Syphilis Screening: Traditional vs. Reverse Algorithms, False Positives, and the SLE Connection

 

Syphilis Screening: Traditional vs. Reverse Algorithms, False Positives, and the SLE Connection

Two tests, two algorithms, one disease that's surging back—and the autoimmune pitfall that keeps tripping everyone up.

Syphilis is back. Rates in the US have been climbing steadily, congenital syphilis cases have skyrocketed, and the CDC has made this a public health priority. For NPs in primary care, prenatal care, and urgent care, understanding the screening algorithms isn't optional anymore—it's essential. And the fact that your lab may be using a different algorithm than the one you learned in school makes it even more critical to know what you're looking at.

The Two Categories of Tests

Before we get to algorithms, you need to understand the two fundamentally different types of syphilis tests:

Nontreponemal Tests (RPR, VDRL)

• Detect antibodies to cardiolipin-cholesterol-lecithin antigens released from damaged host cells and from T. pallidum itself
• NOT specific to syphilis—they detect tissue damage, not the organism
• Reported as a quantitative titer (1:2, 1:4, 1:8, etc.)
• Titers correlate with disease activity and are used to monitor treatment response
• A fourfold change in titer (two dilutions, e.g., 1:16 to 1:4) = clinically significant change
• Become negative after successful treatment in most patients (though some remain "serofast" at a low titer indefinitely)
• Can be falsely positive in many conditions (see pitfalls below)

Treponemal Tests (FTA-ABS, TP-PA, EIA/CIA)

• Detect antibodies specific to Treponema pallidum
• Confirm that a nontreponemal positive is real syphilis (traditional algorithm)
• OR serve as the initial screen (reverse algorithm)
• Remain positive for life in 75–85% of patients after treatment—they are NOT useful for monitoring treatment response
• Cannot distinguish active from past/treated infection on their own
• False positives can occur with Lyme disease and other non-syphilis treponemal infections

The Two Algorithms

Traditional Algorithm

Screen with a nontreponemal test, confirm with a treponemal test.

1. RPR or VDRL (screening)
2. ↓ If reactive:
3. Treponemal test (FTA-ABS or TP-PA) to confirm
4. ↓ Both reactive = syphilis (stage based on clinical findings and titer)

Pros: Familiar, cost-effective, fewer false-positive workups in low-prevalence settings. RPR titer immediately available for staging and monitoring.
Cons: Can miss early primary syphilis and late latent syphilis (RPR may be nonreactive). Higher false-positive rate for the screening step.

Reverse Algorithm

Screen with a treponemal test, then get a nontreponemal titer.

1. Treponemal immunoassay (EIA or CIA—automated) as the screen
2. ↓ If reactive:
3. RPR (quantitative titer) to assess disease activity
4. ↓ If RPR is also reactive = syphilis (stage and treat)
5. ↓ If RPR is nonreactive (discordant):
6. Second, different treponemal test (TP-PA) as tiebreaker

Pros: Automated, high-throughput, catches latent syphilis and previously treated cases that RPR would miss. More sensitive overall.
Cons: Generates more discordant results requiring tiebreaker testing. Can identify past treated syphilis (treponemal tests stay positive for life), leading to unnecessary workups and overtreatment if not interpreted carefully.

Know Your Lab

Find out which algorithm your lab uses. Many high-volume commercial labs have switched to the reverse algorithm because of automation. If you're interpreting results without knowing which algorithm generated them, you can easily misread what a "positive syphilis screen" means. Ask your lab.

Interpreting the Results: The Cheat Sheet

Nontreponemal (RPR/VDRL)	Treponemal (FTA-ABS/TP-PA/EIA)	Interpretation
Nonreactive	Not done or nonreactive	No syphilis detected. (But can miss very early primary syphilis—both tests may be negative in the first 1–2 weeks of chancre.)
Reactive	Reactive	Syphilis confirmed. Stage and treat based on clinical findings and RPR titer. Could be active or past inadequately treated infection.
Reactive	Nonreactive	Biologic false positive RPR. The patient does NOT have syphilis. Investigate causes (see pitfalls).
Nonreactive	Reactive	Discordant (reverse algorithm scenario). Could be: (1) past treated syphilis (most common), (2) late latent syphilis with waning RPR, (3) very early primary syphilis, or (4) false-positive treponemal screen. Resolve with a second, different treponemal test (TP-PA).
Low-titer reactive (1:1–1:2)	Reactive	Could be serofast state (patient previously treated, RPR won't fully decline) or early/late infection. Correlate with treatment history and clinical exam.

