aPTT Reagents Research Articles

Comparison of the ecarin chromogenic assay and different aPTT assays for the measurement of argatroban concentrations in plasma from healthy individuals and from coagulation factor deficient patients

Introduction The aim of this study was to assess the usefulness of different aPTT assays and the ecarin chromogenic assay reaction time (ECA) for measurement of argatroban concentration in plasma from healthy persons as well as in different patient subgroups. Methods We spiked plasma samples from healthy individuals, patients under oral anticoagulation (OAT) or with liver dysfunction (LD) with increasing argatroban concentrations (0 – 2000 ng/ml) and performed 4 different aPTTs assays and the ECA. Results Depending on argatroban concentrations aPTTs increased in a curvilinear fashion; in plasma from healthy individuals means of calculated argatroban concentration at 2-fold aPTT differed extensively depending on the aPTT reagent used (725 ng/ml to 1136 ng/ml) and were even more pronounced in plasma from coagulation factor deficient patients (460 ng/ml in patients with LD vs. 1172 ng/ml in patients with OAT), whereas ECA showed linear argatroban influence and reliable results in all subgroups. Conclusions Because of wide differences in aPTT measurements depending on the aPTT reagent used, interindividual variations and different clinical conditions the aPTT is not the method of choice for monitoring argatroban and the ECA should be preferred.

Performance of Platelin LS and dilute Russell's viper venom for the screening of lupus anticoagulant in patients with venous thromboembolism

Lupus anticoagulant is associated with thrombosis and pregnancy morbidity, and its detection is of major clinical importance. The nature and concentration of phospholipids strongly influence the sensitivity of activated partial thromboplastin time (aPTT) reagents to lupus anticoagulant. We investigated the ability of Platelin LS, an aPTT reagent, to screen lupus anticoagulant among 94 patients with venous thromboembolism by comparing its performance with the dilute Russell viper venom time (dRVVT). Twenty-four patients had an abnormal aPTT and dRVVT, whereas 37 only had a prolonged dRVVT. In users of oral anticoagulants (n = 56), the dRVVT prolonged more frequently than the aPTT (98.2 vs 39.3%, P < 0.0001). After the mixing study, seven patients maintained abnormal aPTT and dRVVT ratios, five of whom had prolonged mixture with both tests. The agreement in the mixing study between aPTT and dRVVT was substantial (kappa = 0.78, 95% confidence interval = 0.48-1.00). Except for one patient, the aPTT screened all cases that demonstrated phospholipid dependency of their inhibitor during the confirmatory procedure with the dRVVT. In conclusion, the aPTT using Platelin LS was highly associated with the presence of lupus anticoagulant detected by the dRVVT among patients with venous thromboembolism, and could be reliably employed as a screening assay for lupus anticoagulant.

Detection of shortened activated partial thromboplastin times: An evaluation of different commercial reagents

Introduction Abnormally shortened activated partial thromboplastin times (aPTT) are associated with significantly increased risk of thrombotic disorders and in-hospital mortality. Shortened aPTTs have been related to increased levels of factor (F) VIII and thrombin-antithrombin complex (TAT). In the current study, four different commercial aPTT reagents were evaluated for their performance to detect shortened aPTTs. Materials and methods aPTT of 400 patients was determined using Actin-FS (Dade Behring), APTT-SP (Instrumentation Laboratory), Automated-APTT (bioMerieux) and Platelin-LS (bioMerieux) reagents. FVIII, FIX, FXI and TAT levels were measured in shortened and normal aPTT samples. Results An association between shortened aPTTs and elevated levels of coagulation factors (FVIII, FIX and FXI) and thrombin generation (TAT) was found with all tested aPTT reagents. Method-comparison studies demonstrated good agreement between Instrumentation Laboratory and bioMerieux reagents. However, 53 to 59% of the patients with a shortened aPTT measured with Actin-FS reagent was determined as a normal aPTT with APTT-SP, Automated-APTT and Platelin-LS reagents. These patients had increased levels of FVIII, FIX and FXI and moderately increased levels of TAT. Conclusion Overall, an acceptable agreement between the different commercial reagents was found with respect to detection of short aPTTs. However, a disparity between some of reagents existed. Actin-FS reagent appeared to be more sensitive in inducing shortened aPTT reactions than APTT-SP, Automated-APTT and Platelin-LS reagents.

