Comparison of six dilute russell viper venom time lupus anticoagulant screen/confirm assay kits.

BACKGROUND: The normalized dilute Russell viper venom time (DRVVT) ratio provides a robust assay methodology for lupus anticoagulant (LA) detection. OBJECTIVES: We evaluated six normalized DRVVT LA screen and confirm systems for inter-method consistency. Reagents were purchased from Diagnostica Stago, Inc. (Parsippany, NJ); Precision BioLogic Inc. (Halifax, Nova Scotia, Canada); Siemens Healthcare Inc. (Deerfield, IL); TCoag (Parsippany, NJ); Instrumentation Laboratories (Bedford, MA); and Sekisui Diagnostics (Pfungstadt, Germany). METHODS: For all assays, we employed the STA-R Evolution automated coagulometer, adhering to manufacturers' instructions. LA-positive and LA-negative plasma controls were purchased from Diagnostica Stago and pooled normal plasma (PNP) was purchased from Precision BioLogic. We computed the mean of the reference interval (MRI) and action limits for all kits using LA-negative aliquots from locally sourced normal subjects (n = 42). We then assayed locally sourced LA-positive plasmas (n = 43) and using analysis of variance compared uncorrected screen/confirm ratios and screen/confirm ratios that were normalized using MRI and mean PNP results. RESULTS: The grand mean action limit, MRI + 3 SD, derived from the local normal plasmas, was 1.2, confirming the manufacturers' recommended limits; however, limits must be locally computed. The all-sample p value was less than 0.001, indicating heterogeneity among ratios. When Sekisui ratios were excluded, the p value was 0.14, thus indicating that this method introduced the major difference among methods. Mean screen/confirm ratios computed from LA-positive specimens were 1.91 to 2.04 for reagent systems other than Sekisui, which instead yielded a mean ratio of 1.198, indicating that this method was relatively insensitive to LA. A negative bias was recorded by two lots from the Sekisui system for LA-positive specimens. Screen/confirm ratios from combined LA-positive and LA-negative samples generated a combined range of 1.59 to 1.67 for all reagents except Sekisui, which instead yielded 1.09. The within-run percent coefficient of variation (CV%) was less than 5.0% using all samples. Between-run CV% using Diagnostica Stago LA-positive and LA-negative controls was less than 5.5%. CONCLUSIONS: DRVVT screen/confirm ratios discriminate between LA-positive and LA-negative samples and generally provide acceptable reproducibility. Ratio results may vary among reagent-instrument combinations. In this study, normalization added little to the clinical result interpretation.

Whole blood platelet aggregometry and platelet function testing.

Platelet aggregometry has been the reference method employed to detect, diagnose, and monitor qualitative platelet disorders since the early 1960s. Lumiaggregometry and impedance-based whole blood lumiaggregometry have advantages over light transmittance aggregometry in that they provide for enhanced specimen management and increase the test sensitivity to impairment of platelet granule secretion. Whole blood lumiaggregometry detects and identifies congenital and acquired platelet plasma membrane receptor defects, metabolic pathway secretion disorders, and storage pool deficiency. Whole blood lumiaggregometry is also being applied to antiplatelet therapy monitoring and identifies aspirin and thienopyridine resistance. There is growing interest in using impedance-based whole blood lumiaggregometry for near-patient whole blood platelet analysis and antiplatelet therapy monitoring. This article will also discuss other whole blood testing processes for assessing platelet function, particularly as applied to assessing the effect of antiplatelet medication.

Screening with the activated protein C resistance assay yields significant savings in a patient population with low prevalence of factor V leiden.

Testing for factor V Leiden can be performed with a molecular assay or a test for activated protein C resistance. We noted that physicians in our institution tended to order the molecular test 80% of the time, but the prevalence of the mutation in our patient population was less than 10%. Consequently, we decided to introduce the activated protein C resistance assay in house and consistently use it for screening before the more expensive genetic test and to negotiate a discounted charge for the latter at a reference laboratory. After 6 years since these interventions began, the prevalence of an abnormal screening test result remained low (202/2,475 [8.2%]), even among white patients (10.9%). With this simple approach, the cost to test patients for factor V Leiden decreased by more than 90%, while the productivity of our laboratory increased by the introduction of a high-volume, fully automated assay.

Comparison of four laboratory methods to assess aspirin sensitivity.