The Pitfalls: Where NPs Get Tripped Up

1. The Biologic False-Positive RPR—and the SLE Connection

This is the pitfall that ties directly into the autoimmune series. The RPR and VDRL use cardiolipin as their antigen. Patients with antiphospholipid antibodies—including anticardiolipin antibodies—will have a false-positive RPR/VDRL because the test is detecting their autoantibodies, not a syphilis infection.

Conditions that cause biologic false-positive RPR/VDRL:

• SLE — the classic autoimmune culprit; up to 10–20% of SLE patients may have a false-positive RPR
• Antiphospholipid syndrome — by definition, these patients have anticardiolipin antibodies
• Pregnancy
• HIV infection
• IV drug use
• Other infections: hepatitis, EBV, malaria, tuberculosis, endocarditis
• Chronic liver disease
• Advanced age
• Recent vaccination

The Autoimmune Pearl

A positive RPR with a negative treponemal test in a young woman should make you think about SLE and antiphospholipid syndrome, not just dismiss it as a lab error. In fact, a biologic false-positive syphilis test was historically one of the earliest recognized clues to antiphospholipid antibody positivity. If you see this pattern, check an antiphospholipid panel (lupus anticoagulant, anticardiolipin, anti-β2GPI) and consider ANA testing.

2. The "Discordant Reverse Algorithm" Trap

With the reverse algorithm, you'll frequently encounter the scenario: treponemal screen reactive, RPR nonreactive. This generates a lot of confusion and unnecessary treatment. In most cases, this represents a patient with past, adequately treated syphilis whose treponemal antibodies persist for life. The key is the tiebreaker TP-PA:

• TP-PA reactive: The patient had real syphilis at some point. If no history of treatment, treat for late latent syphilis. If previously treated, no action needed (unless reexposure is suspected).
• TP-PA nonreactive: The original treponemal screen was a false positive. No syphilis. No treatment.

3. RPR Titers Are Not Interchangeable with VDRL

RPR and VDRL are both nontreponemal tests, but their titers are not directly comparable. An RPR of 1:8 and a VDRL of 1:8 may not represent the same antibody level. Always use the same test (and ideally the same lab) for serial monitoring. A fourfold change (two dilutions) is required to call a result clinically significant.

4. The Prozone Phenomenon

In secondary syphilis, antibody levels can be so high that they overwhelm the nontreponemal assay, causing a false-negative RPR (or a paradoxically low titer). This is called the prozone effect. If you have a patient with classic secondary syphilis symptoms (diffuse rash including palms/soles, mucous patches, condylomata lata) and a nonreactive or weakly reactive RPR, request that the lab perform serial dilutions to unmask a high-titer positive.

Clinical Pearl

If it looks like secondary syphilis clinically but the RPR is negative or suspiciously low, call your lab and ask for dilution testing. The prozone phenomenon is rare but real, and missing secondary syphilis has serious consequences.

5. Treponemal Tests Stay Positive for Life

Once treated, treponemal tests (FTA-ABS, TP-PA, EIA) remain reactive in 75–85% of patients forever. This does NOT indicate reinfection or treatment failure. Only 15–25% of patients treated during primary syphilis will eventually revert to seronegative. Do not use treponemal tests to monitor treatment response. Use the RPR titer.

6. Very Early Primary Syphilis Can Be Seronegative

In the first 1–2 weeks after a chancre appears, both nontreponemal and treponemal tests may still be negative. If you see a painless genital ulcer consistent with a chancre, treat empirically and retest in 2–4 weeks. Don't let a negative serology stop you from treating a clinical primary syphilis.

