Activated Partial Thromboplastin Time Reagents Research Articles

Variability in combinations of APTT reagent and substrate plasma for a one-stage clotting assay to measure factor VIII products.

An investigation of the suitability of reagents for measuring FVIII products in a one-stage clotting assay (OSA) showed variations in their FVIII activity (FVIII:C). Most studies have focused on the activated partial thromboplastin time (APTT) reagent rather than FVIII-deficient plasma (F8DP), even though the APTT-based OSA is comprised of APTT reagents and factor-deficient plasma. A single-centre study was conducted to clarify variations in measurements of FVIII products in an OSA using a total of 12 reagent combinations, including four APTT reagents and three types of F8DP. FVIII:C in nine types of FVIII product-spiked plasma was measured using an OSA with four different APTT reagents and three types of F8DP. F8DP-dependent variations were found in addition to differences derived from APTT reagents. Variations in target recovery (TR) were observed for NovoEight®, Eloctate®, and Jivi®. Reduced TR for Jivi was found only for Pathromtin SL in combination with congenital F8DP (F8DP-3). This lower TR was not observed with alternative manufacturing lots of F8DP-3. The reduced TR for Jivi might be related to impaired contact activation due to lower factor XI activity in F8DP-3. In addition to APTT reagents, variations in F8DPs used for OSAs can also affect FVIII:C results. F8DPs as well as the APTT reagent used for OSA should be chosen with caution, and laboratories should evaluate reagents for F8DPs as they currently do for APTT reagents, especially when lot changes occur.

International Council for Standardization in Haematology Field Study Evaluating Optimal Interpretation Methods for Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies.

The prothrombin time (PT) and activated partial thromboplastin time (APTT) are screening tests used to detect congenital or acquired bleeding disorders. An unexpected PT and/or APTT prolongation is often evaluated using a mixing test with normal plasma. Failure to correct ("noncorrection") prolongation upon mixing is attributed to an inhibitor, whereas "correction" points to factor deficiency(ies). To define an optimal method for determining correction or noncorrection of plasma mixing tests through an international, multisite study that used multiple PT and APTT reagents and well-characterized plasma samples. Each testing site was provided 22 abnormal and 25 normal donor plasma samples, and mixing studies were performed using local PT and APTT reagents. Mixing study results were evaluated using 11 different calculation methods to assess the optimal method based on the expected interpretation for factor deficiencies (correction) and noncorrection (inhibitor effect). Misprediction, which represents the failure of a mixing study interpretation method, was assessed. Percentage correction was the most suitable calculation method for interpreting PT mixing test results for nearly all reagents evaluated. Incubated PT mixing tests should not be performed. For APTT mixing tests, percentage correction should be performed, and if the result indicates a factor deficiency, this should be confirmed with the subtraction III calculation where the normal pooled plasma result (run concurrently) is subtracted from the mixing test result with correction indicated by a result of 0 or less. In general, other calculation methods evaluated that performed well in the identification of factor deficiency tended to have high misprediction rates for inhibitors and vice versa. No single method of mixing test result calculation was consistently successful in accurately distinguishing factor deficiencies from inhibitors, with between-reagent and between-site variability also identified.

Differentiation of Acquired Hemophilia a from Lupus Anticoagulant Using Global Coagulation Assays and Magnesium-Dependent Activated Partial Thromboplastin Time Assay

