Chromogenic Assay Research Articles

The Efficacy of Andexanet Alfa for the Reversal of Factor Xa Inhibitors Is Not Influenced by Hemodilution with Different Volume Expanders

Background: Andexanet alfa is a specific antidote for factor Xa (FXa) inhibitors. It is licensed to treat patients under FXa inhibitor therapy with life-threatening bleeding. Concomitantly, volume expanders are used to compensate for blood loss and maintain circulation. The competitive binding of andexanet to FXa inhibitors may be disrupted due to hemodilution, as shown by laboratory assays with high sample dilution. This study investigated the efficacy of andexanet for the reversal of FXa inhibitors under hemodilution. Methods: Blood from 10 healthy volunteers was anticoagulated with rivaroxaban and subsequently treated with four different volume expanders (Ringer’s solution, 4% gelatine, 5% and 20% human albumin (HA)) at two dilution levels (20% and 50%). After anticoagulation and hemodilution, andexanet was added according to the high-dose protocol. Blood samples were analyzed using a Russell’s viper venom (RVV) test on a Clot Pro® device, a thrombin generation assay, a fully automated coagulation analyzer and a chromogenic anti-FXa activity assay. Results: After anticoagulation, the median rivaroxaban concentration was 272 ng/mL (IQR 254–353). Anticoagulation with rivaroxaban caused a significant impairment of all coagulation parameters, which was further aggravated by hemodilution. After the administration of andexanet, coagulation parameters in anticoagulated samples were reversed to near baseline in all groups. Andexanet administration decreased the rivaroxaban plasma concentration in all groups to a median of <10 ng/mL. In the anticoagulated, non-hemodiluted samples, anti-FXa activity was reduced by 98%. The anti-FXa activity in the anticoagulated, hemodiluted samples was reduced by approximately 96% in the 20% diluted samples and by about 93% in the 50% diluted samples. Conclusions: Our data indicate that FXa inhibitor reversal with andexanet is about 5% less effective with 50% hemodilution than in non-hemodiluted samples.

Detection of Alternative Lengthening of Telomeres via Chromogenic in situ Hybridization (ALT-CISH) for the Prognostication of PanNETs and Other Neoplasms

Molecular studies have shown ALT to be an important prognostic biomarker of shorter relapse-free survival (RFS) for patients with pancreatic neuroendocrine tumors (PanNETs) and other neoplasms. However, the preferred method of detecting ALT in tissue is by fluorescence in situ hybridization (FISH), which has several clinical limitations. These issues necessitate the creation of a chromogenic ALT assay that can be easily implemented into routine practice. A CISH assay was developed using genetically modified osteosarcoma cell lines, 20 normal pancreata, 20 ALT-positive PanNETs, and 20 ALT-negative PanNETs. Thereafter, it was validated on a multi-institutional cohort of 360 surgically resected PanNETs and correlated with multiple clinicopathologic features, RFS, and FISH results. Separately, 109 leiomyosarcomas (LMS) were evaluated by both CISH and FISH, and, similarly, the prognostic significance of ALT status was assessed. Upon optimization, ALT-CISH was identified in 112 of 360 (31%) primary PanNETs and was 100% concordant with FISH testing. ALT correlated with several adverse prognostic findings and distant metastasis (all p<0.004). The 5-year RFS for patients with ALT-positive PanNETs was 35% as compared to 94% for ALT-negative PanNETs. By multivariate analysis, ALT was an independent prognostic factor for shorter RFS. Similarly, ALT was associated with shorter RFS in LMS patients and, analogous to PanNETs, a negative, independent prognostic factor. ALT-CISH was developed and validated in not only PanNETs, but also sarcomas, specifically LMS. CISH testing has multiple advantages over FISH that facilitate its widespread clinical use in the detection of ALT and prognostication of patients with diverse neoplasms.

