Chromogenic Factor Research Articles

A Tale of Two Cases: Acquired Hemophilia A and/or Lupus Anticoagulant?

Abstract Distinguishing between acquired hemophilia A (AHA) and lupus anticoagulant (LA) is essential for effective clinical management. This can be challenging due to similarities in laboratory tests and potential interference between their diagnostic tests. Here we present two complex cases. Case 1 is a 69-year-old female with PNH presenting with a subdural hematoma. Prolonged aPTT with no correction on mixing raised clinical suspicion of AHA. Factor VIII activity was low (2%) across three dilutions in the factor assay. A Bethesda assay revealed a low factor VIII inhibitor (1%). LA was negative by DRVVT. Antiphospholipid antibodies (Cardiolipin and Beta-2-Glycoprotein 1) were also negative. Treatment with prednisone and intravenous immunoglobulin was initiated with presumptive diagnosis of AHA. Subsequent samples to check other factor levels uncovered low Factor IX as well. Higher dilutions in factor assays exhibited non-parallelism in Factor IX and VIII, suggesting a non-specific inhibitor. Chromogenic Factor VIII was within the normal range. Another LA test, Staclot-LA was positive. Overall, this case was consistent with LA rather than AHA. The initial ‘positive’ Bethesda assay was attributed to interference by LA. Case 2 is a 20-year-old female with no significant medical history who was admitted elsewhere for a complicated C-section with intraabdominal hemorrhage. She had prolonged aPTT and positive LA by platelet neutralization test there. Upon transfer, the initial assessment confirmed prolonged aPTT and a time-dependent inhibitor pattern in mixing studies. Factor studies revealed significantly diminished factor VIII activity (<1%) using both one-stage clotting and chromogenic assays. A strong factor VIII inhibitor was identified through chromogenic Bethesda assay (126 BU). Non-parallelism was observed in other factors (Factor IX, XI, XII). LA was negative by DRVVT but positive by Staclot-LA. Cardiolipin and beta2-glycoprotein-1 antibodies were negative. The diagnosis of AHA was evident. But is there concurrent LA present? Both Staclot-LA and platelet neutralization tests are PTT-based and susceptible to interference from factor VIII inhibitor. The patient underwent treatment, and factor VIII inhibitor disappeared in 3 months. Staclot-LA also turned negative, suggesting that the initially ‘positive’ LA test was likely a result of interference. In both cases, adopting a holistic approach to differentiate between AHA and LA is beneficial. Relying solely on individual tests may lead to false interpretations, as LA can inhibit FVIII activity assays and produce false positives in the Bethesda assay. It is advisable to include higher dilutions in factor assays and/or chromogenic assays to mitigate interference. The presence of factor VIII inhibitor can also result in false-positive LA results such as Staclot-LA assay. For more reliable results, DRVVT and antibody tests are preferable. If the aPTT-based assay is the sole positive LA test, the presence of LA is suggested when it occurs before and/or after the presence of a factor inhibitor.

Comparison of one-stage and chromogenic factor VIII assays to tailor the dose of recombinant factor VIII-Fc fusion protein (rFVIIIFc, efmoroctocog alfa) in adult patients with haemophilia A: Single-centre, real-world experience of surgery.

Efmoroctocog alfa (rFVIIIFc) is an extended half-life FVIII used notably in surgery for patients with haemophilia A. More information is needed of its usage in real-life. Adult patients with HA followed at the Lyon Comprehensive Hemophilia Care Center who underwent a surgery with rFVIIIFc were included in this retrospective analysis. The pharmacokinetics of rFVIIIFc was assessed by plasma factor VIII clotting activity (FVIII:C) using both one-stage (OSA) and chromogenic substrate (CSA) assays. A total of 39 major and 31 minor surgeries were performed in 49 patients treated with rFVIIIFc. The median dose of rFVIIIFc infused before major and minor surgeries respectively was 67.5 ((interquartile range [IQR] 52.6-76.9) and 48.0 (38.5-51.8) IU/kg. For major surgeries, during the first postoperative week, the median residual FVIII:C was 78 (64.5-101.5) IU/dL with OSA and 99 (71-118) IU/dL with CSA (p<.0001). After surgery, rFVIIIFc doses were adjusted according to CSA results. This led to a significant decrease of rFVIIIFc consumption compared to what would have been proposed according to the OSA assay, without unusual bleeding or appearance of inhibitor. Considering the high price of the molecule, this was also associated with a significant cost reduction. Dose adjustment of rFVIIIFc according to FVIII: C measured by CSA is effective, safe and well tolerated in patients with haemophilia A undergoing invasive surgery.

