Devreese, Katrien

MULLIEZ, SYLVIE

Van den Bogaert, Julie

English

Ghent University Academic Bibliography

Clin Chem Lab Med 2015; 53(2): e47–e50Letter to the EditorSylvie M.N. Mulliez, Julie Van den Bogaert and Katrien M.J. Devreese*Influence of vitamin K antagonist treatment on activated partial thromboplastin timeDOI 10.1515/cclm-2014-0688Received July 4, 2014; accepted July 21, 2014; previously published online August 19, 2014Keywords: activated partial thromboplastin time; inter-national normalized ratio (INR); oral anticoagulant; pro-thrombin time; vitamin K antagonist.To the Editor,Vitamin K antagonists (VKA) perform their anticoagu-lant effect by inhibiting vitamin K epoxide reductase, an enzyme that recycles oxidized vitamin K to its reduced form. Vitamin K is a cofactor for the post-translational carboxylation of several vitamin K-dependent proteins. Consequently, VKA result in defective synthesis of the vitamin K-dependent clotting factors: factor (F) II, VII, IX, and X. The recommended method used for monitor-ing VKA is determination of the international normalized ratio (INR) which is derived from the prothrombin time (PT) [1]. This test is sensitive to the levels of FII, FVII and FX, but is not responsive to reduction of FIX, which can be detected by activated partial thromboplastin time (aPTT). APTT measures the efficacy of both the intrinsic and the common coagulation pathway and is also influenced by vitamin K-dependent clotting factors (FIX, FX and FII). Therefore, among patients treated with VKA, a prolonged aPTT is observed. The relationship between an elevated INR in a patient on VKA treatment with the aPTT is not known and probably depends on the FIX level. Very low FIX in patients on VKA treatment, due to mutation in the FIX gene, with prolonged aPTT have been described, but is very rare [2]. This relationship likely depends also on the combination of aPTT reagent and instrument utilized and aPTT reagents differ largely in sensitivity to coagula-tion factor deficiency [3]. Although aPTT measurement is not indicated in VKA monitoring, the aPTT is widely used as part of a coagulation screening panel. In case of VKA-treated patients a prolonged aPTT is often due to the VKA, although the measure of prolongation of aPTT in VKA-treated patients is unknown. Misinterpretation of a prolonged aPTT should be avoided and therefore the sensitivity of the aPTT reagent towards INR in AVK-treated patients can be informative.In this study, we have evaluated the relationship between INR and aPTT in patients on VKA anticoagu-lant treatment and the sensitivity of three different aPTT reagents, PTT-A (Diagnostica Stago, Asnières-sur-Seine, France), Cephascreen® (Diagnostica Stago) and C.K. Prest® (Diagnostica Stago).Patients from the Ghent University Hospital treated with VKA and monitored by PT/INR were collected. The study was approved by the Belgian regulatory agency (B670201419819). The trial was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Ghent University Hospital (registra-tion no. 2014/101). Samples with an INR  > 1.5 were pro-spectively and consecutively collected from January to April 2014 and frozen at –20 °C in different aliquots within 4 h after sampling. The aPTT with three different reagents (PTT-A, C.K. Prest® and Cephascreen®) and PT/INR (Neo-plastine® CI plus, Diagnostica Stago) were measured. A simple linear regression analysis with 95% and 90% prediction limits between aPTT and INR was made using SPPS® Statistics 20 (IBM Business Analytics, Armonk, NY, USA). Samples with disproportionate aPTT (‘outliers’) were identified as those with an aPTT outside 90% predic-tion limits and were further analyzed. To measure clotting factor activity the one-stage clotting assay was used. STA®-Thrombin 2 (Diagnostica Stago) and Biophen Heparin LRT (Hyphen BioMed, Andresy, France) were used to measure, respectively, thrombin time (TT) and anti-Xa activity. *Corresponding author: Katrien M.J. Devreese, Coagulation Laboratory, Laboratory for Clinical Biology Ghent University Hospital, De Pintelaan, 185 (2P8), 9000 Gent, Belgium,  Phone: +32 9 3326567, Fax: +32 9 3324985,  E-mail: katrien.devreese@uzgent.beSylvie M.N. Mulliez and Julie Van den Bogaert: Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, BelgiumBrought to you by | Universiteit GentAuthenticatedDownload Date | 2/4/15 4:10 PMe48      Mulliez et al.: Influence of vitamin K antagonist treatment on activated partial thromboplastin timeLupus anticoagulant (LA) was detected according to the SSC guidelines using the dilute Russell’s Viper Venom Test (dRVVT) (LA screen and LA confirm, Life Diagnostics, DSRV, Clarkstons, MI, USA) and a sensitive aPTT assay (PTT-LA and Staclot® LA, Diagnostica Stago) [4]. C-reac-tive protein (CRP) was measured on Cobas®8000 (Roche