Measuring Activated Clotting Time during Cardiac Catheterization and PCI: The Effect of the Sampling Site

Background/Purpose. We aimed to investigate the influence of the sampling site on the variability of ACT measurement. Activated clotting time (ACT) has been used for decades in cardiac surgery and interventional cardiology to assess unfractionated heparin activity. However, standardized protocols for the use of ACT measurement in the catheterization laboratory are lacking. Methods/Materials. After elective cardiac catheterization, ACT measurements were collected in simultaneously obtained blood samples from three different sample sites: the arterial catheter, arterial sheath, and peripheral intravenous line. Measurements were performed using the i-Stat® device (Abbott, Princeton, NJ, USA). The study was conducted with approval of the local medical ethical committee. Results. In 100 patients (mean age 67.1, 65% male), no significant differences were observed in ACT values obtained from the guiding catheter and arterial sheath (mean difference (MD) −18.3 s; standard deviation (SD) 96 s; P = 0.067 ). Contrarily, ACT values obtained from the intravenous line were significantly lower as compared to values obtained from the guiding catheter (MD 25.7 s; SD 75.5; P = 0.003 ) and arterial sheath (MD 39 s; SD 102.8; P < 0.001 ). Furthermore, ACT measurements from the arterial sheath showed a statistically significant proportional bias when compared to the other sampling sites (sheath vs. catheter, r = 0.761, P = 0.001 ; sheath vs. IVL, r = 1.013, P < 0.001 ). Conclusions. The present study shows statistical significance and possibly clinically relevant variations between ACT measurements from different sample sites. Bias in ACT measurements may be minimized by using uniform protocols for ACT measurement during cardiac catheterization.

Chromogenic anti-Xa test: the ratio between heparin activity units and concentration of apixaban and rivaroxaban

Introduction. The direct oral anticoagulants (DOC) therapy does not require alaboratory control; however, it may be required to determine the anticoagulationlevel to choose a treatment strategy if alarge bleeding is developing or emergency surgery is needed.The objective of this experimental study was to investigate the relationship between the residual factor Xa (FXa) activity, anti-Xa activity units oflow molecular weight heparins (LMWH), and the apixaban and rivaroxaban plasma concentrations in a chromogenic anti-Xa assay.Material and methods. Concentrated DOC solutions were prepared by extracting apixaban and rivaroxaban from crushed tablets using methanol and dimethyl sulfoxide, respectively. The resulting solutions were added to the donor plasma pool until final inhibitor concentrations are achieved in the range from 10 to 100 ng/ml plasma. Anti-Xa activity was determined using an STA-compact analyser and the Liquid anti-Xa reagent kit, an analysis protocol, and calibrators designed to control the LMWH therapy. The effect on the thrombin formation dynamics was investigated using the thrombin generation test (TGT) and the PPR reagent as a trigger (final concentrations of tissue factor are 5 pM, and those of phospholipids are 4 μM). TGT curves were analysed using the Thrombinoscope program.Results. It was shown that in the anti-Xa activity test version designed to control the LMWH therapy, there is a high correlation (R2 > 0.98) between thelogarithm of the residual factor Xa activity and the content of apixaban and rivaroxaban in the range from 10 to 80 ng/ml. Rivaroxaban shows about 1.5 times more anti-Xa activity than apixaban at equal concentrations. It was also shown that apixaban and rivaroxaban at doses equal both in concentration and in anti-Xa activity differ in their effect on the thrombin formation dynamics and thrombin inactivation in the TGT.Conclusion. In the LMWH anti-Xa activity test version, the measured range of apixaban and rivaroxaban includes 30 ng/ml and 50 ng/ ml concentrations taken as “cut-off points” to determine the treatment tactics in emergency cases. However, thelack of certified DOC calibratorslimits the use of this test in clinical practice.

Endogenous Heparin-Like Substances May Cause Coagulopathy in a Patient with Severe Postpartum Hemorrhage