Accuracy of FVIII: C assay by one-stage method can be improved using hemophilic plasma as diluent.

the basic prerequisite of Factor VIII clotting assay (FVIII:C) by One-Stage Method is that all other than FVIII clotting factors are present in constant concentration in each dilution of both standard reference and patient's plasma curves. On the contrary, the plasma content of each dilution is decreasing as the dilution factor increases. to keep exactly constant the plasma content in each mixture, we performed all dilutions of both standard reference and patient's plasma with FVIII deficient plasma and further with a fixed amount of buffer (method B). To show the discrepancies between this method and regular method A, using buffer to make dilutions, a comparative study was conducted on FVIII: C assay on samples at known FVIII concentration and in patients' plasma. Imidazole or Owren's buffers and five different aPTT reagents were employed, both in method A and B. a discrepancy between FVIII: C assays obtained by method A and B was observed, mainly when Pathrontin SL and Imidazole buffer were used. The assays derived from method B always better fit with the expected, calculated, values of FVIII:C concentrations. Furthermore, FVIII: C was assayed in 60 patients: the outcome of method A was always higher than values of method B. The discrepancy between the two methods was higher at FVIII concentrations below 50 U/dL but null at 100 U/dL. The A slope was steeper than B slope and the difference was statistically significant starting from the 1/10 dilution. Accordingly, FVIII: C of patients' plasma obtained by method A was always higher that those obtained by method B, even 2 or 3 times for FVIII level < or = 10 U/dL or 1.4-1.6 times for FVIII levels between 10 and 25 U/dL. only method B is able to give FVIII: C assays in agreement with the expected values. The dilution of reference standards and samples with FVIII deficient plasma is crucial to accurately evaluate the post-infusion FVIII concentrations in pharmacokinetic studies or the trough level during prophylactic therapy and to investigate the discrepancy among different FVIII: C assays. In addition, the assessment of severity and classification of hemophilia should be reviewed.

Clinical Impact of Increasing the Sensitivity of Activated Partial Thromboplastin Time (aPTT) Reagent for Lupus Anticoagulant.