CONCLUSION Rapid and reliable differentiation between bleeding and thrombotic tendency in cases with prolonged activated partial thromboplastin time (APTT) remains challenging in clinical practice, in particular, distinguishing cases positive for coagulation factor inhibitor or lupus anticoagulant (LA). To differentiate acquired hemophilia A (AHA) from LA using three global coagulation assays: rotational thromboelastometry (ROTEM), thrombin generation assay (TGA), and clot waveform analysis (CWA); also to validate the effectiveness of magnesium-dependent APTT (APTT-Mg) assay. This method uses APTT-Mg and normal APTT using Ca to screen for LA, and APTT-Mg/APTT ratio is smaller in LA. We included patients with prolonged APTT without congenital coagulation factor deficiency who were referred to our department between November 2019 and September 2021. APTT prolongation was defined as >35 s (normal range: 25-35 s) measured routinely with Coagpia APTT-N (Sekisui Medical). The AHA group had a confirmed diagnosis and underwent treatment of AHA. The LA group was divided into LA1 (possible LA) and LA2 (definite LA). LA1 was defined by any of the following conditions: (1) index of circulation anticoagulant ≥15 in the cross-mixing test performed with Coagpia APTT-N; (2) positive dilute Russell's viper venom time (DRVVT) phospholipid neutralization method using Gladipore LA (Medical & Biological Laboratories); (3) positive APTT phospholipid neutralization test using Hemosil SCT (Instrumentation Laboratory); (4) positive for anti-CL antibody; (5) positive for anti-CLβ2GPI antibody; or (6) positive for phosphatidylserine-dependent anti-PT antibody. LA2 was defined by index of circulation anticoagulant >12.4 in the cross-mixing test performed by Thrombocheck APTT-SLA (Sysmex Corporation), or item (2) or (3) above, in accordance with criteria of LA in the International Society of Thrombosis and Haemostasis. LA1 and LA2 did not overlap, because cases that did not meet the conditions of LA2 were treated as LA1, and global coagulation assays were performed in all LA cases. In the global coagulation assays, 12 healthy volunteer samples were tested and regarded as the normal goroup. For the APTT-Mg assay, equal volumes of 25 mmol MgCl 2 and 25 mmol CaCl 2 were used instead of the normally used CaCl 2, and Thrombocheck APTT-SLA was used to measure APTT-Mg, which was then divided by the normal APTT concentration measured with the same APTT reagent. Ten patients in the AHA, 12 in the LA1, and 32 in the LA2 groups were eligible. ROTEM showed that clotting time in the non-activated rotational thromboelastometry assay (NATEM) mode was >3600 s (minimum 1363 s) for AHA, 501 s (IQR: 430-632 s) for LA1, 533 s (IQR: 443-641 s) for LA2, and 500 s (IQR: 429-645 s) for the normal group. TGA showed that peak thrombin concentration was 16 nM (IQR: 13-29 nM) in the AHA group, 242 nM (IQR: 175-273 nM) in the LA1 group, 174 nM (IQR: 129-259 nM) in the LA2 group, and 190 nM (IQR: 125-288 nM) in the normal group. In the CWA, Ad|min1| was 2.1 (IQR: 1.7-3.2) in the AHA group, 8.7 (IQR: 8.0-9.5) in the LA1 group, 6.7 (IQR: 5.8-8.4) in the LA2 group, and 10.3 (IQR: 9.8-10.6) in the normal group. APTT-Mg/APTT was 1.23 (IQR: 1.18-1.29), 1.26 (IQR: 1.23-1.38), and 1.05 (IQR: 0.95-1.20) in the AHA, LA1, and LA2 groups, respectively. ROTEM, TGA, and CWA effectively differentiated between the LA1/2 and AHA groups. Clotting time in non-activated rotational thromboelastometry assay mode (ROTEM) and peak height (TGA) were better at distinguishing high-titer LA cases from LA-positive AHA cases. APTT-Mg/APTT could be suitable for distinguishing AHA from high-titer LA; however, it may not be useful for differentiating AHA from low-titer or suspected LA.

Comparative Assessment of APTT Reagents for Evaluating Anticoagulant Sensitivity of Fucosylated Glycosaminoglycans (FGs) Derived from Sea Cucumbers.

Fucosylated glycosaminoglycans (FGs) derived from sea cucumbers exhibit potent intrinsic Xase (iXase) inhibition, anticoagulation, and antithrombosis. Plasma activated partial thromboplastin time (APTT), a widely used screening test worldwide, is crucial for evaluating anticoagulant efficacy. However, the applicability of these commercially available APTT reagents for assessing anticoagulation of FGs remains unreported. In this study, we investigated the disparity between ellagic acid and colloidal silica APTT reagents in evaluating anticoagulation of dHG-5 and dHLFG-4, two depolymerized FGs, and elucidated the underlying rationale. The results demonstrated that dHG-5 and dHLFG-4 exhibited heightened sensitivity to the ellagic acid APTT reagent both in vitro and in vivo, and did not significantly affect the activation of APTT reagents for plasma. In addition, both ellagic acid and colloidal silica APTT reagents inhibited the anti-iXase of dHG-5 and dHLFG-4, and the inhibition of the ellagic acid APTT reagent was less pronounced compared to the colloidal silica APTT reagent. These findings suggest that the reduced impact of the ellagic acid APTT reagent on the anti-iXase activity of dHG-5 and dHLFG-4 is responsible for the increased sensitivity in plasma APTT analysis. This study offers valuable insights into the characteristics of two APTT reagents applied for assessing the anticoagulant activity of FG-related compounds.

