Same Blood Sample, Different Results: Why Cortisol Levels Can Vary Between Tests

By SWA

When two cortisol tests are conducted from the same blood sample and yield significantly different results—one low and the other within the normal range—it can be both surprising and concerning. Ideally, the same sample should produce consistent results across multiple tests. However, in practice, certain technical and procedural factors can introduce variability. Here’s a closer look at the reasons why cortisol levels might appear low in one test and normal in a subsequent one.

1. Sample Degradation and Delayed Processing

Cortisol, like many hormones, is sensitive to handling and storage conditions. If a blood sample isn’t processed quickly or stored under optimal conditions, cortisol levels may degrade over time, potentially leading to falsely low results.

  • Time Before Testing: Cortisol is relatively stable when samples are kept frozen, but it can degrade if stored at room temperature or even in a refrigerator for extended periods. If the first test was performed immediately after collection and the second test was done hours or even days later, cortisol levels in the sample might have partially degraded by the time of the second test. This could result in a lower reading for one test while the other remains closer to the true level.

  • Freeze-Thaw Cycles: Labs often freeze samples if there’s a delay in processing. However, each freeze-thaw cycle can cause hormone degradation. If the sample was thawed for the first test, then refrozen, and thawed again for the second test, cortisol levels might decline between tests. Such degradation could make the cortisol level appear low in one test and normal in another, depending on the sample’s condition at the time of testing.

2. Inconsistent Handling within the Lab

Small differences in how samples are handled during processing can lead to variations in cortisol results, even when using the same blood sample.

  • Pipetting or Dilution Errors: In hormone testing, precise measurement is crucial. Lab technicians use pipettes to transfer small volumes of blood serum or plasma. If a small error occurred during the first test—such as adding too much or too little reagent—it could result in an inaccurate, lower cortisol reading. If the second test was conducted with more precise handling, the result might be closer to the true level, yielding a normal reading.

  • Evaporation: Blood samples can evaporate if left open or exposed to air for too long before testing. For sensitive hormones like cortisol, exposure to air can lead to degradation, potentially resulting in a lower reading in the first test. If the sample was then properly sealed or stored in an airtight tube before the second test, this might prevent further degradation, leading to a more accurate, normal result.

3. Variability in Testing Methods or Equipment

Differences in testing methods or slight inconsistencies in lab equipment can also produce discrepancies in cortisol test results.

  • Assay Differences: Labs use different types of assays (testing methods) to measure cortisol levels, and each has unique strengths and limitations. For instance, immunoassays are quick and relatively affordable but can sometimes produce slightly inaccurate results due to cross-reactivity with similar molecules. In contrast, mass spectrometry is more precise but less commonly used due to cost and complexity. If the lab used an immunoassay for the first test and mass spectrometry for the second, this could explain why the first result was low and the second was normal.

  • Instrument Calibration and Quality Control: Lab instruments require regular calibration to maintain accuracy. If the machine used for the first test was slightly out of calibration, it might have recorded a falsely low cortisol level. Quality control procedures aim to minimize these discrepancies, but small calibration issues—particularly with sensitive hormones like cortisol—can still impact results. If the lab used a different, well-calibrated machine for the second test, it could produce a more accurate reading within the normal range.

4. Assay Interference or Cross-Reactivity

Certain substances in the blood, including medications, supplements, or even other hormones, can interfere with cortisol assays, particularly immunoassays. This interference can sometimes cause artificially low or high readings.

  • Substance Interference: Some medications, supplements, or even naturally occurring compounds in the blood can interfere with cortisol measurements. For example, high levels of biotin (a common supplement) can interfere with some assays, leading to inaccurate results. If a substance in the blood interfered with the assay during the first test, it might have led to a falsely low cortisol reading. If the interference was somehow minimized or neutralized by the time of the second test (due to dilution or different handling), the second test could reflect a more accurate cortisol level.

  • Cross-Reactivity: Immunoassays can sometimes detect other substances that are chemically similar to cortisol, leading to inaccurate readings. If the first test was impacted by cross-reactivity with a similar compound, it could yield a lower result. A different test that is less prone to cross-reactivity (like mass spectrometry) would likely produce a more accurate result, showing a normal cortisol level.

5. Intra-Assay Variability and Coefficient of Variation

Even in the most rigorous labs, there is always a small margin of error called intra-assay variability. This means that minor fluctuations can occur even when the same sample is tested multiple times with the same method and equipment.

  • Coefficient of Variation (CV): Each assay has an acceptable range of variability, often expressed as the coefficient of variation (CV). For cortisol assays, a CV of 5-10% is typical. This means that if the actual cortisol level is near a threshold (e.g., borderline low or normal), the natural variability in the test could cause one result to be read as low and the other as normal. Although this variability is small, it can make a noticeable difference when hormone levels are near cutoff values.

6. Laboratory Errors or Sample Mix-Ups

Although rare, human error in the laboratory can lead to discrepancies between test results.

  • Mislabeling or Sample Confusion: Labs follow strict protocols to prevent sample mix-ups, but mistakes can happen. If the lab accidentally mislabeled the results or mixed up the sample with another patient’s sample, this could explain why one test shows a low cortisol level and the other shows a normal level.

  • Reporting Errors: Occasionally, transcription or data entry errors can lead to inaccurate results being recorded. For instance, if a lab technician mistakenly transcribed a low result for the first test, the cortisol level might not have been low at all. This could explain why the second test yields a normal result.