Diagnostics, Belgium).Seven hundred samples were collected. In a first step the aPTT with the three different reagents and PT/INR were measured (Figure 1). A Grubb’s outlier test (α = 0.05) on bias between INR measured during the study and the routinely measured INR was performed to exclude samples (n = 45) influenced by the freezing/thawing cycle. Linear regression analysis between aPTT and INR (n = 655) was performed. Samples of patients with disproportionate aPTT were identified by using the 95% and 90% predic-tion limits (Figure 2). In total 55 outliers were identified, five samples with a disproportionate short aPTT and 50 with a disproportionate prolonged aPTT for correspond-ing INR. In Table 1 the mean aPTT of all samples within the prediction limits and the upper limit of aPTT was cal-culated for the three aPTT reagents for INR of 2, 3 and 4.Next, we further analyzed the samples beyond the 90% prediction limit with at least one of the aPTT reagents. Samples with a disproportional short aPTT (n = 5) were examined for FVIII and FIX activity. In four out of the five samples the elevated FVIII level  > 150% (normal range, 60%–150%) could explain the disproportional short aPTT. One sample showed a normal FVIII activity of 103% and the FIX activity was low (23.3%) and thus could not explain the disproportion. FII, FX and FVII were, respec-tively, 46%, 28%, and 2%. FVII influences strongly the PT-INR [5]. The discrepancy between the relatively high FII and FX level and very low FVII level could explain the disproportional low aPTT (respectively, 67.4 s, 48.8 s, and 63.4 s with PTT-A, C.K. Prest® and Cephascreen®) for high INR of 14.1.Samples with disproportional prolonged aPTT (n = 50) were further analyzed for TT and anti-Xa activity to rule out the presence of heparin (UFH) or low molecular weight heparin (LMWH). In 37 out of 50 samples (74%) presence of UFH or LMWH was confirmed. We explored the remain-ing 13 out of 50 samples by consulting the medical and laboratory history of these patients and extra analyses. Five out of 13 samples showed the presence of LA. LA interferes with clotting in phospholipid dependent clot-ting tests, such as aPTT and explains the prolonged aPTT [4]. In four out of the 13 samples CRP was  > 180 mg/dL 700 SamplesExcluding samplesinfluenced byfreezing/thawingcycle (Grubb’soutlier test on biasbetween INR)655 SamplesLinear regressionDisproportionalprolonged aPTT(90% predictionlimits)Disproportionalshort aPTT (90%prediction limits)50 Samples 5 Samples1 Sample:High FII (46%) andFX (28%) and lowFVII (2%)4 Samples:elevated FVIII(>150%)37 Samples: UFH or LMWH5 Samples:LA4 Samples:CRP > 180 mg/dL1 Sample:FXII deficiency3 Samples:No explanationFigure 1 Flowchart of the selection of the samples and the results.aPTT, activated partial thromboplastin time; CRP, C reactive protein; F, factor; INR, international normalized ratio; LA, lupus anticoagulant; LMWH, low molecular weight heparin; PT, prothrombin time; UFH, unfractioned heparin.Brought to you by | Universiteit GentAuthenticatedDownload Date | 2/4/15 4:10 PMMulliez et al.: Influence of vitamin K antagonist treatment on activated partial thromboplastin time      e49(Table 2). CRP interacts with phospholipids and results in an in vitro prolongation of phospholipid-dependent clotting tests, similar to the in vitro prolongation of aPTT–C.K. Prest® , saPTT–CEPHASCREEN® , saPTT–PTT-A, saPTT–PTT-A, saPTT–C.K. Prest® , saPTT–CEPHASCREEN® , s90% prediction limits90% prediction limits 90% prediction limits95% prediction limits 95% prediction limits95% prediction limitsABC ED F1501251007550251251007550251251007550252001501005002001501005001501251007550250 2 4 6 8 10 12 14INR0 2 4 6 8 10 12 14INR0 2 4 6 8 10 12 14INR0 2 4 6 8 10 12 14INR0 2 4 6 8 10 12 14INR0 2 4 6 8 10 12 14INRFigure 2 APTT values in relation to INR for 655 samples.APTT measured with three different reagents, respectively, PTT-A (A, B), C.K. Prest®(C, D) and CEPHASCREEN® (E, F). The linear regres-sion analysis revealed the following relation between INR and aPTT: aPTT (PTT-A) = 35.4+7.1 INR, aPTT (C.K. Prest®) = 30.6+4.3 INR and aPTT (CEPHASCREEN®) = 29.4+6.9 INR. The lines indicate the linear regression line and the upper and lower 90% and 95%, respectively, in A, B, C and D, E and F, prediction limits of the linear regression between the aPTT and INR.Table 1 Mean aPTT for three different aPTT reagents (PTT-A, C.K. Prest® and Cephascreen®) excluding 5% and 10% of the data, with disproportionate prolonged and shortened aPTT and the upper limit of the expected aPTT with the 95% and 90% prediction limits of the linear regression analysis for INR of 2, 3 and 4.aPTT reagent INR Mean aPTT, s– 5% dataMean aPTT, s– 10% dataaPTT, s95% PLaPTT, s90% PLPTT-A   2  46.9  46.8   < 79.1   < 74.6  3  54.8  54.2   < 