Serotonin Release Assay (SRA) Protocol
The serotonin release assay is the gold standard laboratory test for diagnosing heparin-induced thrombocytopenia (HIT), with high sensitivity and specificity when performed correctly. 1
Overview of the SRA
The SRA detects platelet-activating antibodies against PF4/heparin complexes by measuring serotonin release from donor platelets when exposed to patient serum in the presence of heparin. This functional assay is critical for confirming HIT diagnosis, which can lead to life-threatening thrombosis if not properly identified and managed.
Standard Protocol Components
1. Sample Collection and Preparation
- Collect patient serum (not plasma) in standard collection tubes without anticoagulants
- Allow blood to clot completely at room temperature (30-60 minutes)
- Centrifuge at 1500g for 15 minutes
- Separate serum and store at -70°C if testing is delayed 1
2. Donor Platelet Preparation
- Collect blood from healthy donors who:
- Have not taken medications affecting platelet function for 10 days
- Have not consumed serotonin-rich foods (e.g., bananas) for 24 hours 2
- Use ACD (acid-citrate-dextrose) or citrate as anticoagulant
- Prepare platelet-rich plasma via centrifugation
- Load platelets with radiolabeled serotonin (14C-serotonin) or non-radiolabeled serotonin for HPLC detection 3
3. Assay Procedure
- Incubate donor platelets with patient serum under two conditions:
- Low heparin concentration (0.1 U/mL)
- High heparin concentration (100 U/mL)
- Include positive and negative controls
- Measure serotonin release after incubation period
- Calculate percent serotonin release from total platelet serotonin content
4. Result Interpretation
- Positive result: ≥20% serotonin release at low heparin concentration with inhibition (≤20% release) at high heparin concentration
- Negative result: <20% serotonin release at both concentrations
- Indeterminate result: Patterns that don't clearly fit positive or negative criteria 4
Modern Detection Methods
HPLC Method (Non-radioactive)
- Advantages: Equivalent performance to radioactive method (92% concordance) without radioactivity hazards 3
- Procedure:
- Platelets loaded with non-radiolabeled serotonin
- Released serotonin quantified by HPLC with fluorescent detection
- Results expressed as percent release using established cutoffs
Flow Cytometry Method
- Advantages: Allows multiparametric analysis of individual platelets
- Procedure:
- Platelets identified by CD41a surface staining
- Serotonin content measured by specific antiserotonin intracytoplasmic staining
- Activation confirmed by annexin V binding 5
Quality Control Considerations
- Include known positive and negative controls with each run
- Perform between-run imprecision studies to ensure standard deviation <6% for positive, weak positive, and negative samples 3
- Validate sample stability (typically stable for two freeze-thaw cycles or up to one week refrigerated) 3
- Consider parallel testing with enzyme immunoassay (EIA) for PF4/heparin antibodies to reduce false-positive risk 1
Clinical Context and Interpretation
- SRA should be interpreted in the appropriate clinical context using scoring systems like the 4T score
- Indeterminate results require careful evaluation as they may signal non-HIT platelet activation processes associated with increased mortality (49.3% vs. 21.1% for positive SRA) 4
- A positive SRA in the appropriate clinical context is virtually diagnostic of HIT 1
- Heparin-"independent" platelet activation (activation even without heparin) may indicate unusually severe HIT, including delayed-onset HIT 1
Pitfalls and Caveats
- Approximately 4% of test sera show indeterminate reaction profiles 1
- False-positive results can occur; requiring a positive EIA reduces this risk
- Sample handling is critical - improper collection or storage can affect results
- Sonication of blood samples can decrease serotonin levels 2
- Dietary influences (e.g., banana consumption) can elevate whole blood serotonin 2
By following this protocol, laboratories can effectively implement the serotonin release assay for accurate diagnosis of heparin-induced thrombocytopenia, helping to guide appropriate clinical management of this potentially fatal condition.