Comparing One Stage, Chromogenic Assay Results and Discrepancies with Bleeding Phenotype and Genetic Variants in Females with Hemophilia A

Comparing One Stage, Chromogenic Assay Results and Discrepancies with Bleeding Phenotype and Genetic Variants in Females with Hemophilia A

Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

There are currently no effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). However, our research on Prominin-1 (Prom1), a known structural protein in photoreceptors (PRs), has revealed its distinct role in RPE and offers promising insights. While pathogenic Prom1 mutations have been linked to macular diseases with RPE atrophy, the broader physiological impact of dysfunctional Prom1 in RPE loss is unclear. We have shown that Prom1 plays a crucial role in regulating autophagy and cellular homeostasis in human and mouse RPE (mRPE) cells in vitro. Nevertheless, a comprehensive understanding of its in vivo expression and function in mRPE remains to be elucidated. To characterize Prom1 expression in RPE in situ, we used RNAscope assays and immunogold electron microscopy (EM). Our use of chromogenic and fluorescent RNAscope assays in albino and C57BL/6J mouse retinal sections has revealed Prom1 mRNA expression in perinuclear regions in mRPE in situ. Immunogold EM imaging showed Prom1 expression in RPE cytoplasm and mitochondria. To confirm Prom1 expression in RPE, we interrogated human RPE single-cell RNA-sequencing datasets using an online resource, Spectacle. Our analysis showed Prom1 expression in human RPE. To investigate Prom1's function in RPE homeostasis, we performed RPE-specific Prom1 knockdown (KD) using subretinal injections of AAV2/1.CMV.saCas9.U6.Prom1gRNA in male and female mice. Our data show that RPE-specific Prom1-KD in vivo resulted in abnormal RPE morphology, subretinal fluid accumulation, and secondary PR loss. These changes were associated with patchy RPE cell death and reduced a-wave amplitude, indicating retinal degeneration. Our findings underscore the central role of Prom1 in cell-autonomous mRPE homeostasis. The implications of Prom1-KD causing aAMD-like RPE defects and retinal degeneration in a mouse model are significant and could lead to novel treatments for aAMD.

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 (&amp;lt;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.

Endotoxin Detection in Magnetic Resonance Imaging Contrast Agent Using Optimising Chromogenic Limulus Amebocyte Lysate Assay.

Endotoxin contamination in magnetic resonance imaging (MRI) contrast agents can pose a risk to patient safety causing immune reactions. Strict endotoxin limits are enforced for implants and catheters inserted into the body, but there are not clear rules for MRI contrast agents. Here, we investigated the efficacy of chromogenic LAL assay test for screening endotoxin activity in MRI contrast media manufactured in Malaysia. The powdered agent was dissolved in water for injection and endotoxin levels were measured. The coefficient of efficiency value for the standard curve, exhibiting r 2 ≥ 0.98, along with the absence of interfering substances and endotoxin activity below the regulatory threshold of 0.5 EU/mL, support the conclusion that the agent is unlikely to be pyrogenic or induce pyrogenic effect.

A-171 Novel Fluorometric Assay for Pediatric Anti-Factor Xa Testing: Minimizing Bilirubin and Hemolysis Interference in Whole Blood