Introduction: Postpartum hemorrhage (PPH) is associated with maternal morbidity and mortality. Accurate diagnosis of the cause of puerperal hemorrhage is as important as treatment strategies for resuscitation. We report a case of coagulation disorder caused by endogenous heparin-like substances in a PPH patient. Case Presentation: A 30-year-old woman with no medical history of bleeding disorders suffered intractable hemorrhage following spontaneous delivery in a local hospital. The patient was transferred to the department of obstetrics of a superior hospital. On arrival, the patient was found to have severe hemorrhagic anemia, hemorrhagic shock, and disseminated intravascular coagulation. Active treatments were performed, but the patient continued bleeding. Laboratory testing, performed during early treatment, revealed that all coagulation factors were below normal. The differences between CK-TEG R-time (reaction time in citrated kaolin thromboelastography assay) and CKH-TEG R-time (reaction time in citrated kaolin with heparinase thromboelastography assay) suggested the presence of heparin activity. However, the patient’s family denied heparin use prior to presentation. Thus, we deduced that endogenous heparin-like substances were the main cause of the coagulopathy. After receiving treatment with protamine, the patient stopped bleeding. Meanwhile, all coagulation parameters and the TEG assay results improved. Conclusions: In this case report, TEG assay suggested the presence of heparin activity in a PPH patient, and treatment also highlighted the importance of analyzing different parameters in TEG.

Comparison of Low Molecular Weight Heparin Activity Assays with and without Addition of Exogenous Antithrombin in a Pediatric and Young Adult Leukemia Cohort

Introduction: Enoxaparin (low molecular weight heparin, LMWH) is the most commonly prescribed anticoagulant for pediatric patients with venous thromboembolism (VTE) (Goldenberg et al. 2015). The gold-standard LMWH activity (anti-Xa activity) assays differ in whether they add exogenous antithrombin (AT) or dextran sulfate (Ignjatovic et al. 2007). Adding AT would "standardize" results in patients with low antithrombin, such as infants and asparaginase-treated patients (Mitchell et al 2010); however, questions remain about which assay best reflects the patient's anticoagulation effect and the degree of discrepancy between assays. We assessed LMWH activity in residual plasma samples from a cohort of anticoagulated pediatric acute lymphoblastic leukemia and lymphoma (ALL) patients, with history of VTE and variable AT levels, on four platforms (two instruments and their kits +/- exogenous AT). Methods: We analyzed 60 de-identified residual plasma samples from 12 anticoagulated ALL patients (2-19 years. Mean 13.75 yr) who had AT levels obtained for clinical care. All consented to the IRB-approved Oncology Tissue Repository. LMWH activity was assessed on Siemens and Stago instruments using their recommended kits that did or did not contain exogenous AT (Table 1), according to manufacturer recommendations, by experienced laboratorians. Results: Results were interpretable on 236/240 with 4 rejected for lipemia. Mean AT activity was 80 (42-138 ng/ml, lab normal >81%). Correlation was acceptable for the published kit ranges of LMWH activity when comparing kits +AT (Berichrom® to Stachrom®, r=0.82, p<0.0001), -AT (Innovance® to STA®-Liquid Anti-Xa: r=0.93, p<0.0001), and within the same manufacturer (Berichrom® to Innovance®, r=0.92, p<0.0001, Stachrom® to STA®-Liquid Anti-Xa r=0.98, p<0.0001) (Table 2, Figure 1, Figure 2). Comparing +AT or not by manufacturer, there was a nonsignificant trend to higher LMWH activity result with +AT kits. When AT levels were <70 ng/ml (n=19, mean 56 ng/dl) there was a trend to underestimate LMWH activity when AT was not added; this reached significance when the Stago methods were compared (Table 2.) Conclusions: There is acceptable correlation of LMWH activity using kits with or without exogenous AT in anticoagulated pediatric plasma samples; however, in low AT samples, LMWH activity trends lower in platforms without exogenous AT. Use of residual samples precluded conclusions about the clinical impact of this difference; however, there were a few instances where subtherapeutic activity in the -AT assay was in the therapeutic range (0.5-1 activity units) in the +AT assay. Clinicians should know whether exogenous AT is used in their own laboratory's assay and understand that added AT could overestimate the LMWH effect in patients with low AT levels. Disclosures Schaefer: Stago: Research Funding; Siemens: Research Funding.

Sample Dilution Is Not an Effective Mitigation for Hemolysis Interference in Anti-Xa Heparin Activity Assays