A-160 Factor VIII Chromogenic Low Activity Sample Analysis in a Low Calibration Curve Model

Abstract Background Adequate laboratorial diagnostics of hemophilia is essential and could be done by chromogenic and one-stage assays based on APTT methodologies. Adverse effects of medications became less severe recently, but it remains important to be aware of to ensure that treatment is effective and ensure the best possible conditions for affected individuals. Classification of hemophilia according to FVIII activity: mild 25%–5%; moderate, 5%–1% and severe &amp;lt;1%. For new therapies such as emicizumab and some drugs with an extended half-life, the World Federation of Hemophilia recommends the use of a chromogenic assay of FVIII containing bovine FX for monitoring factor FVIII activity. Objetive: evaluate the applicability of a low calibration curve (LCC) in the chromogenic factor VIII (FVIII-Chr) assay by standardizing the dilution of the standard (calibrator) and thus ensuring the accuracy of the analyses in the lower values like severe hemophilia range. Methods The samples were obtained from H&amp;H LAB SAS, which guarantees all requirements. Sysmex® CS 2100i Siemens Healthineers. Calibration was performed with Standard Human Plasma (SHP), together with FVIII-Chr. Quality Control (QC); the accuracy of the LCC was checked. Results Preparation of the LCC: SHP reconstitution, followed by 1:10 dilution between the SHP and the plasma deficient in FVIII. The curve was named FVIII-Chr Low. QC was performed through pathological control (PC) with 2 dilutions: 1) identical to the formation of the low curve, i.e., dilution 1:10 (2.3%–3.9%). 2) dilution 1:30 (0.2%–2%) to accommodate the range of activity comprising severe hemophilia. The values found confirms the curve’s quality. Discussion This study sought to develop a way to detect FVIII activities &amp;lt;1.5% in a reproductive manner, even with a reduced “N” number of samples for severe state. The chromogenic test presents a limit of detection for the optical density of D.O. 0.0080 reporting &amp;lt;0,3% FVIII-Chr activity, which is very close to the most diluted point of the LCC. Conclusion QC performance with PC 1/10 and 1/30 showed excellent reproducibility. With the protocol FVIII Low and LCC (SHP + PDF) it is possible to obtain results of FVIII-Chr &amp;lt;1.0% compatible patients with severe hemophilia clinical condition.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Both one-stage and chromogenic factor VIII assays are required for the diagnosis of mild hemophilia A

Both one-stage and chromogenic factor VIII assays are required for the diagnosis of mild hemophilia A

Treatment changes in hemophilia A with chromogenic factor VIII assay implementation

BackgroundThe one-stage assay (OSA) and the chromogenic assay (CSA) are 2 factor VIII (FVIII) assays used for the diagnosis and classification of hemophilia A. Discrepancies between the 2 assays exist in approximately one-third of patients with mild hemophilia A. ObjectivesThe objectives of this study were to report the proportion of patients with mild or moderate hemophilia A and OSA-CSA discrepancies and to report the observed changes in treatment approach prompted by the presence of assay discrepancy. The study aimed to identify OSA:CSA ratio associated with the highest sensitivity for identification of patients in whom modification of treatment approach may be recommended. MethodsThis is a retrospective cohort study including adult (>18-year-old) patients with mild or moderate hemophilia A who were followed up at the Adult British Columbia Hemophilia Program between January 2013 and March 2019. ResultsA total of 75 patients with mild and 23 with moderate hemophilia A based on baseline OSA were included. Overall, 52% of study patients had OSA-CSA discrepancies, and change in treatment approach was observed in 27% of patients with OSA-CSA discrepancy. The OSA:CSA ratio of 1.8 to 3.5 demonstrated the highest area under the receiver operating characteristics curve and sensitivity for identification of patients in which modification of treatment approach may be recommended (AUC 0.75; sensitivity 71%). ConclusionIn our population, OSA-CSA discrepancy was observed in 52% of patients with mild or moderate hemophilia A, and the treatment approach in 27% of these patients had to be modified.