Summary

Having a low cortisol result on one test and a normal result on a subsequent test from the same blood sample can be due to a range of technical and procedural factors. Here’s a recap of the main reasons:

  • Sample Degradation: Delayed processing, improper storage, or repeated freeze-thaw cycles can lead to cortisol degradation, resulting in lower readings on one test but not the other.
  • Inconsistent Handling: Minor pipetting or dilution errors, as well as sample evaporation, can affect test results.
  • Variability in Testing Methods: Different assay types or slight calibration issues in lab equipment can produce inconsistent results.
  • Interference or Cross-Reactivity: Medications, supplements, or other substances can interfere with cortisol assays, potentially affecting only one test.
  • Intra-Assay Variability: Small, natural variations in assay performance can cause one result to be low and the other normal, especially near cutoff values.
  • Laboratory Errors: Sample mix-ups or data entry errors, though rare, could lead to unexpected discrepancies.

Given these potential issues, doctors often recommend repeating hormone tests if results seem unexpected or don’t align with a patient’s clinical symptoms. This practice helps confirm whether an abnormal result truly reflects a patient’s hormone levels or if it was due to lab variability or handling issues.

Providing reliable references and links on this topic requires a combination of scientific literature, clinical guidelines, and reputable medical resources. Here are some recommended references to further explore the factors contributing to variability in cortisol testing results.

1. Hormone Stability and Sample Handling

  • Reference: Armbruster DA. "The Importance of Quality Sample Handling in Clinical Chemistry Testing." Clin Biochem Rev. 2006;27(1):43–58.

    • Link: PubMed - Clin Biochem Rev
    • This review article discusses how sample handling, storage conditions, and processing times can affect the stability of various biochemical markers, including hormones.

  • Reference: Young DS. "Effects of Preanalytical Variables on Clinical Laboratory Tests." AACC Press. 1997.

    • Summary: This reference provides a comprehensive guide on how pre-analytical factors, such as time before processing, temperature, and freeze-thaw cycles, can impact laboratory test results.

2. Assay Variability and Cross-Reactivity

  • Reference: Taylor RL, Machacek DA, Singh RJ. "Validation of a High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Method for Cortisol and Cortisone in Plasma and Urine." Clin Chem. 2002;48(9):1511-1519.

    • Link: Clinical Chemistry
    • This paper details the use of mass spectrometry as a more specific and accurate alternative to immunoassays for cortisol measurement, helping to reduce cross-reactivity and assay variability.

  • Reference: Miller WG, Jones GR, Horowitz GL, Weykamp C. "Proficiency testing/external quality assessment: current challenges and future directions." Clin Chem. 2011;57(12):1670-80.

    • Link: PubMed
    • This article covers quality control in lab testing, including issues with assay variability and calibration that can affect hormone test accuracy.

3. Intra-Assay and Inter-Assay Variability

  • Reference: T'Sjoen G, De Vos S, Goemaere S, et al. "Sex steroid assays: clinical significance, difficulties, and pitfalls." Eur J Endocrinol. 2005;153(6):871-80.

    • Link: PubMed - Eur J Endocrinol
    • This study discusses intra-assay and inter-assay variability in hormone testing, including cortisol, and the implications for clinical diagnostics.

  • Reference: College of American Pathologists (CAP) Quality Management Programs

    • The CAP provides guidelines and standards for maintaining quality in laboratory testing, including the handling of intra-assay and inter-assay variability. CAP-accredited labs follow stringent quality control protocols to minimize these types of errors.

4. Assay Interference and Cross-Reactivity

  • Reference: Favresse J, Lardinois B, Mucha D, et al. "Interferences with Immunoassays: A Review." Clin Chem Lab Med. 2018;56(5):718-727.

    • Link: PubMed - Clin Chem Lab Med
    • This article reviews how medications, supplements, and other substances can interfere with immunoassays, leading to false readings, and discusses ways to mitigate these effects in clinical practice.

  • Reference: Watson D, Barltrop D. "Biotin interference in clinical immunoassays: a cause for concern." Clin Biochem Rev. 2019;40(4):205–215.

    • Link: PubMed - Clin Biochem Rev
    • This review highlights the issue of biotin interference in immunoassays, which can affect hormone testing results, including cortisol levels.

5. Laboratory Error and Quality Control

6. General Resources on Cortisol Testing and Variability

  • Lab Tests Online - Cortisol Test

    • Link: Lab Tests Online
    • This site, maintained by the American Association for Clinical Chemistry, offers a patient-friendly overview of cortisol testing, including factors that can influence test results and why repeat testing is sometimes recommended.

  • Mayo Clinic - Cortisol Test Overview

    • Link: Mayo Clinic
    • The Mayo Clinic provides a summary of cortisol testing, explaining how cortisol levels are measured and the factors that can affect results, including sample handling and timing.

Summary of the References

These resources can help deepen understanding of the complexities surrounding cortisol testing, including sample stability, assay selection, handling, and quality control. Together, they illustrate why variations can occur even from the same blood sample and highlight the importance of strict adherence to lab protocols to minimize inconsistencies.

© 2000-2025 Sieglinde W. Alexander. All writings by Sieglinde W. Alexander have a fife year copy right.
Library of Congress Card Number: LCN 00-192742 ISBN: 0-9703195-0-9 

 

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