High variability in Factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system.

Haemophilia B (HB) is an X-linked hereditary bleeding disorder characterized by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates modified to extend their half-life have been developed, called extended half-life factors (EHL: extended half-life). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. To diagnose and monitor the treatment of haemophiliac patients, FIX activity is determined with the one-stage clotting assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-PG with 10 different activated partial thromboplastin time (APTT) reagents on three platforms, in samples spiked in vitro with N9-GP, at four different concentration levels. It was measured the recovery of N9-GP with 10 different APTT reagents (polyphenol, ellagic acid, silice dioxide, colloidal silica as APTT activator on three platforms, in sample spiked in vitro with N9-GP. The results show heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific calibrator of each coagulometer. A recovery percentage between 87% and 108% was obtained only with polyphenol and ellagic acid as activator in the three platforms evaluated. The other reagents studied overestimate or underestimate, with no clear profile. When a calibration curve was performed with a calibrator prepared from the N9-GP vial, all APTT reagents met the established recovery requirement. APTT reagents with polyphenol or ellagic acid as activator would be the only ones appropriate when using the commercially available OSA with specific calibrator to monitor patients treated with N9-GP.

False Prolongation of Activated Partial Thromboplastin Time with Aminoglycoside Antimicrobial Agents: A Case Report

BACKGROUND: Activated partial thromboplastin time (APTT) is a clotting time assay for screening bleeding tendency, evaluating coagulation factor production capacity, assessing preoperatively, monitoring anticoagulant drugs, and searching for blood coagulation abnormalities such as hemophilia and antiphospholipid syndrome. CASE PRESENTATION: Here, we present a 77-year-old male patient with dyspnea who was suspected to have a drug-resistant Pseudomonas aeruginosa infection and pulmonary mycosis. The patient had no history of bleeding tendencies or anticoagulant medication use. The laboratory test results revealed an abnormally prolonged activated partial thromboplastin time (APTT) of 120.3 s using the Coagpia® APTT-N reagent. The APTT test is frequently used to evaluate blood clotting function and assess for bleeding disorders. Prolonged APTT can indicate coagulation factor deficiencies or the presence of certain conditions such as von Willebr and disease, hemophilia, and disseminated intravascular syndrome. However, APTT standardization has not been achieved, causing discrepancies in test results due to variations in the reagents used. The prolonged APTT, in this case, was initially suspected to be caused by contamination or other artifacts, but repeat blood collections and cross-mixing tests revealed the Coagpia® APTT-N reagent as the cause of false prolongation. The reagent was changed to HemosIL SynthASil APTT, which revealed a normal APTT result. The patient had been receiving the aminoglycoside antimicrobial agent tobramycin, and the blood sample taken at the peak tobramycin level demonstrated the longest APTT time. The APTT shortened over time, corresponding to the decrease in tobramycin blood levels. CONCLUSION: Overall, this paper reports a case of false APTT prolongation due to a specific APTT reagent in the presence of aminoglycoside antimicrobial agents. The findings underscore the difficulties in standar PTT testing and the importance of considering reagent performance characteristics in result interpretations.

Reagent Effects on the Activated Partial Thromboplastin Time Clot Waveform Analysis: A Multi-Centre Study.