The dosing conundrum of emicizumab: To waste product or not?

The dosing conundrum of emicizumab: To waste product or not?

Chromogenic Factor VIII Assay for Patients with Hemophilia A and on Emicizumab Therapy.

This chapter will describe a method for measuring endogenous and infused Factor VIII (FVIII) in patients on emicizumab therapy (Hemlibra, Genetec, Inc). Emicizumab is a bispecific monoclonal antibody used in patients with hemophilia A, with or without inhibitors. The mechanism of action for emicizumab is novel and mimics the role that FVIII plays in vivo by binding and bridging FIXa and FX. It is vital that the laboratory understands the effect this drug has on coagulation tests and uses a suitable chromogenic assay which is not affected by emicizumab, for determination of FVIII coagulant activity and inhibitors.

Little discrepancy between one-stage and chromogenic factor VIII (FVIII)/IX assays in a large international cohort of persons with nonsevere hemophilia A and B

BackgroundAccurate measurements of coagulation factor activity form an essential part of hemophilia management and are performed by the one-stage or chromogenic assay. Current literature suggests that approximately one-third of persons with nonsevere hemophilia A exhibit assay discrepancy, albeit with a high variability between studies. Such data are scarce in nonsevere hemophilia B. ObjectivesTo investigate the extent of factor VIII/IX one-stage and chromogenic assay discrepancy in moderate and mild hemophilia A and B. MethodsPersons with previously diagnosed nonsevere hemophilia A and B with a factor level of 2 to 35 IU/dL were included from the international DYNAMO cohort study. Central measurements of the factor VIII and IX activity levels were performed by the one-stage and chromogenic assay. Relative and absolute discrepancy definitions were used, with the International Society on Thrombosis and Haemostasis-Scientific and Standardization Committee proposed ratio of >2.0 or <0.5 being the primary outcome. Discrepancy was also evaluated in a subgroup of 13 persons with mutations previously associated with discrepancy (≥3 cases reported in literature). ResultsA total of 220 persons were included, of whom 3 (1%) showed assay discrepancy: 2/175 hemophilia A and 1/45 hemophilia B. Six persons (3%) exhibited an absolute difference >10 IU/dL between the assay results. In addition, with more lenient definitions, over 90% of participants (n = 197) had no discrepant results. Only 1 out of 13 persons with a mutation previously associated with discrepancy had significant assay discrepancy. ConclusionLittle assay discrepancy was observed despite the presence of mutations previously associated with discrepancy, suggesting that the presence and magnitude of assay discrepancy are largely determined by laboratory variables.

A Difficult Task: Determining Oral Anticoagulation Efficacy (OAC) in Patients with HIT Type II

