Practical-Haemostasis.com



A Practical Guide to Haemostasis


Monitoring:
  1. Factor VIII and Factor IX Concentrates
  2. Emicizumab
  3. FEIBA
  4. rVIIa [NovoSeven]
  5. ALN-AT3SC [Fitusiran]
  6. TFPI Inhibitors


Introduction

Monitoring clotting factor concentrates and novel non-clotting factor therapies can pose problems. Some of there are summarised below:

1. Factor VIII and Factor IX concentrates


A. B-Domain Deleted [BDD] Factor VIII Concentrates.
Discrepancies between the 1-stage FVIII assay and the chromogenic FVIII assay when measuring post-infusion levels of B-Domain Deleted [BDD] FVIII concentrates are well recognised.  This discrepancy can be significantly reduced by the use of a product specific standard and although this is widely used in the UK and in most of Europe, in other parts of the world a product specific standard has not been adopted.  In the EU potency assignment of FVIII concentrates uses a chromogenic FVIII assay whereas in other parts of the world potency is assigned using a 1-stage FVIII assay.

B. Extended Half-life [EHL] Factor VIII Concentrates.
A number of approaches have been taken to extend the half-life of Factor VIII and Factor IX and these include:

Methodology Comments Assays
Fc Fusion Protein 1. The Fc portion of IgG1 is fused to the C2 domain of B-Domain deleted FVIII.   The Fc portion of IgG1 is a ligand for the neonatal Fc receptor - the latter re-cycles IgG and significantly extends its half-life.  All 1-stage Factor VIII methods and
Chromogenic assays provide accurate recovery levels
Site directed pegylation Two approaches have been used:

1. Conjugation of a 60 kDa PEG polymer to a cysteine residue introduced in the rFVIII sequence by directed mutagenesis to replace the lysine at  position 1804.

2. The N-terminus and C-terminus parts of the native B-domain are fused to form a 21 amino-acid sequence separating the light and heavy chain of FVIII. This sequence carries a unique O-glycosylation site that is PEGylated by conjugation of a 40 kDa PEG polymer. Upon thrombin activation, this residual sequence is removed resulting in the formation of a native activated FVIII molecule.


1.  All 1-stage Factor VIII methods and Chromogenic assays provide accurate recovery levels

2. Silica-based 1-stage FVIII methods underestimate the activity.

Chromogenic assays provide accurate recovery levels.
Random pegylation Random multi-site PEGylation of lysine residues with 20 kDa PEG polymers. An average of two PEG
molecules are conjugated per molecule of FVIII with more than 60% of the conjugations occurring within the B-domain which is released upon activation by thrombin to generate a native activated FVIII molecule.
Silica-based 1-stage FVIII methods underestimate the activity.

Chromogenic assays provide accurate recovery levels.


C. Extended Half-life [EHL] Factor IX Concentrates.

A number of approaches have been taken to extend the half-life of Factor IX and these include:

Methodology Comments Assays
Fc Fusion Protein rFIX-Fc fusion protein with Fc fragment of IgG1 Ellagic Acid and Silica-based 1-stage FIX assays provide accurate recovery levels.
Kaolin-based 1-stage FIX assays underestimate levels.

Chromogenic assays provide accurate recovery levels.
Albumin Fusion Protein rFIX-Albumin fusion protein Silica-based 1-stage FIX assays provide accurate recovery
Kaolin-based 1-stage FIX assays and Ellagic-based methods underestimate levels.

Chromogenic assays provide accurate recovery levels.
Site directed pegylation rFIX with site-specific glycoPEGylation with 40kDa PEG Ellagic acid-based 1-stage FIX assays provide accurate recovery
Kaolin-based 1-stage FIX assays methods underestimate levels. Silica-based 1-stage FIX assays methods overestimate levels.

Chromogenic assays provide accurate recovery levels.

 

2. Emicizumab

Emicizumab is a humanised monoclonal IgG4 antibody with a bispecific structure. Emicizumab bridges Factor IXa and Factor X replacing the missing Factor VIII in individuals with Haemophilia A. Emicizumab does not require activation by Thrombin [IIa] and is not affected by antibodies directed against Factor VIII. 

As Emicizumab restores 'Tenase activity', assays or tests that are based on the Intrinsic pathway' of coagulation will demonstrate shortening of results. 