Background: Lupus Anticoagulants (LA) are antibodies that interfere with phospholipids dependent coagulation pathways. First indication of the presence of LA is usually provided by prolonged aPTT. The sensitivity of various commercial aPTT reagents for LA varies. For some years, trend has been to shift towards aPTT reagents that are more sensitive to LA. Starting from May 2004, our coagulation laboratory changed aPTT reagent from Thrombosil by Hemoliance to aPTT-SP by Hemosil. During transition, for quality control purpose, we continued to perform aPTT with Thrombosil in patients found to have prolonged aPTT with aPTT-SP. The aPTT-SP is considered to be more sensitive for LA than Thrombosil. Since then we found increased frequency of prolonged aPTT being discovered during screening coagulation tests. As per our laboratory protocol, all the samples with prolong aPTT were further evaluated with mixing studies and Dilute Russel Viper Venom Test (DRVTT) for LA.We did a retrospective study to evaluate the clinical significance of prolonged aPTT by aPTT-SP.Method: Retrospective chart review of all the patients with prolonged aPTT with aPTT-SP from May 2004 to April 2005 was done. Information regarding patient's age, sex, laboratory data, history of any thrombo-embolic phenomenon and other significant medical conditions was collected.Results: In 1 year 50 patients (28 females and 22 males) with age ranging from 19 to 80 years, with prolonged aPTT with aPTT-SP reagent were identified. 29 had prolonged aPTT by both reagents and 21 had prologed aPTT by aPTT-SP only. 13 of 21 patients with prolonged aPTT with aPTT-SP (more sensitive reagent) and normal aPTT with Thrombosil (less sensitive reagent), had positive LA. Only three had vascular thrombosis. Most institution including ours, routinely tests for LA during thrombophilia work up regardless of aPTT result. Thus LA would have been diagnosed in these three patients despite normal aPTT by Thrombosil. At the same time diagnosing LA in the absence of thrombosis does not lead to change in clinical management.Results are shown in table.Conclusion: Use of aPTT reagent more sensitive to LA, does not provide any clinical advantage. In fact it led to extra work-up and delay in invasive procedures. It also had the potential to lead to extra intervention, like administration of FFP in emergency situations, before LA as a cause of prolonged aPTT could be determined.resultsS. NoVARIABLESPATIENTS=n1Increased aPTT by aPTT SP/Normal aPTT by Thrombosil/LA Negative82Increased PTT by aPTT SP/Normal aPTT by Thrombosil/LA Positive/No Thrombosis103Increased aPTT by aPTT SP/Normal aPTT by Thrombosil/LA Positive /H/o Thrombosis3*4Increased aPTT by both Reagents/LA Positive/H/o Thrombosis7**5Increased aPTT by Both reagents/LA Positive/No Thrombosis126Increased aPTT by both Reagents/LA negative10**** 2 had venous thrombosis, 1 had coronary artery disease.**3 had venous thrombosis, 2 had CVA, 2 had coronary artery disease.*** The reasons for prolonged aPTT by both reagents were varied like coumadin intake, LMWH, 1 patient had factor 11 deficiency etc

Discrepant factor VIII activity in a family with mild haemophilia A andArg531Hismutation using various FVIII assays and APTT reagents

HaemophiliaVolume 11, Issue 5 p. 561-564 Discrepant factor VIII activity in a family with mild haemophilia A and Arg531His mutation using various FVIII assays and APTT reagents J. F. Lucía, J. F. Lucía Department of Haematology, Hospital Universitario Miguel Servet, ZaragozaSearch for more papers by this authorC. Aguilar, C. Aguilar Department of Haematology, Hospital General Santa Bárbara, SoriaSearch for more papers by this authorM. Dobon, M. Dobon Department of Haematology, Hospital Universitario Miguel Servet, ZaragozaSearch for more papers by this authorJ. A. Aznar, J. A. Aznar Haemophilia Unit, Hospital Universitario La Fe, ValenciaSearch for more papers by this authorE. Tizano, E. Tizano Department of Genetics, Hospital de la Santa Creu y Sant Pau, BarcelonaSearch for more papers by this authorC. Borés, C. Borés Dade Behring, BarcelonaSearch for more papers by this authorR. Cornudella, R. Cornudella Department of Haematology, Hospital Clinico Universitario, ZaragozaSearch for more papers by this authorM. T. Calvo, M. T. Calvo Department of Genetics, Hospital Universitario Miguel Servet, Zaragoza, SpainSearch for more papers by this author J. F. Lucía, J. F. Lucía Department of Haematology, Hospital Universitario Miguel Servet, ZaragozaSearch for more papers by this authorC. Aguilar, C. Aguilar Department of Haematology, Hospital General Santa Bárbara, SoriaSearch for more papers by this authorM. Dobon, M. Dobon Department of Haematology, Hospital Universitario Miguel Servet, ZaragozaSearch for more papers by this authorJ. A. Aznar, J. A. Aznar Haemophilia Unit, Hospital Universitario La Fe, ValenciaSearch for more papers by this authorE. Tizano, E. Tizano Department of Genetics, Hospital de la Santa Creu y Sant Pau, BarcelonaSearch for more papers by this authorC. Borés, C. Borés Dade Behring, BarcelonaSearch for more papers by this authorR. Cornudella, R. Cornudella Department of Haematology, Hospital Clinico Universitario, ZaragozaSearch for more papers by this authorM. T. Calvo, M. T. Calvo Department of Genetics, Hospital Universitario Miguel Servet, Zaragoza, SpainSearch for more papers by this author First published: 26 August 2005 https://doi.org/10.1111/j.1365-2516.2005.01122.xCitations: 8 Dr Carlos Aguilar, Department of Haematology, Hospital General Santa Bárbara, Paseo de Santa Bárbara s/n, 42.002-Soria, Spain. Tel.: +34 975 214156; fax: +34 975 234305; e-mail: [email protected] Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume11, Issue5September 2005Pages 561-564 RelatedInformation