Since 1937, heparin has remained the anticoagulant of choice for a wide variety of clinical scenarios. There remains a relatively high prevalence of heparin-induced thrombocytopenia (HIT), an immune-mediated syndrome in which antibodies (most frequently IgG) bind to a complex of heparin and platelet factor IV, resulting in platelet destruction and the release of prothrombotic platelet derived microparticles. Serious complications include thrombocytopenia (usually occurring between 6-12 days after initiating therapy) and thrombosis. The risk of thrombosis in patients with HIT is estimated at 30% and mortality rates have been reported as high as 20-30%. In cases of HIT type II when patients require continuous anticoagulation, such as following implantation of mechanical heart valves, alternative anticoagulants such as direct thrombin inhibitors can be used. The difficulty of using drugs such as argatroban for bridging therapy to warfarin is that they have been found to elevate both the aPTT as well as INR. Therefore, is it difficult to assess whether patients will maintain therapeutic INRs when argatroban is held and they are continued on warfarin monotherapy. The standard treatment regimen calls for argatroban to be started until INR reaches some therapeutic level at which time argatroban is held and INR is rechecked 4-6 hours later. The risk of major bleeding associated with supra-therapeutic INR's is substantial, and as such patients should be continued on bridging therapy to the lowest INR level that is still found to be therapeutic once argatroban is held. Previous research has examined the efficacy of transitioning from argatroban to warfarin in patients with HIT type II. This case report reviews the care of a patient following a diagnosis of HIT, the methods used to measure the efficacy of oral anticoagulation (OAC), and the complications that arose with management. The patient is a 51-year-old woman who received a mechanical mitral valve replacement for mitral valve regurgitation. Two weeks later, she returned to the hospital with lower extremity swelling. On admit, the patient's INR was sub-therapeutic at 1.2. Enoxaparin was initiated with plans to bridge to warfarin until the INR reached 2.5-3.5. Within four days of enoxaparin initiation, platelets decreased from 238,000/uL to 114,000/uL. On day four, a new non-occlusive right internal jugular thrombus was found. A serotonin release assay (SRA) and HIT antibody with reflex were ordered. While labs were in process, enoxaparin was held and argatroban initiated. The PTT was checked and found to be 62.6 seconds, and it was determined that the patient was adequately anticoagulated. A 4-T score of 6 indicated a ~64% probability of HIT with the patient requiring indefinite anticoagulation due to the mechanical mitral valve. When the patient's INR reached 2.5 argatroban was held, however the following INR was found to be subtherapeutic at 1.3. Argatroban was restarted with plans to continue argatroban therapy until INR>4, at which time argatroban was to be held and INR rechecked in 4-6 hrs. If the INR remained therapeutic off argatroban then it was determined that the patient was appropriately anticoagulated. When the INR reached 4.9 and argatroban was held, the recheck 5 hours later was 3.1. Using this algorithm, the patient ultimately achieved a therapeutic INR with platelet counts of >300,000/uL at time of discharge without any major bleeding events or other serious complications. For patients in whom HIT is diagnosed, immediate discontinuation of heparin infusions and elimination of heparin from all flushes and ports is a necessity as the first step of treatment. Argatroban may be used as a bridging agent, but OAC efficacy cannot be monitored solely using the parameter of INR due to argatroban's INR-elevating effect. By trending the aPTT to monitor anticoagulation status and only utilizing the INR once argatroban therapy was removed, we ensured that the patient had achieved OAC efficacy prior to discharge. In our patient an INR of 5 did not result in any major bleeding incident; however, by initially discontinuing prematurely at an INR of 2.5, the patient's hospital stay was extended. The chromogenic factor X assay has shown some accuracy in predicting INR free of argatroban influence by measuring the amount of vitamin x, but further studies are warranted to establish an optimal method by which to convert patients from argatroban to warfarin. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Resource Utilization and Cost Analysis of a Chromogenic Factor X Assay for Anticoagulation Monitoring and Management