Abstract Background High bilirubin levels are common in pediatric patients and are noted in patients undergoing extracorporeal membrane oxygenation (ECMO). Acute anticoagulation therapy necessitates frequent monitoring of anti-Factor Xa activity (aFXa), and high bilirubin levels cause interference in chromogenic assays. Moreover, large volumes of blood lead to iatrogenic anemia in pediatric and neonatal patients. We developed a fluorescence assay to measure aFXa activity on a near-patient digital microfluidic (DMF) platform using low volume whole blood samples (&amp;lt; 50 μL) and present the results of interference of bilirubin and hemolysis on aFXa activity. Methods aFXa assay was performed on a DMF cartridge wherein 50 μL whole blood was loaded into the cartridge which separates plasma droplets through agglutination within the cartridge followed by incubation with a droplet containing exogenous FXa and a fluorogenic substrate. The fluorescence is inversely proportional to the concentration of heparin in the sample. All required reagents for the aFXa assay are dried within the cartridge, allowing for integrated sample preparation and assay automation for point of care use. To test the interference of bilirubin, we spiked into whole blood different concentrations of unconjugated bilirubin, ranging from 0-40 mg/dL. We also tested hemoglobin interference by testing 4 different concentrations of hemolysate (0-1,000 mg/dL) on the aFXa assay. All experiments were performed with 6 replicates of each concentration. Results Across each bilirubin concentration, we saw uniform measurements of aFXa activity (see Table). We observed &amp;lt; 5% CV in aFXa activity with 0, 13.33 and 26.67 mg/dL bilirubin levels. All measured unfractionated heparin values for hemolysate interference were &amp;lt;5% CV. Conclusions Our interference results suggest that high bilirubin and hemolysate levels do not interfere with our novel aFXa fluorescence assay. Further clinical studies are underway to establish clinical performance.

A-294 Exocytosis Inhibitor Increases yield of Limulus Amebocyte Lysate (LAL), Used for adjunct diagnosis of Invasive Fungal infection

Abstract Background The limulus amebocyte lysate (LAL) was widely used in endotoxin detection in vitro and the clinical diagnosis of invasive fungal infection. The previous data showed that a caffeine collection solution could prevent degranulation during the blood collection procedure, and caffeine-derived LAL could work in both the chromogenic test and turbidimetric method assays. However, the cell pellets collected by caffeine collection solution aggregates after re-suspension in 5 mM CaCl2. No similar phenomenon was observed with PBS collection buffer. The purpose of this study is to set up a PBS-caffeine bleed solution to improve the yield of LAL Methods During the 2023 bleed season, experiments were carried out to test the results of PBS-caffeine solution on the degranulation during bleed process. 6 crabs were bled once a week for a total of 60 crabs by the PBS-caffeine collection solution. The blood of each crab was collected by PBS caffeine collection solution. The cell pellets were finally re-suspended into 5 mM CaCl2 to promote degranulation. The LAL activity was tested by both chromogenic and turbidimetric assay methods. Results The data demonstrates that PBS-caffeine collection solution could prevent degranulation for over 4 hours, and the activity yield of PBS-caffeine-derived LAL was higher. There was no discernable impact on the activity yield of the crab’s size during the bleed season. PBS-caffeine-collection solution could reduce amebocyte aggregation/clot formation during processing. Studies described the biochemical characteristics of PBS-caffeine-derived LAL. Moreover, PBS-caffeine-derived LAL could work in both the chromogenic and turbidimetric assays. Conclusions First, PBS-caffeine collection solutions prevent amebocyte degranulation during blood collection and processing. Next, a PBS-caffeine collection solution could reduce cell aggregation/clotting during processing, and the activity of PBS-caffeine LAL is higher. Finally, PBS LAL is functional in both the chromogenic test and turbidimetric assays

A-274 The Yield of the Limulus Amebocyte Lysate (LAL), Which is Used for Endotoxin Detection and Fungal Diagnosis is Increased by Caffeine Collection Buffer

Abstract Background Limulus Amebocyte Lysate (LAL), which is a major component of Fungal infection diagnosis kit, is used for the adjunct diagnosis of invasive fungal infection. This study used different blood collection buffers to find the optimal blood collection buffer to maximize the yield of LAL. The PBS collection buffer was found to prevent degranulation during blood collection procedure and the PBS-derived Limulus Amebocyte Lysate (LAL) was observed to work in both the chromogenic test and the turbidimetric method assays. A similar phenomenon was observed with the caffeine collection buffer. Methods To further confirm this observation, we used a caffeine collection buffer to collect the blood of horseshoe crabs in the 2023 bleeding season (from July to October). Once a week, 3 crabs were bled for a total of 30 crabs. The blood of each crab was collected using the caffeine collection buffer and the 3% NaCl collection buffer, respectively. The cell pellets were resuspended into LAL reagent water (LRW) or 5 mM CaCl2. The final LAL activity was tested by both chromogenic and turbidimetric assay methods. Results The results showed that a caffeine collection buffer could prevent degranulation for over 1 hr and the activity yield of caffeine-derived LAL is much higher than that of the 3% NaCl solution. There was no discernible impact upon activity yield of crab size, period of the bleed season, or the vendors. Interestingly, caffeine-derived LAL was observed to work in both the chromogenic and turbidimetric assays. The enzyme characteristic of the LAL was also described. Conclusions Caffeine collection buffer prevents amebocyte degranulation during blood collection and processing. The activity of caffeine LAL is much higher than that of 3% NaCl. Caffeine LAL works in both chromogenic test and turbidimetric assay.

