratios (INR) may be corrected by assigning an International Sensitivity Index (ISI) specific for the local combination of coagulometer and thromboplastin reagent (System ISI). In the present study, 57 laboratories determined the INR of eight frozen coumarin plasmas. There was considerable variation in INR reported at the different centres, with coefficients of variation (CV) ranging from 11.30% to 17.29%. In addition, their consensus mean INR values were consistently higher than those obtained manually. The overall mean percentage deviation was 20.06%, indicating that correction was required to reduce the over-estimation of INR that occurred even in those Systems incorporating a thromboplastin reagent with an instrument-specific ISI. Local correction using coumarin plasma calibrants reduced this value to < or = 7% with current reference thromboplastins BCT 441, OBT 79 and RBT 90 and also lowered the CV to < 5%. In contrast, when adsorbed plasma calibrants were employed, mean percentage deviations were 7.87% with BCT 441, 12.47% with OBT 79 and 77.37% with RBT 90 as reference respectively. This difference may be due, in part, to the difficulty in assigning an INR to adsorbed plasma calibrants, which contain no protein induced by vitamin K antagonists (PIVKA). This study demonstrates that coumarin plasma calibrants are superior in System ISI calibration.

Calculation of System International Sensitivity Index: how many calibrant plasmas are required?

AIMS: To determine the minimum number of calibrant plasmas required to assign an accurate System International Sensitivity Index (ISI). METHODS: Eighteen lyophilised adsorbed and eight frozen coumarin plasmas were despatched to 57 participating laboratories. Each centre determined the prothrombin time of all of the plasmas using their routine system of coagulometer and thromboplastin reagent and provided their system mean normal prothrombin time (MNPT). For each laboratory, the slope of the line relating local system results, including MNPT, to calibrated values obtained with BCT 441, RBT 1303 or RBT 90 was derived by orthogonal regression. The number of adsorbed calibrant plasmas included in the calculation was reduced, two at a time, from 18 to two. The validity of the System ISI was tested using the frozen coumarin plasmas. RESULTS: All systems tested gave higher International Normalised Ratios (INR) than those obtained manually with the reference thromboplastins. All systems therefore required correction. No changes were seen in the System ISI as the number of calibrant plasmas was reduced from 18 to 12. Below 12, inconsistent results were obtained with some systems and were dependent on plasma selection. CONCLUSIONS: Although System ISI could be assigned using as few as two calibrant plasmas, to avoid imprecise calibration, a minimum of 12 calibrant plasmas is recommended.

Soluble P selectin in peripheral vascular disease: relationship to the location and extent of atherosclerotic disease and its risk factors.

Circulating levels of the adhesion molecule P-selectin (CD62P) are increased in the plasma of patients with atherosclerosis, but its relationship to the anatomical location of symptomatic disease or extent of symptomatic disease is unknown. The influence of the risk factors for atherosclerosis on soluble P-selectin is also unclear. To clarify these questions we analysed plasma samples from 170 patients with symptomatic peripheral vascular disease and 119 asymptomatic controls who were, as a group, age- and sex-matched. Soluble P-selectin (ELISA) was increased in 83 patients with symptomatic disease of the iliac and/or femoral arteries alone (P < 0.05, ANOVA) but not in 37 patients with symptomatic carotid artery disease alone compared with controls. Soluble P-selectin was equally raised in 120 patients with disease at one arterial site and in 50 patients with disease at two or more arterial sites (both P < 0.05) compared with controls. Smoking and atherosclerosis were both independent predictors of raised soluble P-selectin. We conclude that increased soluble P-selectin may have value as a marker of peripheral vascular disease of the iliac and/or femoral arteries in group comparisons only, as the poor discrimination and wide variation of data make comparisons at the individual level difficult.

Haematology and coagulation indices in paired samples of arterial and venous blood from patients with arterial disease.

To investigate whether there are differences in haematology and coagulation indices in arterial and venous plasma, and whether those changes related to damage to the endothelium in atherosclerosis, we obtained blood samples from 22 subjects undergoing diagnostic angiography. There were no differences in any of the 15 routine haematological indices measured. There were no differences in prothrombin time, activated partial thromboplastin time, fibrinogen, tissue plasminogen activator, D-dimer, leucocyte elastase, soluble P-selectin or von Willebrand factor. In venous samples, von Willebrand factor was lower in serum than in plasma (p < 0.0001). Levels of the tissue plasminogen activator/plasminogen activator inhibitor-1 (tPA/PAI-1) complex were markedly higher in arterial blood than in venous blood (p = 0.004) and plasma viscosity was higher in venous blood (p = 0.0014). Consequently, with the exception of viscosity and the tPA/PAI complex, we can find no differences in arterial blood compared to venous blood which can contribute to the debate regarding the mechanism of damage to arterial endothelial cells but the relative protection of venous endothelial cells from injury in atherosclerosis.

An easier alternative to orthogonal regression for calculation of International Sensitivity Indexes.