Introduction High risk venous thrombotic states such as heparin-induced thrombocytopenia (HIT) and antiphospholipid antibody syndrome (APLS) contribute to significant patient morbidity and mortality, and financial toxicity. Traditional coagulation assays (prothrombin time [PT], activated partial thromboplastin time [aPTT], and international normalized ratio [INR]) guide anticoagulation but are unreliable in the presence of a lupus anticoagulant and/or disseminated intravascular coagulation (DIC). Further, some agents used for treatment, such as direct thrombin inhibitors (DTI), elevate the PTT, PT, and INR. Collectively, this highlights the challenge of maintaining a therapeutic index with aberrant PTT values. The chromogenic factor X (CFX) assay depends on an enzymatic reaction that generates photometrically (colorimetric) distinct signals that are proportional to factor X levels and therapeutic INR levels. Rationale for the use of CFX assay is to abrogate difficulty in guiding treatment decisions in HIT with DIC, APLS, and with DTI use. It remains unclear how the use of CFX testing and need for hematology consultation impacts clinical and financial resources at an institutional level seeking to incorporate novel coagulation tests. Here we aim to understand how involving a hematologist in decision-making when using this test can improve healthcare utilization and expenditure associated with thrombotic disease and its complications. Methods This study design was reviewed by the Institutional Review Board and met the criteria for exempt review. This is a single institution retrospective cohort analysis. All patients who had CFX assays performed between July 1, 2019 and June 30, 2021 were included. Each encounter was categorized by location (inpatient vs. outpatient), the indication prompting use of the CFX assay, and if hematology was consulted. Costs were aggregated by evaluating individual decentralized de-identified patient accounts. In particular, variable costs were evaluated to capture all costs from services rendered directly related to the anticoagulation management of the patient (i.e. cost of operations). Descriptive analysis was done for aggregated variable costs and expenditures relevant to the CFX-guided anticoagulation treatment episode. A two-sample t-test was used to identify association between numbers of tests ordered when hematology was consulted in patient care. Statistical analysis to identify differences was set at α=0.05 or less. Maximum type I error rate of p<0.05 was used. Results In total, 179 patient charts were analyzed, of which 90 patients met inclusion criteria (Table 1). The majority of patients had the CFX assay performed inpatient; 33 had the test as an outpatient. A breakdown of testing indication is presented in Table 1, with the majority of tests ordered for APLS, HIT, and recurrent thrombosis. As show in Table 2, for inpatients, fewer tests per patient were ordered when hematology is consulted compared (3.4 vs. 5.4 tests/patient, p=0.005 [α = 0.05]). The mean direct variable cost (DVC) was not statistically different, however there was a median $1800 cost savings when hematology was consulted. There is no difference in length of stay (LOS). All outpatient tests were ordered by a hematologist. In total, 40 tests were ordered for 33 patients (1.2 tests/patient) with a mean DVC of $602.75. Conclusion Our study highlights that there was a statistically significant decrease in tests ordered when a hematologist is involved. For institutions adopting novel coagulation testing, expert hematology consultation helps improve fiscal resource utilization and clinical practice. The discordance of mean and median DVC suggest that variation is not normally distributed. This is often seen in healthcare finance where macro-economic or supply-side factors dictate variation in medically ill patients. Further, the wide ranges in DVC and in LOS suggest a possibly more complex patient population for whom hematology was consulted. Therefore, we highlight that consultation with a hematologist should be considered in complex patients, particularly for anticoagulation given the significant implications inadequate treatment can have. Hematologist can help with anticoagulation management, and their involvement appears to correlate with decreased testing and improved overall savings even with the cost incurred of the consultant. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discrepancy between the Results of One-Stage and Chromogenic Factor VIII Assays: Switch in Diagnosis from Mild to Moderate Hemophilia a in Three Cases

Introduction Factor VIII (FVIII) activity can be measured with different methods including one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Some persons with mild hemophilia A (HA) have a significant discrepancy between the assays which has been previously reported. Discrepancy between these two methods, albeit less frequent, can also lead to a switch in diagnosis from mild to moderate HA. Herein, we report 3 persons with HA (PwHA) whose diagnosis changed from mild to moderate HA after obtaining FVIII levels assessed by CSA. Materials and Methods Data were extracted from the medical records of the PwHA including demographics, bleeding rates, and factor levels. FVIII activity was measured using the OSA on an ACL Top 500 with SynthaSil aPTT reagent (Instrumentation Laboratories Bedford, MA, USA). Factor VIII activity was also determined photometrically via the Chromogenix Coatest® SP4 FVIII chromogenic assay kit (bovine chromogenic FVIII activity, Diapharma Group, West Chester, OH). Results Data were collected from 3 PwHA who were previously diagnosed with mild HA yet were noted to have a bleeding phenotype worse than expected for mild HA (Table-1). Case 1 is 26 years old and had been experiencing multiple joint bleeds. His OSA FVIII level was 6.8%. He was on clotting factor concentrate (CFC) on demand. After having severe hemophilic arthropathy affecting his social life, prophylaxis with CFC was commenced. Significant improvement was observed after the initiation of prophylaxis. His CSA FVIII level was 2.7%, consistent with his bleeding phenotype, and he has been on emicizumab prophylaxis for a year without any bleeding events. Case 2 is 22 years old and had been experiencing frequent muscle bleeds. His OSA FVIII level was 6.1% and CSA FVIII level was 2.7%. He remains on CFC on demand based on his preference despite the frequent bleeding. Case 3 is 15 years old and was diagnosed with mild HA at the age of 8 after recurrent joint bleeds. His OSA FVIII level was 13.8% and CSA FVIII level was 1.5%. He has been on emicizumab prophylaxis for 10 months without any bleeding event. Conclusion While discrepancies between assays in mild hemophilia have been widely reported, there are fewer reports on an assay discrepancy changing a diagnosis from mild to moderate HA. As such, we strongly suggest performing both OSA and CSA for the diagnosis and classification of HA especially if the PwHA has more bleeding episodes than expected. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FACTOR VIII – CHROMOGENIC - LOW ACTIVITY SAMPLE ANALYSIS IN A LOW CALIBRATION CURVE MODEL

