When reference ranges and clinical cutoffs diverge: sex, gender and the practical limits of diagnostic thresholds

Why the gap between reference intervals and clinical cutoffs matters

Laboratory reference intervals and device cutoffs are often presented as neutral numbers, but recent studies and regulatory guidance make clear that sex and gender can change where those lines should be drawn. This matters not as an academic exercise but because thresholds drive real decisions — triage in the emergency department, admission vs observation, and downstream testing or treatment. Understanding when biologic distributions diverge from operational cutoffs helps clinicians, laboratorians and patients weigh risks and benefits carefully.

New assay data show clear sex differences — but not yet a universal call to change practice

A large global reference study for the Roche high‑sensitivity cardiac troponin T Gen‑6 assay (REF‑TSIX) measured 99th‑percentile upper reference limits (URLs) in 4,147 healthy adults and found materially different distributions by sex: female 99th percentile 18 ng/L (95% CI 16–23), male 32 ng/L (28–35); a pooled uniform URL was 27 ng/L (24–31) (REF‑TSIX). The authors and accompanying commentary emphasize that those assay‑level findings need prospective clinical outcome validation before routine clinical thresholds are changed (PERFORM‑TSIX and related outcome studies are ongoing).

Clinical pathway evidence gives a different perspective

Large emergency‑department pathway studies testing rapid rule‑out algorithms using high‑sensitivity troponin have shown that a single, very low uniform presentation threshold (for some hs‑cTnI assays, <5 ng/L) safely identifies many low‑risk patients regardless of sex, even though sex‑specific cutoffs would modestly increase the proportion of women classified as low risk (High‑STEACS/HISTORIC, JACC 2024). That work highlights a common tension: population reference distributions and analytically derived 99th percentiles are not the same thing as operational rule‑out thresholds validated against outcomes. Changing practice requires marrying assay distribution data to pathway outcome data.

Regulatory and implementation realities

The FDA updated device guidance in 2025 to explicitly expect sex‑specific enrollment, analysis and reporting in medical device and IVD studies where differences are anticipated. Sponsors selecting cutoffs should include both sexes at selection and validation and pre‑specify sex subgroup analyses. In short, regulators now expect sex‑aware evidence, or a clear scientific justification for a single cutoff.

Transgender and gender‑diverse patients add complexity

Data in transgender people remain limited but growing. A population cohort study of transgender adults presenting with suspected acute coronary syndrome suggested thresholds aligned with affirmed gender may be usable, but authors warned the evidence is scarce and recommended caution. A recent Clinical Chemistry review recommends using affirmed‑gender reference ranges for many analytes (including hs‑cTn and NT‑proBNP) after ≧3 months of stable gender‑affirming hormone therapy (GAHT), while explicitly noting the evidence base is small and that interpretation should be individualized.

Algorithmic and laboratory sources of bias

Diagnostic systems are not only single numbers; modern tools and decision support often embed demographic information. Imaging and algorithmic studies show models can learn and rely on sex or gender as a shortcut, producing fairness gaps that may not generalize across sites. Aggregation bias from sex‑imbalanced training datasets has been documented in image segmentation work. Those findings argue for sex‑stratified evaluation, out‑of‑distribution testing, and transparent reporting when algorithms influence test interpretation.

What this means for patients and clinicians

• For clinicians: Ask which assay and which cutoff your laboratory uses. Beware of equating a laboratory 99th percentile with an evidence‑based operational rule‑out threshold — they are related but distinct. Expect device labels and manufacturer materials to increasingly report sex‑stratified reference data, and look for outcome‑linked validation when pathways change.
• For laboratorians and device makers: Plan sex‑stratified enrollment and analyses early (the FDA now expects this when differences are likely). When reporting multiple reference intervals or cutoffs, provide clear guidance about clinical validation, timing of GAHT for transgender patients, and how a lab report should be interpreted by clinicians.
• For patients, including transgender and gender‑diverse people: If you're concerned about how a lab value was interpreted, ask whether the lab used sex‑specific ranges and whether any gender‑affirming hormones were considered. Shared decisions matter when evidence is uncertain.

Limitations, uncertainties and next steps

Key evidence gaps remain: outcome‑linked trials showing improved patient outcomes from adopting sex‑specific cutoffs are limited and context dependent; transgender and nonbinary patient data are sparse; and implementation challenges (LIS changes, clinician education, possible confusion at handoffs) are real. Algorithmic fixes that reduce bias at one site can worsen generalization elsewhere, so mitigation strategies must be tested across diverse settings.

Adopting sex‑specific thresholds is not simply a matter of arithmetic; it is an implementation and outcome problem that must balance analytic accuracy, clinical validation, equity for gender‑diverse patients, and the operational realities of care.

Practical checklist

1. Confirm the assay and cutoff used whenever a critical decision depends on a lab value.
2. Prefer pathways that base thresholds on outcome validation (not only population percentiles).
3. Labs and device sponsors should report sex‑stratified data and document how transgender care was handled; clinicians should translate that into individualized care.
4. Audit algorithms and decision support for sex/gender performance and generalizability before deployment.

Bridging the gap between reference ranges and clinical cutoffs will require coordinated science, regulatory clarity, and careful implementation — all grounded in transparency for clinicians and patients.