Heparin-Bead Extraction Enhanced Fluorogenic Aptamer-Thrombin Composite Reporter Enables Sensitive and Rapid Detection of Functional Antithrombin.

Antithrombin (AT) deficiency in the extracorporeal circulation during cardiac surgery leads to uncontrolled inflammation and vascular damage in patients. AT levels decrease in sepsis, major trauma, extracorporeal membrane oxygenation, and eclampsia. Monitoring plasma AT levels facilitates the accurate restoration of AT to baseline values through precise supplementation. Traditional methods of chromogenic assay and enzyme-linked immunosorbent assay (ELISA) kits encounter challenges, such as interference, inconsistency, and delayed response times, making real-time, reliable antithrombin monitoring a clinical gap. To address this critical need, we develop a heparin-bead extraction enhanced fluoroGenic aptamer-thrombin composite reporter (HExGATOR) for the rapid, sensitive, and precise detection of functional AT in plasma. Our design employs thrombin-binding aptamers and a fluorescence "turn on" technology such that a signal is produced upon the interaction of AT with the otherwise "turned off" aptamer-thrombin complex. The prominent signal-background interference originating from plasma is remarkably diminished by using a heparin-bead solid-phase extraction of AT. We achieved highly sensitive and rapid detection of AT in 5 to 20 min with a limit of detection of 15.11 nM. This approach offers a promising alternative to traditional AT tests in clinical settings, potentially facilitating personalized anticoagulant therapy.

Plasma adenosine deaminase-1 and -2 activities are lower at birth in Papua New Guinea than in The Gambia but converge over the first weeks of life.

Dynamic cellular and molecular adaptations in early life significantly impact health and disease. Upon birth, newborns are immediately challenged by their environment, placing urgent demands on the infant immune system. Adenosine deaminases (ADAs) are enzymatic immune modulators present in two isoforms - ADA-1 and ADA-2. Infants exhibit low ADA activity, resulting in high plasma adenosine concentrations and a consequent anti-inflammatory/anti-Th1 bias. While longitudinal studies of plasma ADA have been conducted in infants in The Gambia (GAM), little is known regarding ADA trajectories in other parts of the world. Herein, we characterized plasma ADA activity in an infant cohort in Papua New Guinea (PNG; n=83) and compared to ontogeny of ADA activity in a larger cohort in GAM (n=646). Heparinized peripheral blood samples were collected at day of life (DOL) 0, DOL7, DOL30, and DOL128. Plasma ADA-1, ADA-2, and total ADA activities were measured by chromogenic assay. Compared to GAM infants, PNG infants had significantly lower ADA-1 (0.9-fold), ADA-2 (0.42-fold), and total ADA (0.84-fold) activities at birth which converged by DOL30. Overall, discovery of a distinct baseline and a consistent pattern of increasing plasma ADA activity in early life in two genetically and geographically distinct populations validates and extends previous findings on the robustness of early life immune ontogeny.

Newly diagnosed multiple myeloma with bleeding and coagulation abnormalities caused by a thrombin-inhibiting substance.

