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Journal of Peking University (Health Sciences) ›› 2026, Vol. 58 ›› Issue (2): 332-336. doi: 10.19723/j.issn.1671-167X.2026.02.017

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False positives in female testosterone testing and the diagnostic value of mass spectrometry

Man WU1,2, Yingying LUO1,3, Linong JI1,3,*()   

  1. 1. Department of Endocrinology and Metabolism, Peking University People's Hospital, Beijing 100044, China
    2. Department of Endocrinology, The Third Hospital of Xiamen, Xiamen 361100, Fujian, China
    3. Peking University Diabetes Center, Beijing 100044, China
  • Received:2024-11-28 Online:2026-04-18 Published:2025-12-29
  • Contact: Linong JI
  • Supported by:
    the 2024 National Clinical Key Specialty Construction Program of China

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Abstract:

Objective: To investigate the proportion of pseudo-elevated testosterone levels detected by chemiluminescent immunoassay (CLIA) in female patients and to evaluate the application value of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in improving the accuracy of testosterone detection in this population. Methods: A retrospective analysis was conducted on data collected from female patients who presented at Peking University People' s Hospital from January 1, 2020 to May 1, 2024. These patients were initially identified with elevated testosterone levels through CLIA and subsequently underwent additional blood sampling for confirmatory testing using LC-MS/MS. Based on LC-MS/MS results, the patients were categorized into the pseudo-elevated group and the true-elevated group. Dif-ferences in the testosterone levels between the two groups were compared, the false-positive rate of CLIA was calcula-ted, and further evaluations conducted in the pseudo-elevated group were analyzed. Results: A total of 287 female patients with elevated testosterone levels detected by CLIA were included in the study. According to the LC-MS/MS results, 178 cases (62.0%) were classified into the pseudo-elevated group, while 109 cases (38.0%) were classified into the true-elevated group. The mean testosterone levels measured by CLIA were (3.63±1.60) nmol/L in the pseudo-elevated group and (4.13±2.20) nmol/L in the true-elevated group, showing a statistically significant difference (P < 0.05). The false-positive rate of CLIA in detecting testosterone levels in women was 62.0%, and the area under the receiver operating characteristic (ROC) curve (AUC) for CLIA was 0.601 (95%CI: 0.534-0.668) indicating low diagnostic accuracy. Among the pseudo-elevated group, 40.45% of patients underwent multiple CLIA retests, and some patients were subjected to unnecessary clinical evaluations, including imaging and hormonal testing, which significantly increased patient burden. Conclusion: This study highlights the high false-positive rate of CLIA in detecting testosterone levels in female patients, which can result in misdiagnosis, repeated testing, and unnecessary clinical evaluations, thereby increasing patient burden and resulting in inefficient use of medical resources.LC-MS/MS significantly enhances the accuracy of testosterone detection in women and is strongly recommended as the confirmatory test for female patients with elevated testosterone levels initially detected by CLIA.

Key words: Hyperandrogenism, Testosterone, Chemiluminescent immunoassay, Liquid chromatography-tandem mass spectrometry, False positive

CLC Number: 

  • R588

Figure 1

ROC curve for measuring female testosterone levels using CLIA CLIA, chemiluminescent immunoassay; ROC, receiver operating characteristic; AUC, area under the curve."

Table 1

Additional evaluations in testosterone level pseudo-elevated group (n=178)"

Evaluation items Cases, n (%) Results or special conditions
Testosterone retesting (CLIA) 72 (40.45) Up to 9 retests
Diagnosed with PCOS 61 (34.27) 28 cases (45.90%) with polycystic ovaries on ultrasound, 33 cases (54.10%) without
BG 149 (83.71) 128 normal cases (85.91%), 21 cases (14.09%) diagnosed with diabetes or elevated blood sugar
Insulin 121 (67.98) 115 normal cases (95.04%), 3 elevated cases (2.48%), 3 decreased cases (2.48%)
Cortisol 178 (100.00) 18 elevated cases (10.11%), retests normal; 42 decreased cases (23.60%), retests normal
ACTH 63 (35.39) 7 elevated cases (11.11%), retests normal
IGF-1 19 (10.67) 3 elevated cases (15.79%), 2 retests normal
GH 10 (5.62) 9 normal cases (90.00%), 1 elevated (10.00%)
Thyroid function 142 (79.78) 22 cases (15.79%) with abnormal thyroid function
Gynecological ultrasound 132 (74.16) 28 cases (21.21%) with polycystic changes, 30 cases (22.73%) with uterine fibroids
Adrenal ultrasound 18 (10.11) No significant abnormalities in all cases (100.00%)
Adrenal CT 18 (10.11) 10 cases (55.56%) with no significant abnormalities, 8 cases (44.44%) with adrenal nodules or thickening
Thyroid ultrasound 30 (16.85) 19 cases (63.33%) with thyroid nodules, 9 cases (30.00%) with diffuse changes, 6 cases (20.00%) with heterogeneous echogenicity, 4 cases (13.33%) with no abnormalities
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