Effect of argatroban on the activated partial thromboplastin time: a comparison of 21 commercial reagents

Argatroban is a direct thrombin inhibitor used for the treatment of heparin-induced thrombocytopenia. The drug is administered by continuous infusion, at a recommended initial dose of 2 microg/kg per min, to achieve activated partial thromboplastin times (aPTTs) 1.5-3.0 times baseline. We evaluated the effect of argatroban, at clinically relevant concentrations, on aPTTs using 21 commercially available reagents. The aPTTs of plasma containing argatroban at 0.125-8.0 microg/ml (final concentration) were assessed using each reagent and an ACL 3000+ coagulation analyzer. Argatroban increased aPTTs (and aPTT ratios relative to control) in a broadly comparable fashion among reagents. Concentration-aPTT ratio profiles linearized well using logarithmic-logarithmic transformation (r > 0.98), with the regression slope taken as the reagent's sensitivity to argatroban. Sensitivity ranged from 0.304 +/- 0.006 to 0.364 +/- 0.007. Only the least and two most sensitive reagents (all now unavailable in the United States) differed significantly in sensitivity from the other reagents (P < 0.05). aPTT ratios of 2.25 occurred for all reagents at 0.41-0.92 mug/ml argatroban, and for 14 (67%) reagents at 0.53-0.67 microg/ml. This corresponds to a approximately 0.5 microg/kg per min dose difference in healthy subjects. We conclude that most aPTT reagents are similarly sensitive to argatroban, and reagent choice is unlikely to significantly affect argatroban monitoring in patients with heparin-induced thrombocytopenia.

Using a Single Calibration Curve With the Anti-Xa Chromogenic Assay for Monitoring Heparin Anticoagulation

bration hybrid curve could be used to calibrate the chromogenic anti-Xa assay for monitoring patients on either unfractionated heparin (UFH) or low-molecular weight heparin (LMWH). The APTT is the assay most commonly used to monitor the effects of UFH therapy. Unfractionated heparin potentiates the activity of antithrombin and covalently neutralizes thrombin and factor Xa.1 Low-molecular weight heparin such as enoxaparin selectively catalyzes the neutralization of factor Xa over thrombin and the drug effect cannot be effectively measured using the APTT assay.1 The method of choice for monitoring LMWH and other heparin analogues is the anti-Xa chromogenic assay. This procedure can also be used to measure the amount of UFH present.1,2 Previous publications have cited the interference of different anticoagulant concentrations in evacuated collection tubes, factor deficiencies, interfering substances, specimen collection problems dealing with time of processing and specimen handling, APTT reagent sensitivity, and instrumentation on the APTT results.3-12 Other studies have shown situations where a therapeutic anti-Xa level of UFH was achieved but dosage changes may have been indicated due to a non-therapeutic APTT level result.11,13 The antithrombin in these studies were sometimes from different sources. Some were from the assay and other studies used an assay where the patient’s antithrombin was the source for the test. This protocol used different concentrations of sodium citrate anticoagulant in the evacuated collection tubes and amount of the blood to anticoagulant ratio to see if these variables affected the chromogenic anti-Xa assay results in samples from patients receiving either a UFH or LMWH dosing regimens.

