What is the difference between HLA (Human Leukocyte Antigen) B27 DNA test and HLA B27 antigen test in diagnosing ankylosing spondylitis?

HLA-B27 Testing: DNA vs Antigen Methods

Both HLA-B27 DNA (PCR-based) and antigen (flow cytometry) tests are clinically equivalent for screening spondyloarthritis, but DNA testing is increasingly preferred due to superior technical reliability, faster turnaround, and ability to be performed directly on whole blood without DNA extraction.

Technical Differences Between Methods

DNA-Based Testing (PCR)

• Uses real-time PCR with sequence-specific primers targeting exon 2 or exon 3 of the HLA-B gene 1
• Can be performed directly on whole blood after heating at 95°C for 10 minutes, eliminating DNA extraction steps 1
• Demonstrates 100% concordance with traditional DNA extraction methods in validation studies of over 200 patients 1
• More reliable in patients with low lymphocyte counts or those on immunosuppressive therapy 1

Antigen-Based Testing (Flow Cytometry)

• Detects HLA-B27 protein expression on cell surface using microlymphocytotoxicity assays 2
• Requires viable lymphocytes for accurate results 2
• May yield false negatives in patients with altered immune cell populations 2

Clinical Application for Screening

When to Order HLA-B27 Testing

Order HLA-B27 as a screening parameter (not diagnostic confirmation) in patients with chronic back pain >3 months with onset before age 45 years 3, 4

The test demonstrates:

• 90% sensitivity and 90% specificity for axial spondyloarthritis 3
• Superior to inflammatory back pain criteria alone (75% sensitivity/specificity) 3
• Positive predictive value: 30-40% of HLA-B27 positive patients with chronic back pain ultimately receive an AS diagnosis 3, 4

Referral Algorithm Using HLA-B27

Refer to rheumatology if the patient has chronic back pain (>3 months) with onset before age 45 AND:

• Four or more of these criteria: back pain before age 35, nocturnal pain requiring movement for relief, buttock pain, improvement with exercise or within 2 days of NSAID use, first-degree relative with spondyloarthritis, current/previous arthritis/enthesitis/psoriasis 3, 4
• Three of the above criteria PLUS positive HLA-B27 3, 4

Critical Pitfalls to Avoid

Do Not Use HLA-B27 to Rule Out Disease

• Never exclude spondyloarthritis based solely on negative HLA-B27, as approximately 10% of AS cases are HLA-B27 negative 5
• HLA-B27 negative patients can have equally severe disease requiring the same treatment approach 5
• Normal inflammatory markers (ESR/CRP) have limited sensitivity (≤50%) and should not influence decision-making 5

Proper Test Interpretation

• HLA-B27 provides probability statements, not absolute confirmation or exclusion of disease 6
• The test is most valuable when pre-test probability is 30-50% based on clinical parameters 3
• Testing should be performed only once in a patient's lifetime 3, 4

Cost-Effectiveness Considerations

• HLA-B27 testing costs approximately €40 (equivalent to chest or pelvis X-ray) 4
• One-time testing with high sensitivity/specificity makes it cost-effective for appropriate screening 3, 4
• Number needed to screen to identify one case: 3 patients (compared to 7 for inflammatory back pain criteria alone) 3

Practical Recommendation

Use DNA-based HLA-B27 testing via real-time PCR as the preferred method due to technical advantages, particularly the ability to test directly on whole blood with 100% concordance to traditional methods 1. The choice between DNA and antigen testing does not affect clinical decision-making, as both detect HLA-B27 status with equivalent clinical utility when properly performed 2, 1.