Suorien oraalisten antikoagulanttien laboratorioseuranta

Direct oral anticoagulants (DOACs), the thrombin inhibitor dabigatran and the anti-Xa inhibitors rivaroxaban and apixaban are are simple to use as standard dosing is recommended and no routine monitoring of coagulation is advocated. However, in certain clinical situations, e.g. acute thrombosis or bleeding, emergency surgery and renal or hepatic failure, assessing anticoagulant bioactivity is essential. The aims of this study were to firstly assess the effects of these DOACs in commonly used coagulation screening tests (PT, INR and APTT) using both spiked samples and samples collected from patients on treatment. Secondly, the availability of specific methods for drug effect assessment, namely drug-calibrated thrombin time (dabigatran) and anti-Xa (rivaroxaban, apixaban) assays were assessed in laboratory surveys. Thirdly, the effects of DOACs on these specific assays were explored using patient samples. Finally, the effect of DOACs on the global coagulation assay thrombin generation (TG) was assessed in patients using dabigatran, rivaroxaban or apixaban. The spiked sample laboratory surveys included a total of 86 European laboratories. The effects of all DOACs on INR were modest, but large reagent variability was noted in the responses (p<0.001). In APTT, rivaroxaban and apixaban prolonged the APTT only modestly, but with dabigatran there was a clear prolongation. Only about a fourth of the laboratories were able to provide more specific methods, TT or anti-Xa assays for drug quantification. Dabigatran effects in patient samples were studied 241 unselected patient samples. The PT was rarely prolonged and the APTT was in curvilinear relationship (R2 = 0.71) with dabigatran. However, the sensitivity effects among patients varied, with some patients having a normal APTT at dabigatran levels up to 160 ng/mL. Several specific assays, (diluted thrombin time, ecarin clotting assay and anti-IIa assay) accurately quantified dabigatran concentrations. In TG, a paradoxical increase was observed in the endogenous thrombin potential (ETP) and Peak TG, while lag time also prolonged with increasing concentrations of dabigatran. Rivaroxaban and apixaban effects on coagulation in patient samples were further assessed in a well-characterised group of patients using these drugs for thromboprophylaxis after orthopaedic surgery. Rivaroxaban peak drug levels were higher and trough levels lower than with apixaban, reflecting the once daily vs twice daily dosing of the drugs. In TG, the ETP response at peak drug levels was strong with rivaroxaban, whereas with the trough drug levels, the TG parameters were close to the baseline levels. With apixaban the responses were more stable. The RVVT, used as a qualitative test, detected prolongation only at rivaroxaban peak levels. In conclusion, DOACs present significant challenges in the clinical laboratory, as a wide variety of reagents are used in coagulation assays, and the drug responses also vary widely between individual patients. Many factors other than drug concentration influence the responses in coagulation assays and TG. In clinical practice, care and diligence is required when assessing the effects of DOACs on patients.Suorat oraaliset antikoagulantit trombiini-inbiittori dabigatraani ja hyytymistekijä Xa (FXa) -inhibiittorit rivaroksabaani ja apiksabaani ovat uusia lääkeaineita, joita käytettäessä ei tarvita rutiininomaista hyytymisjärjestelmän laboratorioseurantaa. Lääkeaineen vastearvio on kuitenkin tarpeen mm. akuutin tukoksen, vuodon ja munuaisten tai maksan vajaatoiminnan yhteydessä. Tässä tutkimuksessa selvitettiin näiden lääkeaineiden vaikutuksia sekä hyytymisjärjestelmän seulontakokeisiin (TT, INR ja APTT) että lääkeainespesifiin trombiini- (dabigatraani) ja FXa-pohjaisiin (rivaroksabaani ja apiksabaani) hyytymiskokeisiin. Vaikutuksia selvitettiin sekä näytteissä, joihin lääkeainetta oli lisätty jälkikäteen että potilailla, jotka käyttivät kyseistä lääkeainetta. Potilasnäytteissä pystyttiin tutkimaan näiden lääkkeiden vaikutuksia myös laajemmin hyytymisjärjestelmässä trombiinigeneraatiotestillä. Hyytymisjärjestelmän seulontakokeet TT, INR ja APTT osoittautuivat epäherkiksi rivaroksabaanille ja apiksabaanille sekä potilasnäytteissä että näytteissä, joihin lääkeainetta oli lisätty. Dabigatraaninäytteissä todettiin selvä vaste APTT-ajassa. Käytetty reagenssi vaikutti vasteen voimakkuuteen. Spesifeistä menetelmistä trombiiniaikapohjaisella menetelmällä saatiin luotettavia dabigatraanipitoisuuksia ja FXa-pohjaisilla menetelmillä rivaroksabaani- ja apiksabaanipitoisuuksia niin potilasnäytteissä kuin näytteissä, joihin lääkeainetta oli lisätty. Potilasnäytteissä nähtiin selkeitä eroja trombiinigeneraatiovasteissa eri antikoagulanteilla. Trombiininestäjä dabigatraanilla todettiin paradoksaalinen trombiinigeneraatiovasteen kiihtyminen lääkeainepitoisuuden kasvaessa. Rivaroksabaanilla oli apiksabaaniin verrattuna voimakkaampi trombiinigeneraatiota hillitsevä vaste huippupitoisuudella, mutta jäännöspitoisuudella rivaroksabaanin vaikutus trombiinigeneraatioon oli vähäisempi kuin apiksabaanilla. Suorien oraalisten antikoagulanttien vaikutukset hyytymisjärjestelmään eivät tämän tutkimuksen perusteella ole helposti ennustettavia. Vaikka lääkeainepitoisuus pystytään arvioimaan spesifeillä menetelmillä varsin tarkasti, vaihtelu trombiinigeneraatiotestissä osoittaa, että muutkin tekijät kuin lääkepitoisuus vaikuttavat vasteisiin hyytymistutkimuksissa. Potilaan kokonaistilanne tuleekin aina huomioida lääkeainevaikutuksia arvioitaessa