Effects of vitamin K antagonist phenprocoumon on activated partial thromboplastin time measurement of direct thrombin inhibitors.

The activated partial thromboplastin time (aPTT) is currently the most common test used to measure the anticoagulation intensity of heparins and direct thrombin inhibitors (DTIs). Vitamin K antagonists variably affect aPTT reagents. Interactions between heparin and DTIs occur during concurrent therapy. Three DTIs (lepirudin, argatroban, melagatran) and one unfractionated heparin (liquemin) were added to normal plasma (NP) samples (n = 23) and to vitamin K antagonist plasma (VKAP) samples (n = 23) of patients treated with phenprocoumon. Lepirudin and argatroban were added at concentrations from 300 to 3000 ng/ml, melagatran from 30 to 1000 ng/ml, and unfractionated heparin from 0.016 to 0.48 IU/ml. Wave parameters of clotting time and aPTT ratio curves were evaluated by multivariate analysis for inhibitors, aPTT reagents and NP and VKAP samples. Normal ranges resulting from NP samples were 34.5 +/- 1.0 s with Pathromtin SL and 33.9 +/- 0.8 s with Platelin LS. Normal ranges using VKAP were 52.8 +/- 2.6 s (Pathromtin SL) and 44.2 +/- 1.1 s (Platelin LS) (P < 0.0001). Variance analysis showed that inhibitors, plasmas (NP versus VKAP) and reagents influenced the wave characteristics of aPTT (s) (P < 0.0001) and aPTT ratios (P < 0.0001). Distinct statistical differences between aPTT reagents on one hand and normal versus vitamin K antagonist plasma on the other hand make a comparison of reported aPTT results difficult, especially during overlapping therapy with vitamin K antagonists.

Heparin and Low-Molecular-Weight Heparin: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy

This article about unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) is part of the Seventh American College of Chest Physicians Conference on Antithrombotic and Thrombolytic Therapy: Evidence-Based Guidelines. UFH is a heterogeneous mixture of glycosaminoglycans that bind to antithrombin via a pentasaccharide, catalyzing the inactivation of thrombin and other clotting factors. UFH also binds endothelial cells, platelet factor 4, and platelets, leading to rather unpredictable pharmacokinetic and pharmacodynamic properties. Variability in activated partial thromboplastin time (aPTT) reagents necessitates site-specific validation of the aPTT therapeutic range in order to properly monitor UFH therapy. Lack of validation has been an oversight in many clinical trials comparing UFH to LMWH. In patients with apparent heparin resistance, anti-factor Xa monitoring may be superior to measurement of aPTT. LMWHs lack the nonspecific binding affinities of UFH, and, as a result, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties. LMWHs have replaced UFH for most clinical indications for the following reasons: (1) these properties allow LMWHs to be administered subcutaneously, once daily without laboratory monitoring; and (2) the evidence from clinical trials that LMWH is as least as effective as and is safer than UFH. Several clinical issues regarding the use of LMWHs remain unanswered. These relate to the need for monitoring with an anti-factor Xa assay in patients with severe obesity or renal insufficiency. The therapeutic range for anti-factor Xa activity depends on the dosing interval. Anti-factor Xa monitoring is prudent when administering weight-based doses of LMWH to patients who weigh > 150 kg. It has been determined that UFH infusion is preferable to LMWH injection in patients with creatinine clearance of < 25 mL/min, until further data on therapeutic dosing of LMWHs in renal failure have been published. However, when administered in low doses prophylactically, LMWH is safe for therapy in patients with renal failure. Protamine may help to reverse bleeding related to LWMH, although anti-factor Xa activity is not fully normalized by protamine. The synthetic pentasaccharide fondaparinux is a promising new antithrombotic agent for the prevention and treatment of venous thromboembolism.

