Anti-Xa

Overview of Anti-Xa Assays

Definition: Anti-Xa assays are laboratory tests used to measure the activity of anticoagulant drugs that inhibit Factor Xa (FXa), a key enzyme in the coagulation cascade
Purpose
- Monitoring Low-Molecular-Weight Heparin (LMWH) Therapy: Used to monitor LMWH therapy, especially in patients with:
  - Renal insufficiency
  - Obesity
  - Pregnancy
  - Children
  - Other conditions where standard dosing may not be reliable
- Monitoring Unfractionated Heparin (UFH) Therapy: May be used as an alternative to the aPTT in certain situations
- Monitoring Direct Oral Anticoagulants (DOACs): Used to measure the plasma concentration of direct oral anticoagulants (DOACs) that inhibit Factor Xa (e.g., rivaroxaban, apixaban, edoxaban) in specific situations
- Assessing Heparin Contamination: Used to detect heparin contamination in blood samples from indwelling catheters
Principle: Anti-Xa assays measure the ability of an anticoagulant drug to inhibit Factor Xa activity in a plasma sample

Heparin (UFH and LMWH)
- Mechanism: Enhances the activity of antithrombin, a natural anticoagulant
- Antithrombin inhibits several coagulation factors, including Factors IIa (thrombin), IXa, and Xa
- LMWHs have a greater effect on Factor Xa inhibition compared to UFH
Fondaparinux
- A synthetic pentasaccharide that selectively inhibits Factor Xa via antithrombin
Direct Oral Anticoagulants (DOACs)
- Direct Thrombin Inhibitors (DTIs): Inhibit thrombin (Factor IIa) directly (e.g., dabigatran)
- Factor Xa Inhibitors: Inhibit Factor Xa directly (e.g., rivaroxaban, apixaban, edoxaban)

Instrumentation
- Automated Coagulation Analyzer: Capable of performing chromogenic or clot-based assays
- Spectrophotometer: Used to measure the absorbance of the colored product in chromogenic assays
Reagents
- Antithrombin: Purified antithrombin reagent
- Factor Xa: Purified Factor Xa reagent
- Chromogenic Substrate: A synthetic peptide substrate that is cleaved by Factor Xa, releasing a colored product
- Buffer: Tris buffer or other suitable buffer to maintain a stable pH
- Calibrators: Standards with known concentrations of the anticoagulant drug
- Control Materials: To perform instrument calibration
Sample Preparation
- Plasma: To isolate the plasma, the sample must be centrifuged
- Specific anticoagulant is required for collection; test specifications should dictate if collection can use heparin or citrate

Chromogenic Method (Most Common)
1. Patient Plasma Preparation: Dilute the patient’s plasma with a specific buffer
2. Add Antithrombin and Factor Xa: Mix the diluted plasma with antithrombin and a known amount of Factor Xa
  - The anticoagulant drug in the patient’s plasma will inhibit Factor Xa, and this amount of inhibition will be measured
3. Incubate: Incubate the mixture for a specific period to allow the reaction to proceed
4. Add Chromogenic Substrate: Add a chromogenic substrate that is specific for Factor Xa
  - Uninhibited Factor Xa will cleave the substrate, releasing a colored product
5. Measure Absorbance: Measure the absorbance of the solution at a specific wavelength
6. Calculation: Calculate the anti-Xa activity based on a calibration curve prepared using standards with known concentrations of the anticoagulant drug
Clotting Method (Less Common)
- Measures the prothrombin time. Is less accurate and sensitive and thus is becoming obsolete

Anti-Xa Activity Level
- Reported in units of activity (e.g., IU/mL or μg/mL)
- Reference Range: Varies depending on the anticoagulant drug and the clinical indication
  - Refer to the laboratory’s established therapeutic range for each drug
Therapeutic Range
- LMWH: Typical therapeutic ranges for LMWH depend on the dosing schedule (once-daily or twice-daily) and the clinical indication (e.g., treatment of VTE, prophylaxis)
- UFH: A target range may be established
- DOACs: Some labs offer DOAC specific testing

Pre-Analytical Variables
- Improper Collection Technique: Tissue thromboplastin contamination or hemolysis
- Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube
- Clotted Sample: Invalidates the results
- Delayed Testing: Anticoagulant drugs can degrade over time
- Improper Storage: Incorrect storage temperatures can affect results
Analytical Variables
- Instrument Malfunction: Ensure proper calibration and maintenance of the coagulation analyzer
- Reagent Problems: Use fresh, properly stored reagents and follow the manufacturer’s instructions
- Interfering Substances: Heparin-induced thrombocytopenia (HIT) antibodies can interfere with the assay
- Specific to each test: Reagents, lots numbers, and expirations
Patient-Related Variables
- Medications: Other medications that affect coagulation can influence anti-Xa results
- Liver Disease: Impairs the synthesis of antithrombin and other coagulation factors
- Kidney Disease: Can affect drug clearance and anticoagulant levels