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Immunology

Confirmatory Testing

Think of diagnostic testing as a two-stage investigation process. The first stage, the screening test, is like sending out a wide-ranging police bulletin. It’s designed to be sensitive and catch any possible suspect, even if it means pulling in a few innocent people who just happen to fit a broad description. A confirmatory test, on the other hand, is the intense, follow-up interrogation under the bright lights. Its job is to separate the truly guilty from the innocent and secure the definitive evidence

The bottom line is this: a screening test suggests a diagnosis, but a confirmatory test proves it. The entire philosophy of this two-tiered approach is built upon a strategic use of tests with different performance characteristics to achieve the highest possible accuracy while remaining efficient

The “Why”: Sensitivity vs. Specificity

To understand confirmatory testing, you must be an expert on two fundamental concepts: sensitivity and specificity

  • Sensitivity: This is the defining characteristic of a good screening test. Sensitivity is the ability of a test to correctly identify individuals who have the disease. A highly sensitive test “casts a wide net” and has a very low rate of false negatives
    • The Power of a Negative: If a highly sensitive screening test is negative, you can be very confident that the patient does not have the disease. (Example: a negative ANA test is excellent for ruling out SLE)
    • The Weakness: The trade-off for this sensitivity is often lower specificity, meaning it can produce false positives
  • Specificity: This is the defining characteristic of a good confirmatory test. Specificity is the ability of a test to correctly identify individuals who do not have the disease. A highly specific test acts like a “sniper rifle,” honing in on the one true target with very few false positives
    • The Power of a Positive: If a highly specific confirmatory test is positive, you can be very confident that the patient does have the disease. (Example: a positive Anti-Sm antibody test is virtually diagnostic for SLE)

The two-tiered strategy is beautiful in its logic: Screen with a sensitive net, then confirm with a specific rifle

Characteristics of a Good Confirmatory Test

  • High Specificity: This is non-negotiable. Its primary job is to eliminate the false positives generated by the screening test
  • Different Principle or Methodology: Ideally, a confirmatory test should use a different technology or target a different analyte than the screening test. This helps ensure that if there was some weird interfering substance causing a false positive on the screen, it won’t affect the confirmation
  • High Positive Predictive Value (PPV): This means that when the test is positive, the probability that the patient actually has the disease is very high

Classic Examples

This is where the theory meets the real world. Let’s look at the classic algorithms that every MLS must know

Syphilis (The Absolute Classic)

  • Screening Test: The RPR (a nontreponemal test)
    • What it does: Detects reagin antibodies, a sensitive but non-specific marker of cellular damage
    • The Problem: Can be falsely positive due to other conditions (lupus, pregnancy, other infections)
  • Confirmatory Test: The TP-PA or a treponemal EIA/CLIA (a treponemal test)
    • What it does: Detects antibodies made specifically against Treponema pallidum antigens
    • The Interpretation: If the RPR is reactive and the TP-PA is reactive, you have a confirmed case of syphilis. If the RPR is reactive but the TP-PA is nonreactive, you have a biological false positive, and the patient does not have syphilis. This is the perfect demonstration of a confirmatory test’s job

Lyme Disease (CDC-Mandated Algorithm)

  • Screening Test (Tier 1): An Enzyme Immunoassay (EIA)
    • What it does: Detects total antibodies to a crude lysate of Borrelia burgdorferi
    • The Problem: It’s very sensitive but can cross-react with antibodies from other infections (like EBV or other spirochetes), leading to false positives
  • Confirmatory Test (Tier 2): The Western Blot
    • What it does: This test is only performed if the EIA is positive. It separates the bacterial proteins by size and looks for antibodies against a specific pattern of key Borrelia proteins. A positive result requires reactivity to a certain number of specific bands (e.g., 5 of 10 for IgG). This high level of pattern specificity proves the antibodies are the real deal

HIV Infection

  • Screening Test: The 4th Generation Combination Immunoassay
    • What it does: A highly sensitive test that simultaneously detects both HIV antibodies and the p24 antigen, shortening the window period
  • Confirmatory Test: The HIV-1/HIV-2 Antibody Differentiation Immunoassay
    • What it does: If the screen is positive, this rapid test confirms the presence of antibodies and, critically, tells you which type of HIV the patient has (HIV-1 or HIV-2), which is important for guiding therapy
    • Note: The Western Blot, while historically the classic confirmatory test for HIV, has been replaced by this more efficient and informative algorithm

A Modern Twist: The Reverse Algorithm

The advancement of technology has flipped some of our classic algorithms on their head. In high-volume labs, it’s now common to use a highly sensitive and automatable treponemal EIA/CLIA as the initial screen for syphilis

  • The Process: Screen with the specific (but very sensitive) treponemal test. If it’s reactive, then you perform the nontreponemal RPR
  • The “Confirmation”: In this case, the RPR’s role shifts slightly. It’s not confirming the diagnosis (the treponemal test already did that), but rather it’s confirming the activity of the disease. A reactive RPR suggests an active or recently treated infection, while a nonreactive RPR in the face of a reactive treponemal test suggests an old, successfully treated infection

The bottom line is that the laboratory is not just running isolated tests. We are executing a deliberate, logical diagnostic strategy designed to provide the most accurate and clinically relevant information possible. Understanding the role of each test in that strategy is the mark of a true Medical Laboratory Scientist