The combination of lupus anticoagulant sensitive and insensitive aPTT reagent together with dRVVT screening reagent for the exclusion of the presence of lupus anticoagulant

The combination of lupus anticoagulant sensitive and insensitive aPTT reagent together with dRVVT screening reagent for the exclusion of the presence of lupus anticoagulant

Suboptimal monitoring and dosing of unfractionated heparin in comparative studies with low-molecular-weight heparin.

Site-specific validation of the activated partial thromboplastin time (aPTT) therapeutic range is required to ensure administration of the optimal dose of unfractionated heparin. Therapeutic ranges of 1.5 to 2.5 times the control value are subtherapeutic for most modern aPTT reagents. To audit the appropriateness of aPTT monitoring in clinical trials comparing unfractionated heparin and low-molecular-weight heparin in patients with venous thromboembolism. Search of PubMed database from 1984 to 2001. Randomized, controlled trials that compared unfractionated and low-molecular-weight heparin for the treatment of venous thromboembolism. Use of unvalidated and potentially suboptimal therapeutic ranges for aPTT in patients assigned to receive unfractionated heparin. Fifteen studies met inclusion criteria. Only 3 studies used a validated aPTT therapeutic range, and 11 studies used a range that included aPTT values 1.5 times the control value. Ten studies reported unfractionated heparin doses, and 7 of these documented a reduction to less than 30 000 U/d in response to aPTT results. Most studies monitored unfractionated heparin inappropriately. This shortcoming could be responsible for the reduced efficacy of unfractionated heparin in clinical trials.

Effects of a synthetic factor Xa inhibitor (JTV-803) on various laboratory tests.

Synthetic direct inhibitors of factor Xa are capable of prolonging the global anticoagulant assay times in a concentration-dependent fashion. The relative degree of thrombin generation inhibition at an equivalent prolongation is not similar to the results observed with heparins and oral anticoagulant drugs. In addition, the direct factor Xa inhibitors prolong the Russell's viper venom test (RWT) and Heptest clotting times. Ecarin clotting time (ECT) and thrombin time (TT) remain unaffected. The kinetics of factor Xa inhibition are markedly different than those observed with pentasaccharide and heparins. Therefore, the methods developed for heparins and pentasaccharides may not be applicable for the monitoring of factor Xa inhibitors. To test the feasibility of using the prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), Heptest, thrombin time, RVVT, ECT, and a modified anti-Xa amidolytic assay, to monitor a synthetic factor Xa inhibitor, normal human pool plasma samples were spiked with a synthetic factor Xa inhibitor in the concentration range of 0 to 1 microg/mL and 0 to 25 microg/mL. Different laboratory tests were performed and INR and other ratios were calculated. The anticoagulant effects on whole blood were measured using the activated clotting time (ACT). Further studies on the effect of factor Xa inhibitor on platelet aggregation; factor II, VII, and X functional levels; and fibrinopeptide A (FPA) generation were carried out at equivalent INR levels in comparison to oral anticoagulant and antithrombin agents. FPA generation at equivalent anticoagulant level in comparison to heparin (twice the baseline) was also carried out. Factor Xa inhibitor produced a concentration-dependent prolongation of the ACT. ACT was doubled at a concentration of 4 to 5 microg/mL. There was a marked difference in the prolongation of the PT by a synthetic factor Xa inhibitor dependent on the ISI of the PT reagent used. When the results were calculated to determine INR, marked variations were noted between the recombinant thromboplastin and rabbit brain thromboplastin. The rabbit brain thromboplastin reagent gave markedly high INR values. Similar results were observed when different aPTT reagents were studied. In the anti-Xa assay, modification of the incubation time was employed to extend the proper sensitivity range. These studies warrant further investigation to understand the mechanism of action of factor Xa inhibitors.