Approximately 7% of patients with newly diagnosed multiple myeloma (MM) experience bleeding complications with varying causes, but few reports have described these complications. Here we report the case of a patient with newly diagnosed MM who presented with a bleeding tendency and various coagulation abnormalities. Chromogenic assays, thrombin time, and reptilase time revealed the presence of a thrombin-inhibiting substance that inhibited release of fibrinopeptide A from fibrinogen. The coagulation abnormalities improved after treatment with daratumumab, lenalidomide, and dexamethasone. As the thrombin inhibition mechanism remains unclear, no previous studies have reported recent treatment outcomes in older patients producing thrombin-inhibiting substances, which can hinder clinical treatment. Therefore, we believe that the diagnosis and the treatment course of this case provide valuable information. Moreover, such case reports provide significant insights into the pathophysiology of bleeding complications associated with MM.

Assays for Monitoring Apixaban and Rivaroxaban in Emergency Settings, State-of-the-Art Routine Analysis, and Volumetric Absorptive Microsamples Deliver Discordant Results.

Our aim was to compare the performance of complementary clinical laboratory approaches to monitoring exposure to apixaban and rivaroxaban, the most prescribed direct-acting oral anticoagulants (DOAC's): an automated commercial anti-Xa chromogenic assay suitable for emergency and pre-surgery testing and a laboratory-developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employed for non-emergency analysis in plasma and in dried blood volumetric absorptive microsamples (VAMS) collectible by the patients in their homes. The full validation of the LC-MS/MS method was performed. Cross-validation of the methodologies was accomplished by processing 60 specimens collected for whole blood count and DOAC monitoring in a central clinical laboratory. For VAMS samples, dried plasma and whole blood calibrators were found to be suitable, and a cycle run for seven days could be implemented for rational and economic sample processing. The anti-Xa chromogrenic assay and the LC-MS/MS method delivered discordant plasma analyte concentrations. Moreover, the lack of agreement between plasma and VAMS concentrations was observed. Clinical laboratories must be aware of the differences between the performance of apixaban and rivaroxaban LC-MS/MS and anti-Xa assays. Hematocrit must always be measured along with VAMS samples to obtain accurate results.

Evaluation of the applicability of a chromogenic assay of changes in antithrombin III activity to monitor the accuracy of thermal effects on blood plasma in thawing and warming systems

Evaluation of the applicability of a chromogenic assay of changes in antithrombin III activity to monitor the accuracy of thermal effects on blood plasma in thawing and warming systems

Coagulation factors VIII and factors IX testing practices in China: Results of the 5-year external quality assessment program

Background and aimsTesting for coagulation factors VIII (FVIII) and IX (FIX) plays significant importance in the diagnostic and treatment of hemophilia A and B. External quality assessment (EQA) scheme aimed to assess the participants’ performance of testing for coagulation factors and identify shortcomings in clinical practice. This study aimed to investigate the performance trends of the participating laboratories in China national external quality assessment Scheme (China NEQAS) for FVIII and FIX over a five-year period (2019–2023). Materials and methodsA total of ten external quality assessment (EQA) rounds were conducted from 2019 to 2023 in the China NEQAS for FVIII and FIX. The distribution of method, reagent and instrument were calculated. The trends of method- specific inter-laboratory coefficient of variation (CV) and pass rates were analyzed over 5 years. The dilutions for coagulation factor testing were also investigated. ResultsAll laboratories use one-stage assays to detect FVIII and FIX activity. The inter-laboratory overall CV decreased year by year (10.9 % to 9.3 % for FVIII and 13.5 % to 10.2 % for FIX), and the laboratory pass rate steadily increased (88.0 % to 93.4 % for FVIII and 81.3 % to 92.7 % for FIX). The majority of laboratories employed a single dilution methodology for the assessment of FVIII and FIX activity. The interlaboratory CV was elevated for the Siemens reagent (Actin FSL) during analysis of moderately abnormal FIX concentrations of EQA samples in most batches. ConclusionsThe implementation of the external quality assessment has contributed to facilitate the enhancement of testing quality. Chromogenic assay is a supplement to accurate determination when necessary. Laboratories may choose to perform dilution tests or direct assays to identify the presence of inhibitors, particularly when they are suspected.