7. The Serofast State

Some patients, even after adequate treatment, will maintain a persistently low-titer RPR (typically 1:1 to 1:4) indefinitely. This is called the serofast state and does NOT indicate treatment failure or reinfection. It's more common in patients treated for late-stage syphilis. A serofast patient should be monitored but does not need retreatment unless there's clinical evidence of active disease or a fourfold rise in titer.

8. Pregnancy: Test Three Times

Given the surge in congenital syphilis, current guidelines (CDC, ACOG) now recommend screening pregnant patients three times: at the first prenatal visit, early in the third trimester (~28 weeks), and at delivery. This is non-negotiable. Congenital syphilis is devastating and entirely preventable with timely treatment.

When Should NPs Order Syphilis Testing?

Test When You See

• Any painless genital, anal, or oral ulcer (suspect primary syphilis)
• Diffuse rash involving palms and soles (suspect secondary syphilis)
• Mucous patches, condylomata lata, patchy alopecia
• All pregnant patients (three times during pregnancy)
• All patients diagnosed with another STI (HIV, gonorrhea, chlamydia)
• MSM (men who have sex with men)—per CDC, screen at least annually
• HIV-positive patients—screen at baseline and annually
• New sexual partner evaluation or sexual assault follow-up
• Unexplained neurologic symptoms in a patient with history of syphilis or risk factors
• Biologic false-positive RPR as part of a broader autoimmune workup (check aPL panel)

The SLE/APS Connection: A Summary for Your Autoimmune Patients

Tying It Together

In a patient with SLE or suspected antiphospholipid syndrome:

• A positive RPR/VDRL + negative treponemal test = biologic false positive, NOT syphilis. This is a clue to check antiphospholipid antibodies.
• Conversely, a positive treponemal test in an SLE patient should be taken seriously—the treponemal test is specific for syphilis and is NOT affected by antiphospholipid antibodies.
• The biologic false-positive syphilis test was historically one of the earliest recognized markers of antiphospholipid antibodies—before aPL assays existed, this was how clinicians first identified the autoantibodies that would later define antiphospholipid syndrome.
• Always confirm a reactive RPR with a treponemal test before diagnosing syphilis in an autoimmune patient. And always investigate a false-positive RPR for underlying autoimmune disease in a young patient.

Quick-Reference: The Decision Matrix

Scenario	What to Do
RPR reactive, treponemal reactive	Syphilis confirmed. Stage and treat. Monitor RPR titers.
RPR reactive, treponemal nonreactive	Biologic false positive. No syphilis. Investigate: SLE? APS? Pregnancy? Infection?
Treponemal screen reactive, RPR nonreactive (reverse algorithm)	Order TP-PA tiebreaker. If TP-PA reactive + no treatment history → treat for late latent. If TP-PA nonreactive → false-positive screen.
Previously treated, low-titer serofast RPR (1:1–1:4)	Monitor. No retreatment unless fourfold rise or clinical symptoms.
Clinical chancre, serology negative	Treat empirically. Retest in 2–4 weeks. Very early primary can be seronegative.
Pregnant patient	Screen at first visit, ~28 weeks, and delivery. Treat immediately if positive. Don't wait for confirmatory testing to treat in pregnancy.

Bottom Line

Syphilis testing is not complicated once you understand the two test types and the two algorithms. The critical points: know which algorithm your lab uses, never diagnose syphilis on a nontreponemal test alone, never use a treponemal test to monitor treatment, and always investigate a biologic false-positive RPR for underlying autoimmune disease. For your autoimmune patients, the RPR is a test that speaks two languages—infection and autoimmunity—and knowing which one it's speaking requires the treponemal test as the translator.

And with congenital syphilis surging, screen every pregnant patient three times. No exceptions.

Stay sharp out there.

Thyroid Antibodies Decoded: Which to Order, When They Matter, and When They Don't

 

Thyroid Antibodies Decoded: Which to Order, When They Matter, and When They Don't

Three antibodies, three different clinical stories—and the autoimmune crossover that ties them to everything else you're managing.

Thyroid antibody testing is one of the most commonly ordered—and most commonly misinterpreted—panels in primary care. Every week I see providers ordering the full antibody panel on every patient with an abnormal TSH, or panicking about a positive anti-TPO in someone with a perfectly normal thyroid. Let's sort out what each antibody actually tells you and when it changes your management.