Prekallikrein deficiency: The characteristic normalization of the severely prolonged aPTT following increased preincubation time is due to autoactivation of factor XII

Hereditary plasma prekallikrein (PK) deficiency was diagnosed in a 71-year-old man with an 8-year history of osteomyelofibrosis. PK deficiency was suspected in view of a severely prolonged activated partial thromboplastin time (aPTT) that nearly normalized following prolonged preincubation (10 min) of patient plasma with kaolin–inosithin reagent. Hereditary PK deficiency was demonstrated by very low PK values in the propositus (PK clotting activity 5%, PK amidolytic activity 5%, PK antigen 2% of normal plasma, respectively) and half normal PK values in his children. Normalization of a severely increased aPTT (>120 s) after prolonged preincubation with aPTT reagent occurred in plasma deficient in PK but not in plasma deficient in factor XII (FXII), high-molecular-weight kininogen (HK), factor XI (FXI), factor IX, factor VIII, Passovoy trait plasma or plasma containing lupus anticoagulant. Autoactivation of FXII in PK-deficient plasma in the presence of kaolin paralleled the normalization of aPTT. Addition of OT-2, a monoclonal antibody inhibiting activated FXII, prevented the normalization of aPTT. We conclude that the normalization of a severely prolonged aPTT upon increased preincubation time (PIT), characteristic of PK deficiency, is due to FXII autoactivation.

Use of a fixed activated partial thromboplastin time ratio to establish a therapeutic range for unfractionated heparin.

The commonly recommended therapeutic range for patients receiving unfractionated heparin of 1.5 to 2.5 times the control activated partial thromboplastin time (aPTT) is not universally applicable. It has been suggested that the therapeutic range for each aPTT reagent should be based on plasma heparin levels. We sought to identify an aPTT ratio that corresponds to therapeutic anti--factor Xa heparin levels for combinations of several reagents and coagulometers that are commonly used. Citrated plasma was collected from 126 unselected patients receiving unfractionated heparin. Four automated coagulometers and 6 commercial aPTT reagents were used to measure the aPTT. Plasma anti--factor Xa levels were measured by means of a commercially available assay. The relationship between the aPTT results and anti-factor Xa heparin levels for each reagent-coagulometer combination was determined by linear regression analysis, and the aPTT results corresponding to therapeutic anti--factor Xa heparin levels were calculated. For all reagent-coagulometer combinations studied, an aPTT ratio of 1.5 resulted in anti--factor Xa heparin levels considerably below the lower limit of the therapeutic range. When the aPTT was performed on any of the coagulometers assessed with the use of Actin (Dade Diagnostics, Aguada, Puerto Rico) and IL Test (Instrumentation Laboratories, Fisher Scientific, Unionville, Ontario) reagents, aPTT ratios necessary to achieve therapeutic anti--factor Xa heparin levels approximated 2.0 to 3.5. For laboratories that cannot perform heparin levels, the use of less responsive reagents and any of the coagulometers studied, along with target aPTT ratio between 2.0 and 3.5, appears to be a reasonable alternative.

76-Year-Old Man With Retroperitoneal Bleeding After Prostatectomy

76-Year-Old Man With Retroperitoneal Bleeding After Prostatectomy

The Lupus Ratio Test – An Interlaboratory Study on the Detection of Lupus Anticoagulants by an APTT-based, Integrated, and Semi-quantitative Test

The Lupus Ratio (LR) test for lupus anticoagulants integrates screening, mixing with normal plasma and confirmation procedures into one assay. The sensitivity and reproducibility of the APTT based version of this assay was tested in an interlaboratory study that was part of the Fifth International Survey of Lupus Anticoagulants (ISLA-5). One LA negative plasma containing heparin, six LA positive plasmas and a normal pooled plasma (NP) were distributed to 31 laboratories world-wide together with two APTT reagents, one with a high and one with a low phospholipid concentration. The laboratories performed two APTTs, one with each reagent, on 1:1 mixtures of test plasma and NP. The ratio between the two clotting times was divided by the corresponding ratio for the NP. This final ratio is the LR of that plasma. The overall sensitivity was found to be 95.1%, and the normal, heparin-containing sample was reported to be negative by all the laboratories. When the results were grouped in low, medium and high positive plasmas, a "consensus" regarding the strength of each plasma was easily found. 85.0% of the results were in agreement with this consensus. This study shows that with the LR test, it is possible to obtain high interlaboratory agreement regarding the presence or absence of LA as well as the semi-quantification of this inhibitor.