Hemophilia is a bleeding disorder that causes affected people to bleed longer and, in severe cases, spontaneously. A genetic defect in the X chromosome causes decreased activity of circulating factors. Adequate lab. diagnostics is essential and could be done by different methodologies, such as the determination of factor activity by chromogenic and one-stage assays based on APTT. Treatment has improved significantly over the past few decades, and state-of-the-art treatment is more efficient, safe, and convenient than ever. Adverse effects of medications became less severe in recently, but it remains important to be aware of to ensure that treatment is effective and ensure the best possible conditions for affected individuals. The classification of hemophilic patients is divided into 3 categories, according to the activity of FVIII: mild 25% - 5%; moderate, 5% - 1% and severe when the activity is < 1% of normal FVIII. New substitutive therapies such as emicizumab and some drugs with an extended half-life, the WFH (World Federation of Hemophilia) recommends the use of a chromogenic assay of FVIII containing bovine FX for monitoring factor FVIII activity. This study aimed to evaluate the applicability of a low calibration curve in the chromogenic factor VIII (FVIII-Chr) assay by standardizing the dilution of the standard (calibrator) and thus ensuring the accuracy of the analyses in the lower values like severe hemophilia range. The samples used in the study were obtained from H&H LAB SAS, which guarantees all requirements. In this study, Sysmex®CS 2100i Siemens Healthineers were used to analyze the samples. FVIII-Chr assay; Standard Human Plasma (SHP); Coagulation FVIII Deficient Plasma; Control Plasma P. Calibration was performed with SHP, together with FVIII-Chr according to the instructions; Internal Quality Control; the accuracy of the calibration curve was checked with appropriate controls. Results: Preparation of the Low Curve: SHP reconstitution according to instructions, followed by 1:10 dilution between the SHP and the plasma deficient in FVIII. The curve was named FVIII-Chr Low. Quality control (QC) was performed through pathological control with 2 dilutions: 1) identical to the formation of the low curve, i.e., dilution 1:10 (2.3 – 3.9%). 2) dilution 1:30 (0.2 - 2%) to accommodate the range of activity comprising severe hemophilia. The values found confirms the curve's quality. This study sought to develop a way to detect FVIII activities < 1.5% in a reproductive manner, even with a reduced “N” number of samples for severe state. The detection of chromogenic methodology suggests a limit of detection for the optical density of D.O. 0.0080: they are below the most diluted point of the calibration curve. Suggestion: to report the values of these cases that can be released as an activity < 0.3% and include the DO. Good performance of QC. Dilution 1/10 and 1/30 is checked; reproducibility in the results can be observed. With the protocol FVIII Low and LCC SHP+PDF it is possible to obtain results of FVIII-Chr < 1.0 % in severe hemophilia patients compatible with the patient's clinical condition in treatment with emicizumab.

Gemological Characteristics and Chemical Composition of a New Type of Black Jadeite and Three Imitations

Because of the increasing price of jadeite, many fake species have appeared on the market. We recognized three pieces of fake black jadeite that had been placed among real black jadeite. In this study we conducted a mineralogical investigation of the three fakes and the real jadeite by using FTIR and XRD techniques; in addition, we performed in situ major, minor and trace element chemical characterization based on EPMA-WDS and LA-ICP-MS techniques. The three imitations have different components, dominated by katophorite (97%), augite (66%) and anorthite (97%). In contrast, the real jadeite sample contains more than 99% jadeite. Unlike previous reports on black jadeite, the dark omphacite exsolution around the jadeite cleavage is the chromogenic factor in the present study, whereas the black color of the imitations comes from light absorption by major melanocratic minerals and widespread fine graphite. We propose that 2–4 sharp bands between 600 and 800 cm−1 of FTIR and the 2.42 and 2.49 Å peaks of XRD can be used to discriminate black jadeite from imitations. Even though natural jadeite deposits are being exhausted, materials of the three natural imitations were determined not to be suitable for jewelry due to low hardness, widespread occurrence and unknown injury of the radioactive elements thorium and uranium. Otherwise, they could enhance value and be ideal for large-sized ornaments of fine design.