86.1   < 81.7  4  62.7  61.6   < 93.2   < 88.7C.K. Prest®   2  37.6  37.5   < 60.3   < 56.6  3  41.9  41.7   < 64.6   < 60.8  4  46.2  45.9   < 68.9   < 65.1Cephascreen®  2  40.8  40.8   < 76.4   < 71.7  3  47.8  47.6   < 83.3   < 78.6  4  54.8  54.5   < 90.2   < 85.5aPTT, activated partial thromboplastin time; PL, prediction limit; PT, prothrombin time.coagulation tests by LA due to interference with accumu-lation of coagulation factors on negatively charged phos-pholipids [6, 7]. The influence is reagent dependent as observed in the present data and explains the dispropor-tional prolonged aPTT with Cephascreen® and PTT-A, reagents sensitive to high CRP concentration and not with C.K. Prest®, a reagent insensitive for CRP interference [7]. Furthermore, we found one sample with a factor FXII deficiency.For three out of the 13 samples we could not iden-tify a cause for the disproportional prolonged aPTT (CRP Table 2 INR and aPTT with three different reagents (PTT-A, C.K. Prest® and Cephascreen®) of four samples with CRP  > 180 mg/L.INR  aPTT PTT-A, s  aPTT C.K. Prest®, s  aPTT Cephascreen®, s  CRP, mg/L4.3  99.9  56.3  91.8  184.46.2  101.2  62.8  105.8  332.411.4  154.9  82.7  139.4  405.912.8  155.7  83.1  145.8   > 400aPTT, activated partial thromboplastin time; CRP, C reactive protein; INR, international normalized ration.Brought to you by | Universiteit GentAuthenticatedDownload Date | 2/4/15 4:10 PMe50      Mulliez et al.: Influence of vitamin K antagonist treatment on activated partial thromboplastin timelevel, respectively, 5, 8 and 42 mg/L). One of these samples showed a FIX activity of only 7.6% for an INR of 3.8. As described by Legnani et  al. vitamin K antagonist can greatly influence the factor IX activity but the sensitivity of factor IX activity to VKA varies greatly between indi-viduals with a similar anticoagulation intensity level as measured by INR [2]. See Supplemental Data, Table 1, for an overview of the aPTT values and INR of the 55 samples with disproportional short and prolonged aPTT and indi-cation of the cause.In conclusion, the rate of aPTT prolongation in VKA-treated patients is reagent dependent. We calculated the expected prolongation of aPTT with three different rea-gents (PTT-A, C.K. Prest® and Cephascreen®) for INR of 2, 3 and 4. An extensively prolonged aPTT in VKA-treated patients may not be due to VKA alone. Disproportional prolonged aPTT in patients on VKA treatment merit further attention and should be investigated to avoid mis-interpretation. In 47 of the 50 samples (94%) we found another cause resulting in a prolonged aPTT: presence of UFH/LMWH (n = 37), LA (n = 5) high CRP (n = 4) and FXII deficiency (n = 1).Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.Financial support: None declared.Employment or leadership: None declared.Honorarium: None declared.Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.References1. Ansell J, Hirsh J, Hylek E, Jacobson A, Crowther M, Palareti G. Pharmacology and management of the vitamin K antagonists: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th ed.). Chest 2008;133(Suppl):S187–98.2. Legnani C, Promenzio M, Guazzaloca G, Coccheri S, Palareti G. Assessment of activated partial thromboplastin time and factor IX in subjects attending an anticoagulation clinic. Blood Coagul Fibrin 2000;11:537–42.3. Lawrie AS, Kitchen S, Efthymiou M, Mackie IJ, Machin SJ. Determi-nation of APTT factor sensitivity – the misguiding guideline. Int J Lab Hematol 2013;35:652–7.4. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, et al. Update of the guidelines for lupus anticoagulant detection. Sub-committee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2009;7:1737–40.5. D’Angelo A, Della Valle P, Crippa L, Fattorini A, Pattarini E, Vigano D’Angelo S. Relationship between international normalized ratio values, vitamin K-dependent clotting factor levels and in vivo prothrombin activation during the early and steady phases of oral anticoagulant treatment. Haematologica 2002;87:1074–80.6. Schouwers SM, Delanghe JR, Devreese KM. Lupus anticoagu-lant (LAC) testing in patients with inflammatory status: does C-reactive protein interfere with LAC test results? Thromb Res 2010;125:102–4.7. van Rossum AP, Vlasveld LT, van den Hoven LJ, de Wit CW, Castel A. False prolongation of the activated partial thromboplastin time (aPTT) in inflammatory patients: interference of C-reactive pro-tein. Br J Haematol 2012;157:394–5.Brought to you by | Universiteit GentAuthenticatedDownload Date | 2/4/15 4:10 PM

Influence of vitamin K antagonist treatment on activated partial thromboplastin time

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Influence of vitamin K antagonist treatment on activated partial thromboplastin time

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