(1) Background: The activated partial thromboplastin time (APTT)- based clot waveform analysis (CWA) quantitatively extends information obtained from the APTT waveform through its derivatives. However, pre-analytical variables including reagent effects on the CWA parameters are poorly understood and must be standardized as a potential diagnostic assay. (2) Methods: CWA was first analysed with patient samples to understand reagent lot variation in three common APTT reagents: Pathromtin SL, Actin FS, and Actin FSL. A total of 1055 healthy volunteers were also recruited from seven institutions across the Asia-Pacific region and CWA data were collected with the Sysmex CS analysers. (3) Results: CWA parameters varied less than 10% between lots and the linear mixed model analysis showed few site-specific effects within the same reagent group. However, the CWA parameters were significantly different amongst all reagent groups and thus reagent-specific 95% reference intervals could be calculated using the nonparametric method. Post-hoc analysis showed some degree of influence by age and gender with weak correlation to the CWA (r < 0.3). (4) Conclusions: Reagent type significantly affects APTT-based CWA with minimal inter-laboratory variations with the same coagulometer series that allow for data pooling across laboratories with more evidence required for age- and gender-partitioning.

Clot Waveform Analysis for Hemostatic Abnormalities.

Clot waveform analysis (CWA) observes changes in transparency in a plasma sample based on clotting tests such as activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Evidence indicates that not only an abnormal waveform but also peak times and heights in derivative curves of CWA are useful for the evaluation of hemostatic abnormalities. Modified CWA, including the PT with APTT reagent, dilute PT (small amount of tissue factor [TF]-induced clotting factor IX [FIX] activation; sTF/FIXa), and dilute TT, has been proposed to evaluate physiological or pathological hemostasis. We review routine and modified CWA and their clinical applications. In CWA-sTF/FIXa, elevated peak heights indicate hypercoagulability in patients with cancer or thrombosis, whereas prolonged peak times indicate hypocoagulability in several conditions, including clotting factor deficiency and thrombocytopenia. CWA-dilute TT reflects the thrombin burst, whereas clot-fibrinolysis waveform analysis reflects both hemostasis and fibrinolysis. The relevance and usefulness of CWA-APTT and modified CWA should be further investigated in various diseases.

Comparative analysis on characteristics of two activated partial thromboplastin time reagents

BackgroundFor the lack of standardized activated partial thromboplastin time (APTT), it has been pointed out that there are differences in values among several reagents. Recently, we have performed a parallel measurement on two reagents, Thrombocheck APTT‐SLA and Coagpia APTT‐n, and resulted with some dissociated samples. The purpose of this study is to clarify the possible factors related to ΔAPTT, the difference in measured values between the two reagents.Materials and MethodsIn order to clarify the factors related to ΔAPTT, multiple regression analysis was performed on 8324 samples, using clinical laboratory data of all test items requested simultaneously with APTT. To confirm the items extracted from the multiple regression analysis, the target substance was spiked to pooled plasma and measured with two APTT reagents. Additionally, by spiking phospholipids, the effect on APTT measurement system was assessed.ResultMultiple regression analysis detected albumin–globulin ratio (AGR), C‐reactive protein (CRP), hematocrit, and prothrombin time as factors related to ΔAPTT (p < 0.001). Results revealed no significant differences when albumin was added to change the AGR. Whereas with the addition of CRP, prolongation of APTT was observed in Coagpia APTT‐n compared to Thrombocheck APTT‐SLA (p < 0.001). This prolongation was canceled by the addition of phospholipids, suggesting the interaction of CRP with phospholipids leads to the pseudo‐prolongation.ConclusionIt is considered that the pseudo‐prolongation of APTT is triggered by the interaction of CRP on the phospholipid in Coagpia APTT‐n, which contributed to the APTT dissociation.

Differences in the Composition of Activated Partial Thromboplastin Time (APTT) Reagents Affect Clot Waveform Analysis.

Clot waveform analysis (CWA) based on activated partial thromboplastin time (APTT) is a useful assay for hemostasis. However, the effects of activators and phospholipid conditions on CWA have not been adequately investigated. Therefore, we characterized CWA using four different APTT reagents. We used 39 archived plasma samples from patients with hemophilia A (HA), 16 samples from patients with HA under emicizumab treatment, and 10 samples from healthy individuals for CWA with four different types of APTT reagents (reagents A, B, C, and D). We then compared Ad|min1|, Ad|min2|, and Ad|max2| from the CWA, which reflect the maximum velocity, maximum acceleration, and maximum deceleration, respectively, among the four reagents. Similar clot waveform shapes were observed for each reagent in the healthy donor group, HA group, and HA under emicizumab group, and the waveform was different for each target group. Significant changes were found in clotting time (CT) (s), Ad|min1| (%/s), Ad|min2| (%/s2), and Ad|max2| (%/s2). The waveform pattern for the coagulation reaction by reagent D, comprising silica and synthetic phospholipids, was the fastest among the reagents examined. Further, the difference in Ad|min1| (%/s) and Ad|min2| (%/s2) was larger than that in CT depending on the reagent used(s), indicating that the measured value of CWA was affected by the reagent composition. Our results showed a significant difference among reagents with varying composition and concentration; this was found to affect the parameters obtained from CWA. Thus, the differences between reagents hinder standardization of quantitative evaluation using these parameters; further, this highlights the necessity of understanding the characteristics of APTT reagents and determining the reference range in individual facilities.