Dried plasma spot as an innovative approach to therapeutic drug monitoring of apixaban: Development and validation of a novel liquid chromatography-tandem mass spectrometry method.

Apixaban, a direct oral anticoagulant drug (DOAC), typically does not require routine therapeutic drug monitoring (TDM), yet recent guidelines propose its use in specific clinical scenarios. While various antifactor Xa (anti-FXa) chromogenic assays serve as useful proxies for measuring plasma exposure to apixaban in emergencies, they lack specificity compared with chromatographic methods. This research project is intended to the development and validation of a standardized protocol of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conformity with the ICH guidelines M10 for the measurement of apixaban in both plasma and dried plasma spots (DPSs). Samples preparation included protein precipitation after the addition of a deuterated internal standard (IS), and the chromatographic separation was carried out on a Thermo Scientific™ Accucore™ Polar Premium column (50 mm × 2.1mm, i.d. 2.6 m). The newly developed LC-MS/MS method for apixaban mesurement from both plasma and DPS resulted linear over a wideconcentration range (31.25-500 ng/mL), accurate, and reproducible without matrix effects, allowing for specific and rapid quantification. Stability was assessed on quality controls and a real sample, allowing the setting up of a robust TDM protocol that was applied to five anonymized plasma samples obtained from adult patients undergoing apixaban treatment at steady-state. In conclusion our novel LC-MS/MS method is adequate for accurate apixaban quantitation from both plasma and DPS matrixes, andmay thus facilitate the guidelines suggested implementation of apixaban TDM, even in peripheral hospitals through shipment of DPS at reference laboratories.

Pharmacokinetics of Efmoroctocog alfa by Two-Compartment Model Highlights Hemophilia A Patients with Biphasic Decay, Long Mean Residence Time, and Beta Half-Life.

Background/Objectives: A compartmental pharmacokinetics (PK) analysis of new extended half-life FVIII concentrates has never been performed in a large cohort of hemophilia patients. An improved PK analysis of individual outcomes may help to tailor hemophilia replacement treatment. Methods: PK outcomes after the infusion of a standard single dose of Efmoroctocog alfa were collected from 173 patients with severe/moderately severe hemophilia A in 11 Italian hemophilia centers. Factor VIII clotting activity (FVIII:C) was measured by one-stage clotting assay (OSA) in all patients, and chromogenic substrate assay (CSA) in a subgroup (n = 52). Fifty patients underwent a comparative PK assessment with standard half-life (SHL) recombinant FVIII (rFVIII) products. Non-compartmental analysis (NCA), one compartment model (OCM), and TCM were used to analyze the decay curves of all patients, and one-way paired ANOVA to compare the PK outcomes. Results: All 173 PKs conformed to the NCA and OCM, but only 106 (61%) conformed to the TCM based on the biphasic features of their decay curves. According to the TCM, the Beta HL and MRT of rFVIIIFc were 20.42 ± 7.73 and 25.64 ± 7.61 h, respectively. ANOVA analysis of the outcomes from the three PK models showed significant differences in clearance, half-life (HL), and mean residence time (MRT) (p < 0.001 for all parameters). As anticipated, the HL and MRT of rFVIIIFc were longer than those of SHL rFVIII. Comparing OSA with CSA outcomes, Cmax resulted higher when measured by CSA (p = 0.05) and, according to TCM, Beta HL resulted longer when measured by OSA (p = 0.03). FVIII:C trough levels obtained with SHL concentrates were significantly lower than those obtained with rFVIIIFc at each post-infusion time point. Conclusions: In a large group of hemophilia A (HA) patients, three different PK models confirmed the improved pharmacokinetic (PK) characteristics of rFVIIIFc, compared with standard half-life rFVIII concentrates. The TCM only fits two-thirds of the PKs, highlighting their biphasic decay and a long Beta half-life. In these patients, the TCM would be preferable to properly evaluate individual PK features.