The purpose of this study was to compare the ability of four commercial platelet function assays to detect aspirin response in normal individuals taking 81 or 325 mg aspirin in a single-dose response and then in a 7-day dosing regimen. We employed the Chronolog 570VS whole-blood aggregometer with agonists 1.0 microgram/ml collagen and 0.5 mmol/l arachidonic acid, the PFA-100 epinephrine/collagen cartridge closure time, the Accumetrics Verify/Now arachidonic acid cartridge, and the urine 11-dehydrothromboxane immunoassay normalized to urine creatinine. Fifty normal individuals who met the inclusion criteria were consented in the single-dose study. Blood and urine were collected at baseline, and then each participant was given a 81 mg enteric-coated aspirin tablet. Blood and urine were collected after 24 h. After a minimum of 14 days the process was repeated with a 325 mg aspirin dose. Forty-five individuals were enrolled in the 7-day study. Blood and urine were collected at baseline. Then each participant was given an 81 mg dose of aspirin daily for 7 days. After 7 days, blood and urine specimens were obtained and tested. After a minimum washout period of 14 days the process was repeated using a 7-day regimen of 325 mg enteric-coated aspirin tablet. Student's t-test indicated statistical significance between baseline and post responses in both dosing regimens (P < 0.05). Individuals were not consistently identified as aspirin responsive across all platforms. All assays discriminated between platelet response and nonresponse to aspirin at both dosages. It may be necessary to employ multiple assays to detect individual platelet response.

Whole Blood Platelet Aggregometry.

Light transmittance aggregometry is the historical reference method for platelet function testing and continues to be used extensively. Whole blood impedance lumiaggregometry represents an updated methodology that provides for simplified specimen management, an assay milieu that replicates in vivo platelet activation conditions, improved reproducibility, and near-patient testing. While the impedance-based whole blood aggregometer with luminescence channel is becoming the standard for platelet function testing using this methodology, at least three near-patient whole blood instruments are available, each employing its unique technology. We provide descriptions of whole blood lumiaggregometry and three near-patient systems. We include the principle of operation, materials, and stepwise example protocols and speculate on the importance of concordance among the platforms.

Measuring dabigatran with the dilute Russell viper venom confirm assay in an anticoagulation clinic population.

The dabigatran dose-response is predictable; however, it is necessary to measure plasma levels in a variety of clinical conditions. We evaluated a novel dabigatran measure - the 'dilute Russell viper venom confirm (DRVVC) assay' - against current developmental assays and a reference method. We measured plasma dabigatran and compared results from the Stago Sta-Clot DRVVC assay, Stago Ecarin Chromogenic Assay, Biophen Hemoclot Thrombin Inhibitor, and liquid chromatography tandem mass spectrometry. We obtained dabigatran calibrators and controls from Biophen, and performed the coagulation assays using a Stago STA-R Evolution coagulometer. Liquid chromatography tandem mass spectrometry method specimens were performed on an AB Sciex instrument at LabCorp. We enrolled 97 anticoagulation clinic patients (mean age 76 years) who were taking 150 mg dabigatran twice daily. All had creatinine clearances above 30 ml/min; patients were not excluded for concurrent medications or health issues. Citrated blood specimens were processed immediately, and stored at -70°C. We did not correlate collection time with medication time. We employed descriptive statistics, analysis of variance, and the Bland-Altman difference plot to assess the data. The range for all assays was 11.6-917 ng/ml. Analysis of variance generated a P value of 0.1 and Bland-Altman differences were all below 4.0% compared with DRVVC. The DRVVC measures dabigatran with validity comparable to other methods.

Clinical pathology consultation improves coagulation factor utilization in hospitalized adults.

Coagulation factor replacement can effectively treat or prevent most hemophilia complications, but it is expensive. Although published data describe how to achieve therapeutic goals through cost-effective selection and dosing of replacement products, criteria are not universally known or followed. A review of our institution's experience revealed overdosing of coagulation factors in the majority of patients treated during a 12-month period, at a cost that approached $700,000. Consequently, we established mandatory clinical pathology consultation before releasing such factors. In the subsequent 30 months, 32 adults received 64 courses of treatment. For patients with hemophilia A, the mean cost per admission was reduced by approximately 27% (total savings, $61,536). For patients with factor VIII inhibitor, there was an approximate 6% cost reduction (total savings, $47,292). The combined savings was $108,828. The mean plasma factor level achieved during the intervention period was 84% +/- 55% compared with 117% +/- 58% for the preintervention period (P = .008). Neither the number of treatment (factor transfusion) days nor the number of RBC transfusions changed significantly. Our data support that pathology consultation yields consistent and appropriate therapy and improves resource utilization.

The effect of lupus anticoagulant in the second-generation assay for activated protein C resistance.

The activated protein C resistance (APCR) assay is the test of choice to screen for factor V Leiden. We evaluated the effect of lupus anticoagulant on the baseline clotting time of the second-generation APCR assay with plasma samples from 54 patients to determine whether a falsely low APCR ratio could be predicted. We also assessed whether a modification of the assay could make it more reliable in the presence of strong lupus anticoagulants. Of 54 plasma samples, 5 yielded a false-positive APCR ratio, and all 5 had a prolonged baseline clotting time. Further dilution (1:40) of the plasma samples in factor V-deficient plasma led to correction of the APCR ratio and did not affect the sensitivity of the test for factor V Leiden. Our data support that the baseline clotting time is a good predictor of a false-positive APCR test result and should be checked before calculating the ratio. The modified APCR assay reliably identified the false-positive ratios and could be used to screen for factor V Leiden in samples with strong lupus anticoagulant.

Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization.

The activated partial thromboplastin time (aPTT) is widely used as a screening coagulation test and for monitoring unfractionated heparin therapy. Various commercial reagents are available, with different performance characteristics, particularly responsiveness to the lupus anticoagulant (LA). Because aPTT reagent selection significantly affects the interpretation of results, we reviewed College of American Pathologists proficiency testing data involving approximately 4,000 coagulation laboratories, and conducted a survey of coagulation laboratories (n = 93) using The Fritsma Factor hemostasis Web site to determine the basis for aPTT reagent selection. The data demonstrate that for routine aPTT testing, most laboratories use reagents with high/moderate responsiveness to LA. Significant misunderstanding was apparent regarding the use of appropriate aPTT reagent for routine testing and LA identification. We recommend aPTT reagents with low LA responsiveness to screen for coagulation factor deficiencies and heparin monitoring, and suggest continued education of laboratory professionals and reagent manufacturers about appropriate aPTT reagent use.

Ability of the INNOVANCE PFA P2Y system to detect clopidogrel-induced ADP receptor blockade in preangiocath individuals.

We compared the ability of four test systems to detect platelet P2Y12 (ADP receptor) blockade by clopidogrel. The systems were the INNOVANCE PFA P2Y cartridge (PFA P2Y), the Accumetrics VerifyNow P2Y12 cartridge (VN P2Y12), whole blood aggregometry (WBA) using 5 (WBA 5) and 10 (WBA 10)  µmol/l ADP, and light transmittance aggregometry (LTA) using 20 (LTA 20)  µmol/l ADP. Blood was collected in 3.2% citrate from 101 preangiography participants who had received 300-600  mg of clopidogrel within 6-24  h or 75  mg daily for at least 7 days. Blood was also collected in 3.8% citrate for the PFA P2Y. Cut-offs indicating blockade were PFA P2Y, more than 106  s; VN P2Y12, less than 20%,  less than  235 Plavix resistance unit (PRU); WBA 5, less than 5  ohms; WBA 10, less than 8  ohms; and LTA 20, less than 50% aggregation. Percentage positives were PFA P2Y (3.2% citrate), 59%; PFA P2Y (3.8% citrate), 95%; VN P2Y12, 60%; VN P2Y12 PRU, 50%; WBA 5, 88%; WBA 10, 89%; and LTA 20, 72%. Percentage agreements were PFA P2Y 3.2% to VN P2Y12, 71%; PFA P2Y 3.2% to WBA 5 and 10, 64 and 65%, respectively; PFA P2Y 3.2% to LTA 20, 69%; PFA P2Y 3.8% to VN P2Y12, 71%, and to VN P2Y12 PRU, 60%; PFA P2Y 3.8% to WBA 5 and 10, 90% for both; PFA P2Y 3.8% to LTA 20, 76%; VN P2Y12 to WBA 5 and 10, 68 and 67%, respectively; and VN P2Y12 to LTA 20, 72%. PFA P2Y (3.2% citrate) detection compared favorably to VN P2Y12. The same system at 3.8% citrate compared more closely to WBA 5 and WBA 10.