Laboratory methods for monitoring argatroban in heparin-induced thrombocytopenia.

The Summary of Product Characteristics for the direct thrombin inhibitor argatroban states monitoring should be by activated partial thromboplastin time (APTT), with a target range of 1.5-3.0 times the patients' baseline APTT. APTT may be influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. Previous studies have shown sensitivity differences of APTT reagents to argatroban. Some recent publications have favoured the use of anti-IIa methods to determine the plasma drug concentration of argatroban. This study aims to compare the anti-IIa assays: Hemoclot thrombin inhibitor assay (HTI) and Ecarin chromogenic assay (ECA) alongside the APTT. Residual plasma of 25 samples from 8 patients (3 with COVID-19 and HIT: n=18, 5 with HIT: n=7) was tested at two sites: site A: Sysmex CS5100 by HTI and APTT (Actin FS and SynthASil), and also on Stago STA Compact Max: ECA and APTT (CK Prest); and site B: Stago STA R Max 2 by ECA and APTT (Cephascreen). Mean APTT ratio was 1.96 (Actin FS), 1.84 (SynthASil), 1.59 (CK Prest) and 2.48 (Cephascreen). Mean argatroban concentration by HTI was 0.60µg/mL and by ECA was 0.65µg/mL (site A) and 0.70µg/mL (site B). There was a poor correlation to HTI in APTT ratios (range r2 =.0235-0.4181) with stronger correlations between ECA methods to HTI (r2 =.8998 site A, r2 =.8734 site B). This study confirms previous publications on the unsuitability of APTT and expands this by being multisited and included APTT reagents that use mechanical clot detection. Both anti-IIa methods are more suitable.

APTT therapeutic range for monitoring unfractionated heparin therapy. Significant impact of the anti‐Xa reagent used for correlation

IntroductionUnfractionated heparin (UFH) therapy is monitored by using the anti‐activated factor X (anti‐Xa) activity, or the activated partial thromboplastin time (APTT), which remains the most widely used assay. One of the main advantages of anti‐Xa relies on its hypothesized standardization, with a unique therapeutic range (0.30‐0.70 IU/ml) for all reagents, whereas APTT is influenced by numerous preanalytical and analytical parameters not related to the anticoagulant activity of UFH. MethodsThe aim of this study was to compare the anti‐Xa‐correlated APTT therapeutic ranges calculated using different combinations of APTT (n = 4) and anti‐Xa reagents (n = 4) in frozen citrated plasmas from 87 inpatients on UFH. ResultsThe median APTT ratio ranged from 2.19 for the less sensitive to 3.23 for the most sensitive reagent, whereas the median anti‐Xa activity was between 0.37 IU/ml and 0.57 IU/ml. The APTT therapeutic ranges calculated to correlate with anti‐Xa activities between 0.30 and 0.70 IU/ml were found to be highly different from one combination of APTT reagent and analyzer to another. The same applied to the therapeutic range of a single APTT reagent calculated using different anti‐Xa assays performed on the same analyzer, leading to a lack of agreement as to whether a sample was classified as subtherapeutic, therapeutic or supratherapeutic in 8.0% to 23.0% of the patients, with kappa coefficients between 0.908 and 0.753. ConclusionsThese results suggest that the APTT therapeutic range calculated to correlate with anti‐Xa activities between 0.30 and 0.70 IU/ml is influenced not only by the APTT reagent, but also by the anti‐Xa reagent used for calculation.