Advances in laboratory assessment of thrombosis and hemostasis.

Technologies in laboratory diagnostics are changing fast with progress in understanding and therapy of diseases. Unfortunately, new analyzers are often needed to be installed in a clinical laboratory to implement such techniques. The demand for new hardware is a bottleneck in improving the diagnostic services for many facilities with limited resources. In this regard, hemostasis laboratories take a slightly different position. Because many in vitro diagnostic tests target the functional aspects of hemostasis, further meaningful information can be obtained from the same analyzers as in current use. Automated coagulometers are good candidates for such further utilization. Clot waveform analysis is a leading example. Behind the simple values reported as clotting time, clotting curves exist that represent the process of fibrin clot formation. Clot waveform analysis examines the clotting curves and derives new parameters other than clotting times. The clot waveform parameters are now in active use in assessing the hemostatic potential of hemorrhagic patients. Clinical application of coagulometers can also be widened by modifying the reagent formulation. For example, the chromogenic factor VIII assay with bovine source reagent compositions has recently been introduced for hemophilia A patients on emicizumab prophylaxis. Also, new immunoturbidimetric functional assays for von Willebrand factor have been developed recently. Thus, new clinically relevant information can be mined from the automated coagulometers that are based on old technology.

Von Willebrand factor assays in patients with acquired immune thrombotic thrombocytopenia purpura treated with caplacizumab.

Acquired immune thrombotic thrombocytopenic purpura (iTTP) is a rare disease with a poor prognosis if undiagnosed. It is caused by autoantibody production to the von Willebrand factor (VWF) cleaving protease, A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). Caplacizumab, an immunoglobulin directed to the platelet glycoprotein Ibα receptor of VWF, has been reported to induce quicker resolution of iTTP compared to placebo. The laboratory measurement of VWF activity was significantly reduced in clinical trials of caplacizumab. Several VWF assays are available in the UK and this study investigated whether differences in VWF parameters were present in 11 patients diagnosed with iTTP and treated with daily caplacizumab. Chromogenic factor VIII activity, VWF antigen, collagen binding activity, VWF multimers and six VWF activity assays were measured prior to caplacizumab therapy and on several occasions during treatment. VWF antigen and collagen binding activity levels were normal or borderline normal in all patients. Ultra-large molecular weight multimers were present in all patients following treatment. VWF activity assays were normal or reduced during treatment, but this was reagent and patient dependant. In the unusual scenario of a caplacizumab-treated patient requiring measurement of VWF activity, it is important that laboratories understand how their local reagents perform as results cannot be predicted.

The edoxaban‐M4 metabolite and measurement of edoxaban by chromogenic assays in human plasma

IntroductionEdoxaban is the only anti‐Xa inhibitor metabolized in pharmacologically active moiety that could interfere with chromogenic anti‐Xa assays, especially in case of drug‐drug interactions or physiological disorders. Materials and methodsWe evaluated the contribution of the main metabolite of edoxaban, edoxaban‐M4 (M4), in 79 plasma samples from patients taking edoxaban. The total anti‐Xa activity was evaluated on three different chromogenic factor Xa‐based assays. Results were compared with a validated ultra‐high‐performance liquid chromatography coupled with a tandem mass spectrometry measurement. Edoxaban and its active M4 metabolite have also been spiked separately in normal pooled plasma to assess the sensitivity of chromogenic anti‐Xa assays to both molecules individually. ResultsSpiked edoxaban or M4 provided different slopes of linear regression models between chromogenic and chromatographic measurement (from 0.97 for STA Liquid Anti‐Xa to 1.10 for Biophen Heparin LRT Low with edoxaban and from 0.70 for Biophen DiXaI High to 0.83 for Biophen Heparin LRT High, respectively). A positive correlation is observed between the increase of the ratio M4/edoxaban with the difference between chromogenic and chromatographic measurements. ConclusionEdoxaban and M4 do not similarly impact chromogenic assays, leading to biased chromogenic estimations of ponderal concentrations. In patient samples, this impact is even more important at low concentrations or in the case of an increase in the M4/edoxaban ratio because of hepatic or renal impairments or in case of drug interactions. This study highlights the limitations and risks of error of expressing results in ponderal concentrations instead of global activity anti‐Xa.