Clinical use and laboratory testing of oral anticoagulation therapy: experience from Finland

Anticoagulation therapy has been undergoing a transition during the last decade with the progressive availability of direct oral anticoagulants (DOACs). While DOACs are increasingly used in common indications for anticoagulation, including non-valvular atrial fibrillation (AF), deep vein thrombosis (DVT) and pulmonary embolism (PE), traditional anticoagulants, either warfarin or heparins, remain the primary choice for patients with cardiac valvular replacement, severe thrombophilia, pediatric, pregnant and many cancer patients. Laboratory monitoring of warfarin and heparins are well established, while under specific emergency conditions DOACs require specific assays, the availability of which may be limited. Finland benefits from centralized laboratory networks, offering harmonized services throughout our rather sparsely populated country. Specific assays for all DOACs are available 24/7 in the core laboratory (Southern Finland). Interaction, collaboration and continuous education between clinic and laboratory is increasingly needed, under the era of rapid change of traditions in anticoagulation management.Peer reviewe

Perorala antikoagulantia - när, var och hur?

English summary

Comparison of novel thrombin generation methods with established techniques is mandatory

Non peer reviewe

Novel Ex Vivo DOAC Removal Methods Reduce Interference in Lupus Anticoagulant Testing

Direct oral anticoagulants (DOAC) interfere in laboratory coagulation testing. The aim here was to study how commercial DOAC removal methods, DOAC Filter® and DOAC-Stop™, perform to eliminate DOAC concentrations and false positive results in lupus anticoagulant (LAC) testing. We acquired 50 patient samples with high concentrations of DOACs: apixaban (n = 18, range 68–572 ng/mL), dabigatran (n = 8, range 47–154 ng/mL), edoxaban (n = 8, range 35–580 ng/mL) and rivaroxaban (n = 16, range 69–285 ng/mL). DOACs were removed ex vivo with either DOAC Filter® (n = 28) or DOAC-Stop™ (n = 22). Additionally, commercial control and calibrator samples were studied (n = 13 for DOAC Filter®, n = 14 for DOAC-Stop™). LAC screening was performed before and after DOAC removal. Both DOAC Filter® and DOAC-Stop™ were effective in removing DOAC concentrations in samples: DOAC concentrations decreased to median of 0 ng/mL (range 0–48 ng/mL). Only one sample had more than residual 25 ng/mL of DOAC (apixaban). Before DOAC removal, 96% (48/50) of patient samples and over 90% (12/13 DOAC Filter®, 13/14 DOAC-Stop™) of control/calibrator samples were positive in the LAC screening. In patient samples, LAC screening turned negative in 61% (17/28) after DOAC Filter® and 45% (10/22) after DOAC-Stop™ treatment. All control samples became negative after DOAC removal. In conclusion, DOAC removal ex vivo reduces false positives in LAC screening. DOAC removal halved the need for confirmation or mixing tests- Although a subset of patients would require further testing, DOAC removal reduces unnecessary repeated LAC testing

Hyytymisaktivaatio ja koronavirus : infektion komplikaatioita voi ennustaa

Hyytymishäiriöissä verenkiertoon vapautuu fibriinin D-dimeeriä, kun sekä hyytymisaktivaatio että fibrinolyysi kiihtyvät. COVID-19-taudissa hoitojakson aikana nousujohteinen FiDD-pitoisuus on yhteydessä inflammaation vaikeusasteeseen, tukostapahtumiin ja kuoleman riskiin. Jopa neljäsosalla tehohoitoon joutuvista potilaista on todettu alaraajojen laskimotukos, joka voi johtaa keuhkoemboliaan