Applying index of circulating anticoagulant to mixing tests with lupus anticoagulant screen and confirm reagents can distinguish with high specificity between lupus anticoagulants and direct factor Xa inhibitors.

Lupus anticoagulants (LA) are detected by prolongation of clotting times for dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) screening tests. Direct oral anticoagulants (DOACs) can interfere with both screening and confirmatory tests. The present study aimed to investigate the influence of direct factor Xa inhibitors (DiXaIs) on screen, confirm and mixing tests and establish a method for differentiation from other sample types. A total of 257 samples including nonanticoagulated LA positive, LA positive with DiXaIs, factor deficiency, FVIII inhibitors, warfarin and non-APS DiXaIs were tested. APTT reagents Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used, respectively, to screen/confirm the study group. Index of circulating anticoagulant (ICA) was calculated from clotting times based on the following formula as ICA screening and ICA confirmation. ICA= (1:1 Mix sample - Normal pooled plasma) / Screen patient x 100. An ICA matrix was established which suggested the presence of a DiXaI when both ICA screening and confirmation were above the cut-off. When only ICA screening is elevated, LA is suspected. Sensitivity and specificity of the ICA matrix were 52.2% and 92.8% for DiXaIs and 38.1% and 96.7% for LA in APTT, and 61.2% and 92.9% for DiXaIs and 22.2% and 88.4% for LA in dRVVT, respectively. The ICA matrix achieved high specificity with a lower apparent sensitivity for DiXaI samples comparatively to other devices but due only to less interferences: the matrix could contribute to differentiating DiXaIs from LA in samples where anticoagulation status is unknown.

Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX‐Padua

BackgroundLimited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX‐Padua gene therapy. ObjectiveAssess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX‐Padua gene transfer. MethodsFIX:C was compared between central (n = 1) and local laboratories (n = 5) in the study, and across four commonly used FIX:C one‐stage assays and one FIX:C chromogenic assay. For comparison, samples of pooled congenital FIX‐deficient plasma spiked with purified recombinant human FIX (rHFIX)‐Padua protein or rHFIX (nonacog alfa) to obtain FIX:C concentrations from ~20% to ~40% were tested. ResultsFIX:C results at local laboratories strongly correlated with central laboratory results. However, absolute values at the central laboratory were consistently lower than those at local laboratories. Across five different FIX:C assays, a consistent pattern of FIX:C was observed for subjects receiving fidanacogene elaparvovec‐expressed gene transfer. Use of Actin FSL activated partial thromboplastin time (APTT) reagent in the central laboratory resulted in lower FIX:C values compared with other APTT reagents tested. The chromogenic assay determined lower FIX:C than any of the one‐stage assays. The rHFIX‐Padua protein–spiked samples showed similar results. In contrast, FIX:C results for rHFIX‐nonacog alfa measured within 25% of expected for all one‐stage assays and below 25% in the chromogenic assay. ConclusionsAssay‐based differences in FIX:C were observed for fidanacogene elaparvovec transgene product and rHFIX‐Padua protein, suggesting the variable FIX:C determined with different assay reagents is inherent to the FIX‐Padua protein and is not specific to gene therapy–derived FIX‐Padua.

The Evaluation of APTT Reagents in Reference Plasma, Recombinant FVIII Products; Kovaltry® and Jivi® Using CWA, Including sTF/7FIX Assay.

The FVIII activity in patients treated with several extended half-life FVIII (EHL-FVIII) agents different when various activated partial thromboplastin time (APTT) reagents were used. The present study examined the difference in clot waveform analysis (CWA) findings and FVIII activity when various APTT reagents and CWA were used. The CWA including FVIII activity was measured using 12 APTT reagents, and the FIX activation based on a small amount of tissue factor assay (sTF/FIX) were examined in reference plasma (RP), EHL-FVIII (Jivi®) and Kovaltry®. The 3 APTT reagents were associated with high variation in the peak time and height in the CWA when analyzing low concentrations of FVIII. The peak time and height could not be measured with one APTT reagent, and there were marked differences in the CWA findings between Jivi® and Kovaltry® among APTT reagents. Several APTT reagents showed a markedly lower FVIII activity with Jivi® than with Kovaltry®. In the FVIII assay, the peak time measured with sTF/FIX did not differ markedly between Jivi® and Kovaltry®; however, the FVIII activity in Jivi® (as measured by the peak height) tended to be higher than in Kovaltry®. The CWA findings for monitoring Jivi® varied for monitoring Jivi® depending on the APTT reagents used, and sTF/FIX assay may be able to measure the EHL-FVIII.