Comprehensive laboratory assessment of lonoctocog alfa versus octocog alfa in severe haemophilia A.

Lonoctocog alfa is a single-chain factor VIII (FVIII) molecule with high binding affinity to von-Willebrand-factor. While it is well known that its plasma activity is underestimated by one-stage clotting assays (OSCA), there is a lack of knowledge on the post-infusion performance of lonoctocog alfa in global coagulation assays or its potential impact on the haemostatic balance in vivo. To characterize lonoctocog alfa versus octocog alfa in pre- and post-infusion samples obtained from patients undergoing repeated investigation of incremental recovery (IR). Eighteen patients with severe haemophilia A (lonoctocog alfa: 10, octocog alfa: 8) were included. A panel of factor-specific and global coagulation assays was applied, comprising a FVIII OSCA, two FVIII chromogenic substrate assays (CSA), rotational thrombelastography and thrombin generation (TG). Potential activation of coagulation was assessed by measuring plasma thrombin markers and levels of activated protein C. Comparable IRs were found for lonoctocog alfa and octocog alfa (2.36 [IU/dL]/[IU/kg] vs. 2.55 [IU/dL]/[IU/kg], respectively). Lonoctocog alfa activities were found to be underestimated by the FVIII OSCA while also the two FVIII CSAs showed statistically significant assay discrepancies on lonoctocog alfa. Effects of both FVIII products on rotational thrombelastography were less distinct than those on TG parameters. No elevated pre- or significantly shifting post-infusion plasma levels of coagulation biomarkers were detected. Lonoctocog alfa and octocog alfa showed comparable recovery and safety in vivo as well as similar impacts on TG in vitro. Observed assay discrepancies on lonoctocog alfa demonstrated variability of results also between different FVIII CSAs.

Damoctocog Alfa Pegol, a PEGylated B-domain Deleted Recombinant Extended Half-life Factor VIII for the Treatment of Hemophilia A: A Product Review.

Damoctocog alfa pegol (BAY 94-9027, Jivi®), is a site-specifically PEGylated, extended half-life recombinant factor VIII (FVIII) that is approved in several European and non-European countries for on-demand treatment and prophylaxis of bleeding in previously treated patients aged ≥ 12 years with hemophilia A. Reliable measurements can be obtained using most one-stage and chromogenic FVIII assays over a wide concentration range. The efficacy, safety and pharmacokinetics (PK) of damoctocog alfa pegol have been studied extensively in the PROTECT VIII clinical trials, and its long-term safety and effectiveness profile is continuing to build through observational and interventional real-world studies. The PK of damoctocog alfa pegol was shown to be improved as compared with that of sucrose-formulated rFVIII (rFVIII-FS, Kogenate®), and was also demonstrated to be non-inferior to and, for some variables, more favorable than rFVIII-Fc fusion protein, efmoroctocog alfa (Elocta®; NCT03364998), rurioctocog alfa pegol (BAX 855, Adynovate®/Adynovi®; NCT04015492), and antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM, Advate®; NCT02483208). Damoctocog alfa pegol was generally well tolerated and none of the patients in any of the clinical trials, including the PROTECT VIII clinical program, HEM-POWR, or ongoing single-center studies, developed FVIII inhibitors. Efficacy for perioperative hemostasis has been demonstrated. Low bleeding rates were achieved across the studies, with twice weekly, every 5-day and every 7-day prophylaxis offering patients ≥ 12 years and their clinicians the chance to tailor treatment to individual needs and lifestyles, while maintaining long-term protection from bleeds and their consequences.

An Inhibitory Single-Domain Antibody against Protein Z-Dependent Protease Inhibitor Promotes Thrombin Generation in Severe Hemophilia A and FXI Deficiency.

Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia. To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity. We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay in normal plasma, factor VIII- and FXI-deficient plasma. Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency. ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders.