Jianbo Yang, PhD, DABCC, Assistant Professor, Section Director of Automated Chemistry, Department of Pathology, Anatomy, & Laboratory Medicine, West Virginia University
Yusheng Zhu, PhD, DABCC, FAACC, Professor, Medical Director, Clinical Chemistry and Automated Testing Laboratory, Director of Postdoctoral Clinical Chemistry Program, Department of Pathology & Laboratory Medicine, Penn State University Hershey Medical Center.
Most of the testosterone in the body binds to sex hormone-binding globulin (SHBG) or albumin, while only 1-4% is free. Bioavailable testosterone (BioT) includes free testosterone (FT) and albumin-bound testosterone. FT is used for diagnosing testosterone deficiency when the total testosterone (TT) is near the lower limit of normal or SHBG is altered. Measuring FT and BioT can be technically challenging and time-consuming, requiring equilibrium dialysis and ammonium sulfate precipitation, respectively. Most laboratories utilize algorithms to calculate FT and BioT based on measured TT and SHBG. The accuracy of calculated FT and BioT depends on the accuracy of TT, the accuracy of SHBG, and the validity of algorithms.
When the Core Reference Laboratory at the Penn State University Hershey Medical Center brought FT and BioT tests in-house, we evaluated four different calculation algorithms to estimate FT and BioT using the in-house TT and SHBG assays, by comparing with a proprietary method used at our reference laboratory. The in-house TT assay demonstrated a negative bias of -11.5% ± 7.5% (mean ± SD) relative to the reference lab assay, although both were certified by the CDC Hormone Standardization (HoSt) Program. The in-house SHBG assay showed no significant bias relative to the reference lab assay. In comparison with the reference lab FT results, the observed percent biases were -9.3% ± 7.8% (mean ± SD) for Vermeulen FT, 15.6% ± 11.1% for Sodergard FT, and -6.6% ± 23.4% for Emadi-Konjin FT. Compared with the reference lab FT% results, the observed percent biases were 2.6% ± 4.8% (mean ± SD) for Vermeulen FT%, 30.9% ± 11.0% for Sodergard FT%, and 5.3% ± 23.5% for Emadi-Konjin FT%. The Vermeulen algorithm demonstrated the best agreement with the reference laboratory in calculated FT or FT%. In comparison with the reference lab BioT results, the observed percent biases were -21.1% ± 9.1% (mean ± SD) for Vermeulen BioT, 13.0% ± 14.4% for Sodergard BioT, -68.5% ± 8.0% for Emadi-Konjin BioT, and -23.3% ± 28.8% for Morris BioT. The Sodergard algorithm showed the best agreement with the reference laboratory BioT, followed by the Vermeulen algorithm. Compared with in-house measured BioT% by (NH4)2SO4 precipitation, the observed percent biases were 12.9% ± 15.1% for Vermeulen BioT%, 62.4% ± 28.0% for Sodergard BioT%, -55.6% ± 7.5% for Emadi-Konjin BioT%, and 10.2% ± 38.1% for Morris BioT%. The Vermeulen algorithm showed the best agreement with measured (NH4)2SO4 BioT%. Overall, the results for the Vermeulen algorithm based on the in-house TT and SHBG assays showed favorable agreement with the reference laboratory results.
Although the CDC HoSt program standardizes different methods for TT, there is no such standardization program for FT or BioT. Our comparison of different algorithms for the calculation of FT or BioT showed significant discrepancies, stressing the importance of revalidating calculation algorithms locally. Clinicians and laboratorians should be aware that FT and BioT calculated using different algorithms or different TT and SHBG assays may not be exchangeable.
游离睾酮和生物可利用睾酮的四种计算方法的比较
体内大部分睾酮与性激素结合球蛋白 (SHBG) 或者白蛋白结合，只有 1-4% 以游离状态存在。游离睾酮（FT）和白蛋白结合睾酮都属于生物可利用睾酮（BioT）。 当总睾酮（TT）接近正常值下限或SHBG浓度偏离正常值时，FT常用于诊断睾酮缺乏综合征。 直接测量 FT 和 BioT 分别需要平衡透析法和硫酸铵沉淀法，这两种方法在技术上难度较大且耗时长。大多数实验室根据血清总睾酮浓度 和 SHBG浓度计算 FT 和 BioT的浓度和百分率。 FT 和 BioT计算 方法的准确性取决于总睾酮 、SHBG血清浓度的准确性以及算法的有效性。
当宾州州立大学Hershey医学中心实验室在建立 FT 和 BioT 检测方法时，我们对照参考实验室评估了四种不同的计算方法，包括Vermeulen, Sodergard, Emadi-Konjin，和Morris。本实验室总睾酮浓度测定方法相对于参考实验室存在 -11.5% ± 7.5%的偏差，尽管两者均为液相色谱串联质谱法且都通过 CDC 激素标准化 (HoSt) 认证。SHBG 浓度测定方法相对于参考实验室没有显著偏差。 与参考实验室 FT 结果相比，Vermeulen FT浓度的偏差为 -9.3% ± 7.8%，Sodergard FT 为 15.6% ± 11.1%，Emadi-Konjin FT 为 -6.6% ± 23.4% 。 与参考实验室 FT百分比结果相比，Vermeulen FT%的偏差为 2.6% ± 4.8%，Sodergard FT% 为 30.9% ± 11.0%，Emadi-Konjin FT% 为 5.3% ± 23.5%。 Vermeulen 算法在计算 FT 或 FT% 方面与参考实验室方法表现出最佳一致性。 与参考实验室 BioT 浓度结果相比，Vermeulen BioT 的偏差为 -21.1% ± 9.1%，Sodergard BioT 为 13.0% ± 14.4%，Emadi-Konjin BioT 为 -68.5% ± 8.0%， Morris BioT 为-23.3% ± 28.8%。 Sodergard 算法与参考实验室 BioT 的一致性最佳，其次是 Vermeulen 算法。 与本实验室硫酸铵沉淀法测量的BioT百分比结果相比， Vermeulen BioT% 算法的偏差为 12.9% ± 15.1%，Sodergard BioT% 为 62.4% ± 28.0%，Emadi-Konjin BioT%为 -55.6% ± 7.5%，Morris BioT% 为 10.2% ± 38.1%。 Vermeulen 算法与硫酸铵沉淀法测量的BioT% 结果具有最佳一致性。 总体而言， Vermeulen 算法得到的结果与参考实验室测定的结果显示出良好的一致性。
尽管 CDC HoSt 项目实现了多种血清总睾酮浓度测定方法的标准化，但目前还没有FT 和 BioT 测定方法标准化项目。 我们的研究显示不同计算方法得到的 FT 和 BioT 结果存在显著差异，这也强调了每个实验室应该验证自己算法的重要性。临床医生和检验人员应注意，使用不同算法得到的FT 和 BioT 结果之间不具有可比性。
Reference:
Yang J, Hamilton C, Robyak K, Zhu Y. Discrepancies in Four Algorithms for the Calculation of Free and Bioavailable Testosterone. Clin Chem. 2023 Dec 1;69(12):1429-1431. doi: 10.1093/clinchem/hvad177.