Warfarin dose requirement in patients having severe thrombosis or thrombophilia

Aims Warfarin dose requirement varies significantly. We compared the clinically established doses based on international normalized ratio (INR) among patients with severe thrombosis and/or thrombophilia with estimates from genetic dosing algorithms. Methods Fifty patients with severe thrombosis and/or thrombophilia requiring permanent anticoagulation, referred to the Helsinki University Hospital Coagulation Center, were screened for thrombophilias and genotyped for CYP2C9*2 (c.430C>T, rs1799853), CYP2C9*3 (c.1075A>C, rs1057910) and VKORC1 c.-1639G>A (rs9923231) variants. The warfarin maintenance doses (target INR 2.0-3.0 in 94%, 2.5-3.5 in 6%) were estimated by the Gage and the International Warfarin Pharmacogenetics Consortium (IWPC) algorithms. The individual warfarin maintenance dose was tailored, supplementing estimates with comprehensive clinical evaluation and INR data. Results Mean patient age was 47 years (range 20-76), and BMI 27 (SD 6), 68% being women. Forty-six (92%) had previous venous or arterial thrombosis, and 26 (52%) had a thrombophilia, with 22% having concurrent aspirin. A total of 40% carried the CYP2C9*2 or *3 allele and 54% carried the VKORC1-1639A allele. The daily mean maintenance dose of warfarin estimated by the Gage algorithm was 5.4 mg (95% CI 4.9-5.9 mg), and by the IWPC algorithm was 5.2 mg (95% CI 4.7-5.7 mg). The daily warfarin maintenance dose after clinical visits and follow-up was higher than the estimates, mean 6.9 mg (95% CI 5.6-8.2 mg, P <0.006), with highest dose in patients having multiple thrombophilic factors (P <0.03). Conclusions In severe thrombosis and/or thrombophilia, variation in thrombin generation and pharmacodynamics influences warfarin response. Pharmacogenetic dosing algorithms seem to underestimate dose requirement.Peer reviewe

Anaemia and enhancement of coagulation are associated with severe COVID-19 infection

Coagulation disturbances are common in severe COVID-19 infection. We examined laboratory markers in COVID-19 patients during the first wave of the pandemic in Finland. We analysed a wide panel of coagulation tests (IL ACL TOP 750/500) from anonymously collected samples of 78 hospitalized COVID-19 patients in intensive care units (ICUs; n = 34) or medical wards (n = 44) at Helsinki University Hospital in April-May 2020. These coagulation data were supplemented with the laboratory information system results, including complete blood count and C reactive protein (CRP). Coagulation and inflammatory markers were elevated in most: FVIII in 52%, fibrinogen 77%, D-dimer 74%, CRP 94%, platelet count 37%. Anaemia was common, especially in men (73% vs. 44% in women), and overall weakly correlated with FVIII (women R-2 = 0.48, men R-2 = 0.24). ICU patients had higher fibrinogen and D-dimer levels (p < .01). Men admitted to the ICU also had higher platelet count, leukocytes and FVIII and lower haemoglobin than the non-ICU patients. None of the patients met the disseminated intravascular coagulation (DIC) criteria, but 31% had a D-dimer level of at least 1.5 mg/L. Presence of both anaemia and high D-dimer together with FVIII is independently associated with ICU admission. Antithrombin was reduced in 47% of the patients but did not distinguish severity. Overall, CRP was associated with coagulation activation. Elevated FVIII, fibrinogen and D-dimer reflected a strong inflammatory response and were characteristic of hospitalized COVID-19 patients. The patients were often anaemic, as is typical in severe inflammation, while anaemia was also associated with coagulation activity.Peer reviewe

Platelet function and filamin A expression in two families with novel FLNA gene mutations associated with periventricular nodular heterotopia and panlobular emphysema

Pathogenic variants of the X-linked FLNA gene encoding filamin A protein have been associated with a wide spectrum of symptoms, including the recently described pulmonary phenotype with childhood-onset panlobular emphysema. We describe three female patients from two families with novel heterozygous FLNA variants c.5837_2del and c.508C > T. Analysis of immunofluorescence of peripheral blood smears and platelet function was performed for all patients. FLNA-negative platelets were observed, suggesting that these variants result in the loss of a functional protein product. All three patients also had periventricular nodular heterotopia and panlobular emphysema. However, they had considerably milder symptoms and later age of onset than in the previously reported cases. Therefore, patients with pathogenic FLNA variants should be studied actively for lung involvement even in the absence of pronounced respiratory symptoms. Conversely, any patient with unexplained panlobular emphysema should be analyzed for pathogenic FLNA variants. We also suggest that immunofluorescence analysis is a useful tool for investigating the pathogenicity of novel FLNA variants.Peer reviewe