What is the proper procedure for taking a blood sample for cortisol measurement?

Blood Sample Collection Procedure for Cortisol Measurement

Optimal Timing for Blood Draw

Blood samples for cortisol measurement must be drawn between 8:00-9:00 AM to capture the physiologic peak of cortisol secretion and ensure accurate interpretation of adrenocortical function. 1

• This timing window provides optimal sensitivity (68%) and specificity (100%) for detecting ACTH-dependent hypercortisolism when evaluating Cushing's syndrome 1
• The 8:00 AM reference point is the standard used in all diagnostic algorithms, including interpretation of overnight dexamethasone suppression tests 1
• For patients requiring both ACTH and cortisol measurement, both must be drawn simultaneously at 8:00-9:00 AM from the same blood draw 1

Critical Exception: Shift Workers

• Do not perform standard AM cortisol testing on shift workers or patients with disrupted circadian rhythms, as their cortisol peaks occur at different times and will yield unreliable results 1
• For these patients, the dexamethasone suppression test is the preferred diagnostic approach 2

Pre-Collection Patient Preparation

Physical Activity Restrictions

• Patients must abstain from strenuous physical activity for 24-48 hours before blood collection, as exercise significantly elevates cortisol levels 3, 4
• Physical stressors increase salivary cortisol and chromogranin A levels, which can confound interpretation 3

Mental Stress Avoidance

• Avoid cognitive assessments or neurocognitive testing immediately before blood draw, as mental and cognitive stressors acutely elevate cortisol and amylase levels 3, 4
• Acute psychological stress increases cortisol secretion, potentially causing transiently elevated values 4

Substance Restrictions

• No smoking, vaping, or nicotine-containing products for 4 hours before sampling 3
• No alcohol consumption for 12 hours before sampling 3

Infection Status

• Document any recent acute infection, especially upper respiratory tract infections within the past 2 weeks, as immune markers are elevated and may interfere with measurements 3, 4
• Ideally, reschedule sampling for 2 weeks after infection resolution 3

Sleep Considerations

• Document sleep disturbances, as they can affect morning awakening cortisol patterns, though effects vary by population 3, 4

Medication and Physiologic State Documentation

Critical Medications to Document

The most important clinical pitfall is interpreting elevated total cortisol as pathologic without considering cortisol-binding globulin (CBG) status—always inquire about the following before blood draw: 4

• Oral contraceptives and estrogen-containing medications: Estrogen increases CBG levels, which can raise total cortisol measurements dramatically while free cortisol remains normal 4
• Pregnancy status: Increases CBG levels, leading to elevated total cortisol that does not reflect true hypercortisolism 4
• Chronic active hepatitis: Can increase CBG production, falsely elevating total cortisol 4
• Topical hydrocortisone preparations: Can contaminate samples and cause falsely elevated results 4

Medications Affecting Test Interpretation

• CYP3A4 inducers: Accelerate dexamethasone metabolism, causing false-positive suppression test results 4, 2
• CYP3A4 inhibitors: Increase dexamethasone levels, causing false-negative suppression test results 4
• Exogenous steroids (oral prednisolone, dexamethasone, fluticasone inhaler): Confuse interpretation of serum cortisol levels 4

Sample Handling Requirements

For ACTH Measurement (When Ordered Concurrently)

• ACTH is extremely labile and requires immediate processing on ice—coordinate with the laboratory before drawing to ensure proper handling 1
• Both ACTH and cortisol must be drawn from the same blood draw at 8:00-9:00 AM 1

Assay Considerations

• Automated immunoassays are most widely used in routine clinical laboratories but lack specificity and show significant inter-assay differences 5
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers improved specificity and sensitivity, though cortisol cut-offs for diagnostic tests are yet to be validated for these assays 5
• Direct immunoassays have problems if extraction and prepurification are not carried out before the assay 6

Diagnostic Thresholds for Interpretation

Morning Cortisol Reference Values

• Normal morning serum cortisol: 5-23 μg/dL (138-635 nmol/L), with most values between 10-20 μg/dL (276-552 nmol/L) 4
• Morning cortisol >14 μg/dL (>386 nmol/L) effectively rules out adrenal insufficiency 4
• For outpatients, morning cortisol <275 nmol/L identifies subnormal adrenal function with 96.2% sensitivity 7

For Suspected Adrenal Insufficiency

• Cortisol <250 nmol/L with elevated ACTH in acute illness is diagnostic of primary adrenal insufficiency 4
• Cortisol <400 nmol/L with elevated ACTH in acute illness generates strong suspicion of primary adrenal insufficiency 4
• Treatment of suspected acute adrenal insufficiency should never be delayed by diagnostic procedures 4

For Suspected Cushing's Syndrome

• After overnight 1-mg dexamethasone suppression test, cortisol ≥1.8 μg/dL (≥50 nmol/L) at 8:00 AM is abnormal 4, 2
• Cortisol >5 μg/dL (138 nmol/L) after dexamethasone indicates overt Cushing's syndrome 4

Common Pitfalls to Avoid

• Never interpret a single cortisol value without knowing the exact time of collection, as the same value may be normal at 9:00 AM but pathologically elevated at midnight 1
• Do not use spot urine specimens for cortisol assessment due to excessive variability 4
• Avoid blood draws within 1-2 hours of teeth brushing or dental work, as blood contamination can lead to falsely elevated biomarker levels 3, 4
• Document pseudo-Cushing's states (depression, alcoholism, severe obesity, polycystic ovary syndrome) that can cause mild hypercortisolism mimicking true disease 4, 2