The Three Thyroid Antibodies

Anti-TPO (Thyroid Peroxidase Antibody)

Target: Thyroid Peroxidase Enzyme

The workhorse antibody for Hashimoto's thyroiditis. TPO is the enzyme that catalyzes thyroid hormone synthesis. Anti-TPO antibodies activate complement and are directly involved in thyroid tissue destruction. Present in ~95% of Hashimoto's patients and ~70% of Graves' patients. This is the most sensitive thyroid antibody for detecting autoimmune thyroid disease (AITD).

The catch: anti-TPO is also positive in about 10% of the general population who have no thyroid disease—and this prevalence rises with age and female sex. A positive anti-TPO alone, without thyroid dysfunction, doesn't require treatment.

Anti-Tg (Thyroglobulin Antibody)

Target: Thyroglobulin Protein

Present in 30–50% of Hashimoto's patients and at about half that rate in Graves' disease. Less sensitive than anti-TPO for diagnosing AITD. Per Mayo Clinic Laboratories guidance, anti-Tg should only be ordered if anti-TPO is negative but clinical suspicion for AITD remains high.

Critical second role: Anti-Tg is essential in the monitoring of differentiated thyroid cancer after treatment. Thyroglobulin (Tg) is used as a tumor marker post-thyroidectomy, but anti-Tg antibodies interfere with the Tg assay—causing falsely low results on immunometric assays. If anti-Tg is present, the Tg tumor marker is unreliable, and rising anti-Tg titers themselves may indicate recurrence.

TRAb / TSI (TSH Receptor Antibodies / Thyroid-Stimulating Immunoglobulin)

Target: TSH Receptor

The Graves' disease antibody. TSI binds to the TSH receptor and mimics TSH, causing unregulated thyroid hormone production. TSI is ~96% sensitive and ~99% specific for Graves' disease. There are actually three types of TSH receptor antibodies—stimulating (TSI), blocking, and neutral—but TSI is the clinically actionable one.

Nomenclature warning: TRAb, TBII, TSI, and LATS all refer to related but distinct assays. TRAb/TBII measures all receptor antibodies (stimulating + blocking + neutral). TSI measures only the stimulating type. In most clinical contexts, TSI is what you want when confirming Graves' disease.

The Master Decision Table: Which Antibody, When

Clinical Scenario	What to Order	Why
Elevated TSH, suspect Hashimoto's	Anti-TPO	Most sensitive marker; confirms autoimmune etiology. Anti-Tg adds little if TPO is positive.
Elevated TSH, anti-TPO negative, still suspect AITD	Anti-Tg	~5% of Hashimoto's patients are TPO-negative but Tg-positive
Subclinical hypothyroidism (mildly elevated TSH, normal T4)	Anti-TPO	Positive anti-TPO predicts progression to overt hypothyroidism; may influence treatment decision
Suppressed TSH, suspect Graves' disease	TSI (or TRAb)	Confirms Graves' vs. other causes of hyperthyroidism (toxic nodule, thyroiditis, exogenous T4)
Hyperthyroidism when radioactive iodine uptake is contraindicated or unavailable	TSI	Can confirm Graves' without imaging
Pregnant with Graves' disease (or history of Graves')	TRAb/TSI	Maternal TRAb crosses the placenta; high titers predict neonatal thyrotoxicosis risk
Post-thyroidectomy thyroid cancer monitoring	Anti-Tg (with every thyroglobulin measurement)	Anti-Tg interferes with Tg tumor marker assays; rising anti-Tg may signal recurrence
Positive ANA workup, looking for cause	Anti-TPO + Anti-Tg	Thyroid antibodies may be the sole explanation for ANA positivity (speckled or homogeneous pattern)
Euthyroid patient, no symptoms	Nothing	Don't screen asymptomatic patients for thyroid antibodies

The Pitfalls: What Trips Up Clinicians

1. Positive Anti-TPO in a Euthyroid Patient ≠ Disease

About 10% of the general population—and up to 25% of women over 60—will have positive anti-TPO with completely normal thyroid function. This does not require treatment. It does mean the patient has a higher risk of developing overt hypothyroidism over time (about 2–4% per year if TSH is already in the high-normal range). The appropriate response is periodic TSH monitoring, not levothyroxine.

Clinical Pearl

The combination of positive anti-TPO + mildly elevated TSH is where the treatment decision gets nuanced. Many endocrinologists will recommend starting levothyroxine in this scenario, especially if the patient is symptomatic, has a goiter, is trying to conceive, or has TSH >10. Antibody-negative subclinical hypothyroidism is less likely to progress and may be observed.

2. Don't Routinely Order Anti-Tg for Hashimoto's Diagnosis

Anti-TPO is more sensitive and has equal specificity. Ordering both together for initial diagnosis adds cost without meaningful diagnostic yield. Save anti-Tg for when TPO is negative but suspicion is high, or for thyroid cancer monitoring.

3. Thyroid Antibodies Can Explain a Positive ANA

This was highlighted in the Bellocchi autoimmune serologic testing review that started this whole blog series. Anti-TPO and anti-Tg antibodies can cause ANA positivity, typically with a speckled or homogeneous pattern. In a patient with positive ANA but no clinical features of CTD, always check thyroid antibodies. Hashimoto's may be the sole explanation—and it's far more common than lupus.

Connecting the Dots

If you're working up a positive ANA and the patient has no joint pain, no rash, no Raynaud's, and no oral ulcers—but they do have fatigue and a TSH of 6.2—check anti-TPO before chasing an ENA panel. You may save the patient a rheumatology referral.

4. TSI vs. TRAb: Know What You're Ordering

The nomenclature is genuinely confusing. TRAb (or TBII) assays measure all TSH receptor antibodies—stimulating, blocking, and neutral. TSI assays specifically measure only stimulating antibodies. For confirming Graves' disease, TSI is more specific. Some labs report "TRAb" when they mean TBII; others use TRAb to mean TSI. Know what your lab is actually running, and when in doubt, specifically order TSI.

5. Graves' Disease Patients Can Be TRAb/TSI-Negative (Rarely)

About 5% of Graves' patients are seronegative for TSI. If the clinical picture is classic (suppressed TSH, elevated free T4, diffuse goiter, ophthalmopathy) but TSI is negative, a radioactive iodine uptake scan will confirm the diagnosis. Don't exclude Graves' based solely on a negative antibody.

6. Biotin Interference

This is a practical pitfall that catches people. Biotin (vitamin B7)—found in many hair, skin, and nail supplements—can interfere with immunoassays used for thyroid testing, including antibody tests. It can cause falsely low TSH and falsely high free T4 (mimicking hyperthyroidism), and can interfere with antibody measurements. Patients should stop biotin supplements for at least 12 hours (some labs recommend 48–72 hours) before thyroid testing.

7. Pregnancy: When Antibodies Really Matter

Thyroid antibodies take on special significance in pregnancy:

• Positive anti-TPO in euthyroid pregnant women increases risk of miscarriage and preterm birth. Some guidelines recommend levothyroxine in this population, though this remains debated.
• TRAb/TSI in pregnant women with Graves' disease (active or history of Graves'): these antibodies cross the placenta and can cause neonatal thyrotoxicosis. Check TRAb/TSI in early pregnancy and again at 18–22 weeks; high titers warrant neonatal monitoring.
• Postpartum thyroiditis: anti-TPO positive women have a significantly higher risk of developing postpartum thyroiditis. Persistently elevated antibodies after postpartum thyroiditis predict permanent hypothyroidism.

8. Immune Checkpoint Inhibitor Thyroiditis

For NPs managing oncology patients: immune checkpoint inhibitors (pembrolizumab, nivolumab, ipilimumab) frequently cause thyroid adverse effects. Patients with pre-existing thyroid antibodies are at higher risk. Checking anti-TPO before starting immunotherapy can help predict who will develop thyroiditis, and thyroid function should be monitored throughout treatment.

The Autoimmune Cluster

Autoimmune thyroid disease rarely travels alone. If you diagnose Hashimoto's or Graves', keep these associated conditions on your radar:

• Type 1 diabetes — screen if symptomatic
• Celiac disease — consider if GI symptoms, anemia, or weight changes
• Pernicious anemia — check B12 and anti-intrinsic factor if macrocytic anemia
• Vitiligo, alopecia areata — skin manifestations of shared autoimmune susceptibility
• Addison's disease — rare but part of autoimmune polyglandular syndrome
• SLE, Sjögren's, RA — broader CTD spectrum
• Primary Raynaud's — may be associated with autoimmune thyroiditis

Quick-Reference: The Ordering Algorithm

TSH Result	Clinical Question	Order
Elevated (hypo)	Is this autoimmune?	Anti-TPO. If negative, consider anti-Tg.
Mildly elevated (subclinical)	Will this progress?	Anti-TPO (positive = higher risk of progression)
Suppressed (hyper)	Is this Graves'?	TSI (or TRAb). Do NOT use anti-TPO to diagnose Graves'.
Normal	ANA positive, no CTD features	Anti-TPO + anti-Tg (thyroid antibodies may explain ANA)
Normal	No symptoms, no clinical indication	Don't order thyroid antibodies
Any	Pregnant with Graves' (active or history)	TRAb/TSI (1st trimester and 18–22 weeks)
Any	Thyroid cancer monitoring post-treatment	Anti-Tg (with every thyroglobulin tumor marker)

Bottom Line

Thyroid antibody testing is powerful when used correctly. Anti-TPO is your go-to for Hashimoto's and subclinical hypothyroidism risk stratification. TSI is what confirms Graves'. Anti-Tg is a niche player—reserve it for TPO-negative suspected AITD and thyroid cancer monitoring. And remember: a positive antibody in a euthyroid patient is not a diagnosis. It's a risk factor that requires monitoring, not reflexive treatment.

Most importantly for this series: thyroid antibodies are the hidden explanation behind many "unexplained" positive ANAs. If you take nothing else from this post, remember to check anti-TPO and anti-Tg when you're chasing a positive ANA in a patient who just doesn't look like lupus.

Stay sharp out there.

The Celiac Serologic Panel: Getting It Right the First Time

 

The Celiac Serologic Panel: Getting It Right the First Time

Two tests, one massive pitfall, and the number-one mistake that makes the whole workup useless.

Celiac disease affects roughly 1 in 100 people, but up to 80% of cases remain undiagnosed. It masquerades as IBS, iron deficiency anemia, unexplained osteoporosis, chronic fatigue, infertility, and even "normal" GI symptoms that patients have lived with for years. The serologic panel is straightforward—but there are pitfalls that will make you miss the diagnosis entirely if you don't know them.

Let's get this one right.

The Tests: What to Order and Why

The First-Line Screen: tTG-IgA + Total IgA

That's it. For most patients over age 2, you need exactly two tests:

1

tTG-IgA (Tissue Transglutaminase IgA Antibody)
The preferred screening test per ACG, AGA, and ESPGHAN guidelines. Sensitivity of 93–98%, specificity of 96–98%. This is the workhorse of celiac diagnosis. If it's positive, you're on the right track. If it's strongly positive (>10× upper limit of normal), that alone may be sufficient for diagnosis in children without biopsy (per ESPGHAN criteria).

2

Total Serum IgA
This is the test that gets forgotten—and it's the one that saves you from a false negative. Selective IgA deficiency occurs in 2–3% of celiac patients (compared to 1 in 400–800 in the general population). If your patient is IgA-deficient, every IgA-based celiac test will be falsely negative. You need this result to know whether you can trust the tTG-IgA.

The #1 Mistake

Ordering tTG-IgA without a total IgA level. If your patient happens to be IgA-deficient, a negative tTG-IgA means nothing. You've just given them a false all-clear for celiac disease. Always order both together.

When IgA Is Deficient: The IgG Backup Panel

If total IgA is low or undetectable, switch to IgG-based tests:

• DGP-IgG (Deamidated Gliadin Peptide IgG) — the preferred IgG test in the setting of IgA deficiency. Slightly better sensitivity than tTG-IgG.
• tTG-IgG — less specific than tTG-IgA; should only be used if IgA deficiency is confirmed. In IgA-sufficient patients, tTG-IgG has an unacceptably high false-positive rate.
• EMA-IgG (Endomysial Antibody IgG) — highly specific but expensive and observer-dependent. Available at some centers.