AIMS: To evaluate an easier alternative method to orthogonal regression analysis for calculating International Sensitivity Indexes (ISI). METHODS: ISI for 18 reagents were estimated from reference and test reagent prothrombin times using plasma from 60 stabilised patients undergoing anticoagulation therapy and 20 normal subjects. ISI were also derived for 12 systems (instrument/reagent combination) using lyophilised plasma calibrants. Orthogonal regression and the easier alternative log ratio method were evaluated by comparing resultant International Normalised Ratios (INR) for 58 patients using two test systems. RESULTS: For the reagent calibrations, the differences in the two methods for the sensitivity slopes were very small. For the system calibrations, slope differences were still of little clinical importance. Parallel observations for INR on the 58 patients confirmed that the bias introduced by easier log ratio derivation of ISI was small and of minor clinical importance, although the bias increases for high INR. CONCLUSIONS: The easier method for ISI determination is a useful alternative to orthogonal regression analysis, particularly when computer assistance is not available, for checking for gross errors in computer computation and for use when calculating ISI from INR calibrants.

Activated partial thromboplastin time for automated techniques: a comparison of two commonly used reagents.

In a comparison between two widely used activated partial thromboplastin time reagents (Instrumentation Laboratories, Manchester low-opacity), using one popular instrument (Automated Coagulation Laboratory, model 300R), a wide range of defects and 40 normals were tested. There was generally good agreement between methods, although for samples from patients on high heparin dosages the agreement was poor. The manufacturer's recommendation for the therapeutic range for heparin for the Instrumentation Laboratory reagent was lower than the range in current general use. The Manchester reagent was more sensitive to the lupus anticoagulant.

The correction of coagulometer effects on international normalized ratios: a multicentre evaluation.

One hundred and twenty-nine centres in the U.K. participated in a study to test the reliability of the three methods of correction for coagulometer effects on international normalized ratios (INR). Results from 37 centres which tested three warfarinized plasmas by the manual method were taken as the 'true' INR for the assessment of coagulometers. 63 centres (11 manual and 52 using coagulometers) determined their local International Sensitivity Index (ISI) in a calibration exercise. This was performed with a set of 20 lyophilized plasma calibrants with certified manual prothrombin times for the thromboplastin used in the study. The following methods of INR derivation were compared by assessing the percentage deviation from the three INR values established by 37 manual users: I. No coagulometer correction, i.e. (local PT/reference manual normal PT)manual ISI II. Coagulometer ratio correction, i.e. (local coagulometer PT/local coagulometer MNPT)manual ISI III. Local system ISI, i.e. (local coagulometer PT/local coagulometer MNPT)local system ISI IV. System ISI, i.e. (local coagulometer PT/local coagulometer MNPT)system ISI The local system ISI with the plasma calibrants (method III) gave the most reliable correction (mean deviation from 'true' INR 4.87%). The method which gave the least was with the coagulometer ratio correction, i.e. the manual ISI and local coagulometer MNPT (mean 11.25%). The system ISI tested with ACL coagulometers gave less correction than the local ISI calibration. The local system calibration with lyophilized plasmas also avoids some of the constraints on conventional thromboplastin calibrations.

Effect of the choice of WHO International Reference Preparation for thromboplastin on International Normalised Ratios.

AIMS: To compare the International Normalised Ratio (INR) obtained directly with the two types of WHO plain International Reference Preparation for thromboplastin in patients treated with coumarin. METHODS: Prothrombin times were performed in parallel at four centres using WHO human plain IRP (BCT/253) and rabbit plain IRP (RBT/79). Sixty patients and 20 normal controls were tested at each centre. Differences in INR among the centres were assessed by one factor, analysis of variance. The bias for each centre was assessed by the t test. RESULTS: At all four centres higher INRs were consistently found with the rabbit plain reagent. Two of the centres showed significantly greater bias. CONCLUSIONS: There was a small but significant difference in INR results obtained directly with these two reference reagents at all four centres (mean 7.35%). This in part may result from the different responsiveness of the two IRP to the coumarin defect or to imprecision of the original ISI calibrations of the two plain WHO IRP. The findings support the adoption of a single master IRP, in accord with WHO recommendations, which would resolve the present anomalous situation.

Safety of anticoagulation in the elderly: reasons for discontinuing therapy.

We have conducted a retrospective study on the reasons for discontinuing anticoagulants in 50 patients over the age of 75 years compared with 198 adults under 75 years to determine the safety of therapy in the elderly. Venous thromboembolism and arterial embolization were the most common indications for therapy in the elderly and the median duration of therapy in all patients was 7 months (9 days-22 years). There were no deaths attributable to anticoagulants. There was no significant difference in the proportion of elderly patients who stopped treatment because of bleeding compared with 198 patients under 75 years (5/50 (10%) vs 12/198 (6.1%), P = 0.26), nor in the rate of bleeding between the two groups (5/52.5 (9.5%) treatment-years vs 12/249 (4.8%) treatment-years, P = 0.15). This complication rate does not suggest that age per se is a risk factor in the use of oral anticoagulants.