External quality assessment for one-stage and chromogenic factor IX assays in samples containing Alprolix (rFIXFc) or Idelvion (rIX-FP) and evidence that UK National External Quality Assessment Scheme for blood coagulation samples are commutable with patient samples.

There may be clinically relevant differences between results of different FIX assays in samples containing extended half life FIX concentrates requiring regular surveillance of assay results through proficiency testing exercises. Control materials used in proficiency testing must be commutable, that is have the same inter-assay properties as those demonstrated by authentic clinical samples when measured by different analytical methods. We assessed relationships between results with different FIX assays and commutability of UK National External Quality Assessment Scheme (NEQAS) materials containing rIX-FP (Idelvion) or rFIXFc (Alprolix) by comparing results obtained using widely used one-stage and chromogenic assays during a proficiency testing exercise with results obtained when analysing a series of individual patient samples using the same assay systems. NEQAS samples prepared by addition of either Idelvion or Alprolix to FIX deficient plasma were sent to 76haemophilia centres in Europe. A total of 18 Idelvion and 22 Alprolix patient samples were assayed in a single centre. Two chromogenic and two one-stage assays were compared. The pattern of results obtained for NEQAS samples and patient samples was similar. In all cases, the NEQAS sample data point was within the scatter of patient sample data in plots of patient sample results showing one-stage assay results using Synthasil or Actin FS plotted against chromogenic assay results with Biophen or Rox chromogenic FIX kits. This indicates that the NEQAS samples containing Idelvion or Alprolix were commutable and therefore suitable for use in proficiency testing exercises.

Genetic analysis of non-severe hemophilia A phenotype with A discrepancy between one-stage and chromogenic factor VIII activity assays

IntroductionThe diagnosis of hemophilia A (HA) is based on the measurement of factor VIII activity (VIII:C). About one-third of non-severe HA patients show a discrepancy of VIII:C measured by one-stage (VIII:C 1st) and chromogenic (VIII:C chr) assays. Different mutations in the F8 gene may cause the discrepancy in results of the FVIII activity assay.The aim of this study was to investigate F8 gene mutations in patients with assay discrepancies and to evaluate their impact on the results of VIII:C assays. MethodsMutation analysis was performed on 41 individuals with a discrepancy in VIII:C 1st and FVIII: C chr assays by direct sequencing. In addition, the effect of the variants on FVIII macromolecule structure was investigated by in silico and bioinformatics tools. ResultsGenetic analysis disclosed 22 different variants, of which 19 were identified for the first time to be involved in the phenotype of VIII:C discrepancy. Most of the variants related to the higher VIII:C 1st were found in A1, A2, A3 domains. The variant related to VIII:C chr > VIII:C 1st was located in the thrombin cleavage site. In silico analysis showed the effect of variants on FVIII macromolecule stability, which may be the possible mechanism causing the discrepancy. ConclusionOur data shed light on the impact of genetic defects on VIII:C assay and provided evidence that the consideration of these mutations may open a new window to the proper diagnosis and treatment monitoring of non-severe HA patients.

Evaluation of the effect of apixaban using a viscoelastic coagulation assay with Russell\u2019s viper venom reagent

BackgroundConventional coagulation tests, such as prothrombin time and activated partial thromboplastin time, are not sensitive to anticoagulation by apixaban. We evaluated the antithrombotic effect of apixaban using a Russell viper venom (RVV) test for a patient who underwent posterior spine fusion surgery.Case presentationAn 84-year-old man was scheduled for percutaneous posterior spine fusion. He continued apixaban until the night before surgery and resumed it on the first day after surgery. We performed an RVV test as point-of-care coagulation monitoring in combination with chromogenic anti-activated factor X (anti-Xa) activity, prothrombin time, and activated partial thromboplastin time. Clotting time with the RVV test was prolonged according to the anti-Xa activity of apixaban, which was in the therapeutic range during surgery.ConclusionsAn RVV test might be useful as a point-of-care assay for estimation of the anti-Xa level induced by apixaban during the perioperative period.