Assay Interference By Emicizumab in Aptt and Chromogenic Based FVIII:C and Inhibitor Assays

Background:Emicizumab, a humanized bispecific antibody, has been approved for the prophylactic treatment of hemophilia A in patients with and without inhibitors. Both activated partial thromboplastin time (APTT) based FVIII:C and inhibitor assays, as well as select chromogenic FVIII:C assays have been reported to be affected in the presence of emicizumab. Aims:To assess the effect of emicizumab spiked at 0, 25, 50, and 100 ug/mL into congenital FVIII deficient plasma, containing FVIII inhibitor at 0, 1 and 5 BU, in seven commonly used APTT and chromogenic based FVIII:C assays. Samples were also tested in an APTT and chromogenic based FVIII Nijmegen inhibitor assay. Methods:Four IVD approved, APTT based FVIII:C assays and three chromogenic FVIII:C assays were evaluated (Table 1). In addition, FVIII inhibitor levels were measured by Nijmegen Bethesda assay using either APTT (Actin®FSL, Siemens Healthcare) or chromogenic based (FVIII Chromogenic Assay, Siemens Healthcare) FVIII:C assays. Results:Reagent and dose-dependent increases in FVIII:C were observed for all APTT reagents tested as well as for the BIOPHEN FVIII:C chromogenic assay. The presence of low or high titer inhibitors did not impact APTT based FVIII:C measured. Neither of the two chromogenic FVIII:C assays, using bovine based FX and FIXa (i.e. FVIII Chromogenic Assay and Coatest®SP4) demonstrated measurable FVIII levels at any of the emicizumab concentrations tested. FVIII Nijmegen Bethesda inhibitor results using APTT based FVIII:C, when tested in the presence of emicizumab, consistently produced false negative results (Table 1) whereas results determined using the FVIII Chromogenic Assay measured inhibitor titers at expected concentrations. Conclusion:Confirming previously published results, emicizumab interferes in the APTT based FVIII:C and Nijmegen Bethesda inhibitor assays. Even at low emicizumab concentrations, false negative inhibitor titers are measured in APTT based inhibitor assays. Using a bovine based chromogenic FVIII:C assay is imperative when measuring both FVIII:C and FVIII inhibitors in samples containing emicizumab. Disclosures Tiefenbacher: Laboratory Corporation of America Holdings:Current Employment, Current equity holder in private company;Novo Nordisk:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Shire:Honoraria;Octapharma:Honoraria;Siemens Healthcare:Membership on an entity's Board of Directors or advisory committees;BioMarin:Membership on an entity's Board of Directors or advisory committees.

The traceability of commercial plasma calibrators to the plasma International Standards for factor VIII and factor IX.

Accurate measurement of coagulation factors is essential, especially for diagnosis of deficiency. Clinical laboratories use commercially available plasma calibrators, which should be traceable to the relevant plasma International Standard (IS). This study assessed the relationship between the plasma IS for factors IX (FIX) and VIII (FVIII) and some commonly used commercial calibrators. Calibrators from seven manufacturers were assayed for FIX and FVIII activity by one‐stage clotting assay (OSCA) using different activated partial thromboplastin time (APTT) reagents and deficient plasmas, or chromogenic assay (CA). Results were calculated relative to the 4th IS Factors II,VII,IX,X, Plasma or the 6th IS Factor VIII/VWF, Plasma. Results for each calibrator were similar across the APTT reagents and deficient plasmas used. All calibrators showed a recovery of 90%‐111% of the manufacturers’ values, except calibrator C, which had recovery of around 85%. CA gave similar results, with good recovery for all but calibrator C. Similar low recoveries for OSCA and CA were found for a different lot of calibrator C and for a different calibrator product from manufacturer C. When all calibrators from manufacturer C were assayed by OSCA using the manufacturer's own deficient plasmas and APTT reagents, the mean recovery was still below 90%. Overall, there was good traceability of the international unit between the IS and commercial calibrator plasmas. Calibrators from one manufacturer consistently yielded lower than expected values for FIX and FVIII. This could lead to an over‐estimation of the coagulation factor content in patient samples and demonstrates the importance of careful choice of calibrator.

