2nd generation testosterone is an improved immunoassay method of measuring testosterone in the blood. However, other compounds may still affect accuracy. LC-MS/MS is still the preferred method of measuring testosterone.
Measuring plasma testosterone is confounded by several variables including age, gender, disease state, diurnal fluctuations, interference from other steroids in circulation, bound versus free testosterone, etc. Even the methods used to measure testosterone can affect sensitivity, specificity, and overall accuracy (French 2019, Rosner 2007).
Second-generation testosterone immunoassays were introduced in 2007 and designed to improve the detection of testosterone at low levels, especially below 115.27 ng/mL (4 nmol/L). Research suggests that second-generation assays are comparable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays when levels are above 115.27 ng/mL (4 nmol/L) but needed an extra extraction step to be comparable when levels are below that mark. Cross-reactivity and interference from other compounds, including DHEA, still affected the second-generation assays despite their updated status (Groenestege 2012). Compounds found in oral contraceptive medications also interfere with second-generation testosterone assays, resulting in a falsely elevated total testosterone (Rowe 2018).
The preferred method of measuring testosterone is liquid chromatography-tandem mass spectrometry. The CDC maintains a standardization system to certify that labs produce results with less than 6% deviation from reference using LC-MS/MS. Other assay methods can then be compared to this benchmark program (AACC 2016).
Research confirms that various immunoassays, including second-generation assays, are not comparable to the LC-MS/MS method of measuring testosterone (Debeljak 2020).
References
AACC. Choosing the Right Testosterone Assay. December 2016. Retrieved from https://www.aacc.org/cln/articles/2016/december/ask-the-expert-december-2016
Debeljak, Željko et al. “Analytical bias of automated immunoassays for six serum steroid hormones assessed by LC-MS/MS.” Biochemia medica vol. 30,3 (2020): 030701. doi:10.11613/BM.2020.030701
French, Deborah et al. “Comparison of four clinically validated testosterone LC-MS/MS assays: Harmonization is an attainable goal.” Clinical mass spectrometry (Del Mar, Calif.) vol. 11 12-20. 1 Dec. 2018, doi:10.1016/j.clinms.2018.11.005
Groenestege, Wouter M Tiel et al. “Accuracy of first and second generation testosterone assays and improvement through sample extraction.” Clinical chemistry vol. 58,7 (2012): 1154-6. doi:10.1373/clinchem.2011.181735
Rosner, William et al. “Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement.” The Journal of clinical endocrinology and metabolism vol. 92,2 (2007): 405-13. doi:10.1210/jc.2006-1864
Rowe, Ceri, and Sarah Rabet. “Potential interference in the Abbott Architect 2nd generation total testosterone assay.” Annals of clinical biochemistry vol. 55,5 (2018): 621-622. doi:10.1177/0004563218780888