Short activated partial thromboplastin times are related to increased thrombin generation and an increased risk for thromboembolism.

This prospective study was designed to investigate whether patients with short activated partial thromboplastin times (aPTTs) have increased thrombin generation and are at increased risk for thromboembolism. During a 4-month period, routine coagulation specimens were screened for the presence of a short or normal aPTT, and, accordingly, 250 specimens were collected. Prothrombin fragment F1 + 2 (F1 + 2) was measured to evaluate thrombin activation, and a second aPTT was performed with a different reagent. Diagnoses were obtained from medical records after conclusion of sample collection. Five to 9 months later, patients were questioned on thromboembolic events during the previous 18 months by questionnaire and telephone interview. F1 + 2 and the incidence of venous thromboses were elevated significantly in the short aPTT group. Unexpectedly, patients with acute bleeding had short aPTTs, but 36% of these also had thromboembolic events during the 18 months proximal to blood collection. These findings were confirmed with the second aPTT reagent. Patients with short aPTTs have increased thrombin generation and are at increased risk for thromboembolism, mainly venous thromboses, despite the fact that a short aPTT can occur in the acute setting of bleeding.

The clinical impact of increased sensitivity PT and APTT coagulation assays.

To determine how more-sensitive prothrombin time (PT) and activated partial thromboplastin time (aPTT) reagents affected the number and distribution of abnormal test results and whether the increased sensitivity for deficiencies resulted in improved diagnosis of clinically significant coagulopathies, we retrospectively compared preoperative coagulation screening data for 140 children undergoing open heart surgery after the reagent change with a similar group of 135 before the change. The more sensitive reagents resulted in a higher rate of abnormal values, but no increase in the identification of clinically significant hemostatic abnormalities. Of 67 patients with abnormal aPTTs in the group screened with more sensitive reagents, 63 had no further workup. No patients in either group were diagnosed subsequently with a coagulopathy because of unexpected bleeding. An abnormal test result did not predict the need for perioperative blood products. We hypothesize that the high frequency of abnormal aPTTs led to physician "desensitization" about the merit of coagulation screening. Therefore, we question the usefulness of preoperative coagulation screening of the pediatric cardiac surgery patient, particularly since lasting changes in physician perception regarding the clinical significance of abnormal values may lead to missed diagnoses in other settings.

The Use of Frozen-Thawed Platelet-Derived Phospholipids as a Confirmatory Test for the Diagnosis of Lupus Anticoagulants. Comparison with Two Commercial Confirmatory System Tests

Lupus anticoagulants (LAs) belong to acquired circulating anticoagulants interfering with phospholipid-dependent coagulation tests. Owing to the remarkable variability among patients, SSC guidelines recommend more than one test to detect and confirm the presence of LAs. However, this is an expensive procedure and greatly raises the work load of the laboratory. A standardised platelet-derived phospholipid preparation was obtained and platelet neutralisation (PNP) procedures with APTT and DRVVT reagents were performed on plasmas from 16 patients with LAs and from 41 control subjects. In comparisons, STAclot-PNP® and DVVconfirm® clotting assays were conducted. PNP by using APTT or DRVVT reagents showed intra-assay coefficient of variation of 1.6 and 1.8%, whereas inter-assays coefficient of variations were 6.8 and 5.1%, respectively. A complete concordance was achieved between STAclot-PNP® and PNP by using APTT reagents and between DVV-confirm® and PNP with DRVVT reagents, demonstrating a high reliability of both the PNP-based assays. This consistency indicates that the standardised platelet-derived phospholipid preparation obtained allows for reliable, reproducible, and cheap PNP-based confirmatory assays for LAs testing.