Determination of cut-off threshold of normalized ratio for determining lupus anticoagulant in normal plasma of donors – healthy volunteers (large network laboratory study results)

Laboratory tests for lupus anticoagulant (LA) are necessary for antiphospholipid syndrome (APS) diagnosis. Due to antibody heterogeneity, there are two test systems recommended to maximize detection rates: one of them is the dilute Russell’s viper venom time (dRVVT) and the second one is the activated partial thromboplastin time (APTT). Current guidelines recommend getting the reference ranges and cut-off of the normalized screen/confirm ratio in dRVVT and APTT in every laboratory. The aim of this work is establishing of reference ranges and cut-off for the normalized ratio in dRVVT and APTT reagents using two type of sample preparation. The cut-off values of LA1/LA2 (37 °C, 30 minutes), LA1/LA2 (22–25 °C, 30 minutes), Actin FSL/Actin FS (37 °C, 30 minutes) and Actin FSL/Actin FS (22–25 °C, 30 minutes) pairs in the normalized ratio were 1.21, 1.18, 1.07 and 1.08, respectively.

Unexpected, isolated activated partial thromboplastin time prolongation: A practical mini-review.

A common inquiry in coagulation laboratories is how to interpret an unexpected, isolated prolonged activated partial thromboplastin time (APTT). In this context, isolated means together with a normal prothrombin time (PT) and/or normal international normalized ratio (INR). This finding may lead to contact with laboratory doctors for further advice on a diagnostic strategy. Occasionally, the need for a diagnostic algorithm can be subacute, where surgery has to be postponed until an explanation for the isolated, prolonged APTT has been established. Activated partial thromboplastin time as a coagulation test was developed to monitor patients with hemophilia. Different APTT reagents display considerable differences in their sensitivity to deficiencies of coagulation factors. An isolated, prolonged APTT is seen in (a) individuals/patients with lupus anticoagulants, (b) patients in treatment with anticoagulants, mainly heparin, and (c) patients with deficiencies of specific coagulation factors. In this tutorial review, we summarize what may cause an isolated prolonged APTT and we present a simple diagnostic algorithm to differentiate between lupus anticoagulants (common) and factor deficiencies (rare). The identification of an isolated prolonged APTT as well as the underlying cause can be of the utmost importance in ensuring the correct therapeutic follow-up.

An overview of turoctocog alfa pegol (N8‐GP; ESPEROCT®) assay performance: Implications for postadministration monitoring

Factor replacement therapy with factor VIII (FVIII) concentrates is the current standard of care for patients with haemophilia A. Postadministration monitoring of FVIII activity during on‐demand or prophylactic treatment is important, for example to guide a suitable dosing regimen. While the use of two‐stage chromogenic substrate (CS) assays is increasing, activated partial thromboplastin time (APTT)‐based one‐stage clotting (OSC) assays are most commonly used to measure FVIII activity in clinical laboratories. Substantial variations in activity measurements have been observed in association with some OSC assay reagents when assessing extended half‐life FVIII molecules. Certain silica‐based APTT reagents have previously been shown to underestimate FVIII activity with the polyethylene glycol (PEG)‐conjugated product turoctocog alfa pegol (N8‐GP [ESPEROCT®]; Novo Nordisk A/S). As a wide range of assay reagents are used in clinical laboratories worldwide, it is essential to establish which can be used to accurately measure activity with modified FVIII concentrates. Here, we describe the approach taken by Novo Nordisk to determine the suitability and accuracy of assays and reagents to measure FVIII activity in samples that contain N8‐GP. While accurate activity measurements were possible with all tested CS assays and most of the OSC APTT reagents tested, three APTT reagents that contain silica as a contact activator were found to underestimate N8‐GP recovery (APTT‐SP, TriniCLOT™, STA® PTT‐Automate). The data demonstrate the importance of characterizing the accuracy of each FVIII activity assay. Any limitations should be communicated to treating physicians and the clinical laboratories that test samples containing N8‐GP.