The presence of hemolysis or icterus can interfere with chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity), resulting in falsely lower estimates of heparin activity. Dilution is a common method for mitigating interference in spectroscopic assays that measure a single analyte by endpoint analysis and has been proposed as a solution for hemolysis interference in heparin activity assays. Heparin activity assays measure heparin/antithrombin complex activity using a chromogenic rate of absorbance change method. Heparin activity is a complex equilibrium of heparin binding to multiple proteins, including antithrombin, heparin cofactor II, platelet factor 4, histidine-rich glycoprotein, vitronectin, fibronectin, and others; some enhance while others neutralize heparin activity. The objective of this study was to determine how dilution affects heparin activity with different starting levels of antithrombin. First samples were diluted without interference (no hemolysis). Samples with 1 U/mL heparin in pooled normal plasma (PNP) and 50% PNP (to simulate 50% antithrombin levels) were diluted 1:1 with PNP versus OK buffer. Dilution resulted in nonlinear changes. Samples with 100% antithrombin recovered 88% of expected activity with PNP versus 74% with OK buffer; samples with 50% antithrombin recovered 111% with PNP versus 77% with OK buffer. Samples were then spiked with varying concentrations of hemolysate and 1:1 dilutions with PNP demonstrated nonlinear recoveries ranging from 41% to 84%. We were unable to recover the original heparin activity with or without hemolysis due to the complex interaction of initial heparin, antithrombin, and other binding protein concentrations with diluent antithrombin and other diluent heparin binding protein levels. When PNP is used as the diluent, the recovery will be lower when the initial antithrombin level is normal due to the addition of other heparin binding proteins; when the initial antithrombin level is low, the recovery is higher due to the addition of antithrombin from the diluent plasma. The recovery is always lower when buffer is used due to dilution of sample antithrombin. Careful consideration should be taken in evaluating the utility of sample dilution to mitigate interference from hemolysis and icterus in heparin activity assays, particularly in patients with fluctuating levels of antithrombin, such as those on extracorporeal life support or with liver failure.

Detection and Extraction of Heparin from Camel Lungs

Background: Heparin is an essential drug used as an anticoagulant. Access to raw material suitable for heparin extraction is critical for creating a viable business opportunity. In Saudi Arabia, large amounts of raw material with potential for heparin extraction are wasted. Objective: To extract heparin and low-molecular-weight heparin (LMWH) from the camel lung, and measure its potency and activity. Methods: Heparin preparation included three steps: extraction, electrophoretic identification, and activity measurement. Fresh lung tissue (100 g) was minced and homogenized in a blender. Crude heparin extracts were prepared using Charles’s or Volpi’s method with slight modifications. Heparin was purified by electrophoresis using high-purity agarose gels in barium acetate buffer. The heparin activity of purified samples was assayed spectrophotometrically using commercial heparin kits. Results: Charles’s and Volpi’s extraction methods were simple and easy to establish. The yield was 90 mg crude heparin per 100 g of camel lung tissue following Volpi’s extraction protocol, whereas Charles’s method did not yield any heparin. The separation of heparin and LMWH by gel electrophoresis resulted in sharp and clear product bands using material prepared according to Volpi’s method. The heparin preparation had an anti-factor Xa activity of 37 IU/mg, indicating weak potency. Conclusion: Preparation of active heparin from camel lung tissue is a technology applicable in manufacturing. Further method development is needed to increase heparin purity and potency.

Cartridge-Based Thromboelastography Can Be Used to Monitor and Quantify the Activity of Unfractionated and Low-Molecular-Weight Heparins

Thromboelastography is increasingly utilized in the management of bleeding and thrombotic complications where heparin management remains a cornerstone. This study assessed the feasibility of the cartridge-based TEG® 6s system (Haemonetics Corp., Braintree, Massachusetts, United States) to monitor and quantify the effect of unfractionated and low-molecular-weight heparin (UFH and LMWH). Blood samples from healthy donors were spiked with UFH (n = 23; 0–1.0 IU/mL) or LMWH (enoxaparin; n = 22; 0–1.5 IU/mL). Functional fibrinogen maximum amplitude (CFF.MA), RapidTEG activated clotting time (CRT.ACT), and kaolin and kaolin with heparinase reaction time (CK.R and CKH.R) were evaluated for their correlation with heparin concentrations, as well as the combination parameters ΔCK.R − CKH.R, ratio CK.R/CKH.R, and ratio CKH.R/CK.R. Nonlinear mixed-effect modelling was used to study the relationship between concentrations and parameters, and Bayesian classification modelling for the prediction of therapeutic ranges. CK.R and CRT.ACT strongly correlated with the activity of LMWH and UFH (p < 0.001). Using combination parameters, heparin activity could be accurately quantified in the range of 0.05 to 0.8 IU/mL for UFH and 0.1 to 1.5 IU/mL for LMWH. CRT.ACT was able to quantify heparin activity at higher concentrations but was only different from the reference range (p < 0.05) at >0.5 IU/mL for UFH and >1.5 IU/mL for LMWH. Combination parameters classified blood samples into subtherapeutic, therapeutic, and supratherapeutic heparin ranges, with an accuracy of >90% for UFH, and >78% for LMWH. This study suggests that TEG 6s can effectively monitor and quantify heparin activity for LMWH and UFH. Additionally, combination parameters can be used to classify blood samples into therapeutic ranges based on heparin activity.