Key Rule

Never order tTG-IgG in an IgA-sufficient patient. Per AGA best practice advice, IgG isotype testing for tTG is not specific in the absence of IgA deficiency. It will generate false positives and unnecessary endoscopies.

The Other Tests You'll See on Panels

Test	Role	When to Use
tTG-IgA	First-line screening	All patients ≥2 years with suspected celiac disease
Total IgA	Rule out IgA deficiency	Always order alongside tTG-IgA
DGP-IgG	Best IgG alternative	When IgA deficiency is confirmed; also useful in children <2
tTG-IgG	IgG backup	Only in confirmed IgA deficiency (high false-positive rate otherwise)
EMA-IgA	Confirmatory test (~100% specificity)	To confirm a positive tTG-IgA, especially if tTG is >10× ULN (biopsy-free pathway in children)
DGP-IgA	Secondary test	No advantage over tTG-IgA for initial screening; higher false-positive rate
HLA-DQ2/DQ8	Rule-out test (excellent negative predictive value)	If negative, celiac is virtually excluded. Does NOT confirm celiac if positive (30–40% of the general population carries these alleles)

The Pitfalls: Where Clinicians Go Wrong

1. Testing While the Patient Is Already Gluten-Free

This is the single most destructive mistake in celiac testing. If the patient has already eliminated or significantly reduced gluten, both serology and biopsy may be falsely negative. Antibody levels decline within weeks to months on a gluten-free diet (GFD), and villous atrophy can begin healing.

Critical Rule

Do NOT allow the patient to start a GFD before completing the diagnostic workup. If they've already started one, they need a gluten challenge (consuming gluten daily for at least 2–6 weeks, ideally 6–12 weeks) before testing. Many patients are unwilling to do this after they've felt better gluten-free—which is why getting the testing done first is so important.

2. Forgetting Total IgA

Worth repeating: 2–3% of celiac patients are selectively IgA-deficient. Without checking total IgA, you may reassure an IgA-deficient celiac patient that they don't have the disease. This is a preventable miss.

3. Ordering the Full Panel Instead of the Right Tests

Many labs offer "celiac panels" that include tTG-IgA, tTG-IgG, DGP-IgA, DGP-IgG, and sometimes EMA. Per the AAFP Choosing Wisely recommendation, don't order tTG-IgG or DGP antibodies as initial screening tests. They have higher false-positive rates and add cost without improving diagnostic accuracy in IgA-sufficient patients. Start with tTG-IgA + total IgA. Add IgG tests only if IgA deficiency is confirmed.

4. Low-Positive tTG-IgA Results

False-positive tTG-IgA results occur, typically at low titers (1–2× the upper limit of normal). These can be seen in type 1 diabetes, autoimmune thyroid disease, autoimmune liver disease, heart failure, and other inflammatory conditions. A low-positive tTG-IgA should be confirmed with EMA or repeated before committing the patient to endoscopy. Strongly positive results (>10× ULN) are much more reliable.

5. Assuming a Positive Serology = Celiac Disease

In adults, a positive tTG-IgA should lead to referral for endoscopic duodenal biopsy to confirm the diagnosis before starting a GFD. Serology alone is not diagnostic in adults per current ACG guidelines. In children and adolescents, the ESPGHAN guidelines do allow a biopsy-free diagnosis if tTG-IgA is >10× ULN AND EMA-IgA is positive on a second, separate blood draw—but this decision should be made by a pediatric gastroenterologist.

6. Children Under 2: Use DGP Too

In very young children, tTG-IgA may be less sensitive. ACG guidelines recommend adding DGP-IgA and DGP-IgG to the workup in children under 2 years of age. The immune response to gluten takes time to develop, and these children need to have been eating gluten-containing foods for a sufficient period before testing will be accurate.

7. HLA Testing: Great for Ruling OUT, Not for Ruling IN

HLA-DQ2 and HLA-DQ8 are present in virtually all celiac patients. But they're also carried by 30–40% of the general population, most of whom will never develop celiac disease. The value of HLA testing is its negative predictive value: if a patient is DQ2/DQ8-negative, celiac disease is essentially excluded. This is particularly useful for first-degree relatives being considered for lifelong screening, or for patients already on a GFD where serology is unreliable.

