Standard Operating Procedure: D-Dimer Testing

1. Purpose and Scope

This procedure defines the step-by-step method for performing D-dimer testing to aid in the diagnosis and exclusion of venous thromboembolism (VTE), disseminated intravascular coagulation (DIC), and other thrombotic disorders.

2. Specimen Requirements

• Specimen Type: Platelet-poor plasma.
• Anticoagulant: 3.2% (0.109 M) sodium citrate (blue top tube).
• Collection: Fill tube to proper volume (9:1 blood-to-anticoagulant ratio).
• Volume: Minimum 2 mL whole blood (yields approximately 1 mL plasma).
• Processing: Centrifuge within 2 hours of collection at 1500-2000 × g for 15 minutes at room temperature.
• Storage/Stability:
• Room temperature: Test within 4 hours
• Refrigerated (2-8°C): Stable for 24 hours
• Frozen (-20°C or below): Stable for 1 month (avoid repeated freeze-thaw cycles)
• Rejection Criteria: Underfilled tubes, clotted samples, hemolyzed or lipemic samples, improper anticoagulant.

3. Safety and Precautions

• Wear appropriate PPE (lab coat, gloves, eye protection).
• Treat all specimens as potentially infectious.
• Handle reagents according to manufacturer's safety data sheets.
• Dispose of waste in appropriate biohazard containers.
• Never mouth pipette.

4. Equipment and Reagents

• Equipment: Automated coagulation analyzer or ELISA reader, centrifuge, micropipettes, timer, vortex mixer, 37°C water bath or heating block (if required).
• Reagents: D-dimer test kit (latex-enhanced immunoturbidimetric, ELISA, or fluorescence immunoassay), calibration standards, quality control materials, citrate diluent buffer.

5. Step-by-Step Procedure

A. Specimen Collection and Processing

1. Perform venipuncture using standard technique.
2. Collect blood into 3.2% sodium citrate tube, filling to the indicated volume.
3. Gently invert the tube 3-4 times to mix.
4. Transport to laboratory at room temperature.
5. Centrifuge at 1500-2000 × g for 15 minutes within 2 hours of collection.
6. Carefully remove plasma without disturbing the cell layer.
7. If not testing immediately, aliquot and store appropriately.

B. Instrument Setup and Calibration

1. Turn on the analyzer and allow to warm up per manufacturer's instructions.
2. Load D-dimer reagents, calibrators, and controls.
3. Perform calibration according to manufacturer's schedule (typically with each new reagent lot or every 30 days).
4. Verify calibration is acceptable before proceeding.

C. Quality Control

1. Run at least two levels of quality control material with each batch of patient samples.
2. Controls should be run:

• At the beginning of each shift
• With each new reagent lot
• After instrument maintenance
• As per laboratory QC policy

1. Verify control results are within acceptable ranges before reporting patient results.
2. Document all QC results.

D. Testing Procedure (Immunoturbidimetric Method)

1. Bring all reagents and samples to room temperature (if refrigerated).
2. Label test cuvettes or sample cups.
3. Pipette the specified volume of citrate buffer into the cuvette (typically 100-200 µL).
4. Add the specified volume of patient plasma (typically 50-100 µL).
5. Add the specified volume of D-dimer reagent (latex-coated antibodies).
6. Mix gently and incubate at 37°C for the specified time (typically 5-10 minutes).
7. Measure the change in absorbance/turbidity at the specified wavelength (typically 575-660 nm).
8. The instrument calculates D-dimer concentration from the calibration curve.

E. Testing Procedure (ELISA Method)

1. Bring all reagents to room temperature.
2. Add diluted patient samples and standards to antibody-coated wells.
3. Incubate for specified time (typically 1-2 hours).
4. Wash wells to remove unbound material.
5. Add enzyme-conjugated antibody.
6. Incubate and wash again.
7. Add substrate solution and incubate.
8. Add stop solution.
9. Read absorbance at specified wavelength (typically 405-450 nm).
10. Calculate concentration from standard curve.

F. Result Interpretation

• Normal/Negative: <0.5 µg/mL FEU (or <500 ng/mL FEU)
• Elevated/Positive: ≥0.5 µg/mL FEU
• Note: Cutoff values may vary by manufacturer and clinical application.

G. Units of Measurement

• FEU (Fibrinogen Equivalent Units): Most common; approximately 2× DDU
• DDU (D-Dimer Units): Reports actual D-dimer concentration
• Conversion: 1 µg/mL FEU ≈ 0.5 µg/mL DDU

6. Quality Control

• Run two levels of QC with each batch (normal and elevated).
• Acceptable QC ranges should be established per CLSI guidelines.
• Participate in external quality assessment/proficiency testing programs.
• Monitor delta checks for repeat patients.
• Correlate with clinical presentation and other coagulation tests.

7. Reference Ranges

• The canonical adult negative threshold for D-dimer is shown in the Reference Ranges panel below.
• Age-Adjusted Cutoff (for patients >50 years): Age (years) × 10 ng/mL FEU
• Example: 70-year-old patient: cutoff = 700 ng/mL FEU
• Note: Each laboratory should establish or verify reference intervals.

8. Clinical Significance

Elevated D-dimer may indicate:

• Deep vein thrombosis (DVT)
• Pulmonary embolism (PE)
• Disseminated intravascular coagulation (DIC)
• Recent surgery or trauma
• Pregnancy
• Malignancy
• Inflammation or infection
• Advanced age

Clinical Applications:

• Rule-out VTE: High negative predictive value (>95%) in low-to-moderate risk patients
• Monitor DIC: Serial measurements track disease progression
• Assess thrombotic risk: In various clinical settings

9. Reporting Results

• Report result with units (µg/mL FEU or ng/mL FEU).
• Include reference range on report.
• Flag elevated results (>0.5 µg/mL FEU).
• For VTE rule-out, include comment: "Negative D-dimer has high predictive value for excluding VTE in low-to-moderate risk patients."
• Critical values: Report immediately to physician if >4.0 µg/mL FEU (suggests possible DIC or massive thrombosis).