Tests with strong interference from Emicizumab Comments
APTT Shortens clotting time
Bethesda Assay:  Factor VIII inhibitors A bovine-reagent chromogenic-based inhibitor assay  can be used to quantify FVIII inhibitors in patients with Haemophilia A treated with Emicizumab
1-stage APTT-based Factor Assays Strong interference in APTT-based assays including those for FVIII, FIX, FXI, FXII, Protein C and Protein S.   Leads to falsely elevated results. 

A chromogenic bovine FVIII assay can be used to measure FVIII levels.

A modified one-stage FVIII assay, which uses a specific calibrator and controls for Emicizumab is in development for the measurement of Emicizumab in plasma.
APTT-based APCr Assays Strong interference in results.
Activated Clotting Time [ACT] Shortens the ACT and this includes plasma samples containing UFH.  In such cases an anti-Xa based assay using a chromogenic substrate, should be used to monitor heparin therapy.

Due to the long half-life of Emicizumab, the effects on coagulation may be observed for up to 6 months after cessation of the drug.

Tests with no interference from Emicizumab Comments
Thrombin Time  
PT  
1-stage PT-based Assays  
Chromogenic-based factor assays Excluding FVIII assays.  However, a bovine chromogenic FVIII assay can be used to measure FVIII levels.
ELISA and immunological assays  
Latex-particle based assays  

 

Emicizumab can influence the INR but the effects are minimal.

3. FEIBA [FVIII Inhibitor Bypassing Activity]

FEIBA is a plasma derived, activated Prothrombin Complex Concentrate [APCC] used in management of individuals with Haemophilia A or B who have developed an inhibitor [an alloantibody targeting FVIII or FIX].  It is indicated both for the treatment of a bleeding episode and for prophylaxis to prevent bleeding episodes.  FEIBA is also used in the treatment of individuals with acquired inhibitors [autoantibodies] to Factor VIII, IX or XI.

Due to the complex mechanism of action of FEIBA, no direct monitoring is available. Coagulation tests such as the Whole Blood Clotting Time (WBCT), the TEG and the APTT usually show only a slight shortening but do not correlate with the clinical efficacy.
Thrombin generation has been used to monitor FEIBA and the data shows a correlation between in vivo clinical response and Thrombin-generating capacity. Thrombin generation may, therefore, represent a surrogate marker for monitoring FEIBA in clinical situations.

4. rVIIa [NovoSeven]

NovoSeven is recombinant human coagulation Factor VIIa (rFVIIa), and designed to promote haemostasis via the extrinsic pathway of coagulation. 

rVIIa is licensed for the treatment of:
  i. Individuals with Haemophilia A or B and who have developed an inhibitor [an alloantibody targeting FVIII or FIX]
  ii. Individuals who have developed acquired Haemophilia A ie an autoantibody targeting FVIII
  iii. Individuals with congenital Factor VII deficiency
  iv. Individuals with Glanzmann's Thrombasthenia who have developed antibodies to the GPIIb/IIIa complex following treatment with platelets and which makes them refractory to further treatment with platelets.

Thrombin generation analysis and the ROTEM have been used to monitor rVIIa and the response of individuals to treatment.


5. ALN-AT3SC [Fitusiran]

ALN-AT3SC is an investigational RNA interference (RNAi) therapy that specifically targets Antithrombin mRNA encoded by the Antithrombin gene [SERPINC1] in the liver and which suppresses the production of Antithrombin in the liver. 

Initial data suggests that this agent can be monitored by assessing Thrombin Generation in addition to measuring plasma Antithrombin levels.

6. TFPI Inhibition

TFPI is a Kunitz-type serine protease inhibitor that regulates Tissue Factor (TF)-induced coagulation by forming a quaternary complex with the FXa-FVIIa-TF complex in plasma and on endothelial surfaces.
Concizumab is a monoclonal, humanised antibody that targets the second Kunitz domain of TFPI that binds to and inhibits FXa and so abolishes the inhibitory effect of TFPI.
Concizumab has been monitored by Thrombin Generation Thrombin generation and a dose dependent increase in peak Thrombin and Endogenous Thrombin Potential [ETP] was observed with values in the normal range when plasma TFPI levels were nearly undetectable.