Who Should Be Tested?

Test When You See

• Chronic or recurrent diarrhea, bloating, abdominal pain (especially if labeled "IBS")
• Unexplained iron deficiency anemia (especially if refractory to supplementation)
• Unexplained weight loss or failure to thrive in children
• Unexplained osteoporosis or osteopenia, particularly in premenopausal women or men
• Dermatitis herpetiformis (intensely pruritic blistering rash on elbows, knees, buttocks—this IS celiac disease of the skin)
• Recurrent aphthous stomatitis (canker sores)
• Unexplained elevated transaminases
• Peripheral neuropathy without other explanation
• Dental enamel defects
• Unexplained infertility or recurrent miscarriage
• First-degree relatives of celiac patients
• Type 1 diabetes (associated autoimmune condition—screen if symptomatic)
• Autoimmune thyroid disease, Down syndrome, Turner syndrome, Williams syndrome

Do NOT Test

• As population screening in asymptomatic, low-risk individuals
• While the patient is already on a GFD (results will be unreliable)
• With tTG-IgG or DGP as first-line tests in IgA-sufficient patients

The Autoimmune Connection

This is where celiac testing intersects with the rest of this blog series. Celiac disease clusters with other autoimmune conditions:

• Type 1 diabetes — 3–8% of T1D patients have celiac disease
• Hashimoto's thyroiditis and Graves' disease — increased prevalence
• Autoimmune liver disease — celiac can cause cryptogenic transaminitis
• Sjögren's syndrome, SLE, RA — shared autoimmune susceptibility
• Selective IgA deficiency — both associated with celiac AND causes false-negative testing
• Dermatitis herpetiformis — the skin manifestation of celiac disease; always biopsy the uninvolved skin adjacent to the lesion (direct immunofluorescence shows granular IgA deposits at the dermal papillae)

If you're working up one autoimmune condition, keep celiac in the differential—especially if the patient has unexplained anemia, GI symptoms, or weight changes.

Monitoring After Diagnosis

Once celiac disease is confirmed and a GFD is started:

• Recheck tTG-IgA (or DGP-IgG if IgA-deficient) at 3–6 months after diagnosis
• Continue checking every 6 months until levels normalize
• Then annually to monitor GFD adherence
• Persistent or rising antibodies suggest ongoing gluten exposure (intentional or inadvertent)
• Screen for nutritional deficiencies at diagnosis: iron, B12, folate, vitamin D, calcium, zinc
• Repeat DEXA scan if osteopenia/osteoporosis was present at diagnosis

Quick-Reference: The Celiac Testing Algorithm

Scenario	What to Order	Next Step
Standard screen (age ≥2)	tTG-IgA + total IgA	If tTG-IgA positive → refer GI for biopsy
IgA deficient	DGP-IgG (± tTG-IgG)	If positive → refer GI for biopsy
Children <2 years	tTG-IgA + DGP-IgA + DGP-IgG + total IgA	Refer pediatric GI for interpretation
Low-positive tTG-IgA (1–2× ULN)	Confirm with EMA-IgA or repeat tTG-IgA	If confirmed → refer for biopsy. If negative on repeat → likely false positive
Already on GFD	HLA-DQ2/DQ8 (to rule out), or gluten challenge then serology	If HLA negative → celiac excluded. If HLA positive → need gluten challenge for definitive testing
Monitoring on GFD	tTG-IgA (or DGP-IgG if IgA-deficient)	Every 6 months until normal, then annually

Bottom Line

Celiac testing is deceptively simple: two tests will get you there in most cases. But the pitfalls are real—forgetting total IgA, testing on a GFD, ordering the wrong IgG tests in IgA-sufficient patients, and accepting low-positive results without confirmation. Get the order right, make sure the patient is still eating gluten, and always check that total IgA.

For your autoimmune patients especially, celiac disease should live permanently on your differential diagnosis list. It's common, it's underdiagnosed, and it's one of the few autoimmune conditions where the treatment—a gluten-free diet—can fully control the disease.

Stay sharp out there.