1. Establishment of reference intervals for sex hormone binding globulin, LC-MS/MS derived free androgen index and calculated free testosterone
SN Duignan, MM MacMahon, KJ Mulready, JJ Brady, MC Fitzgibbon, MR Cullen
Department of Clinical Biochemistry & Diagnostic Endocrinology, Mater Misericordiae University Hospital, Dublin
Introduction We have recently introduced a liquid chromatography–tandem e mass spectrometry (LC-MS/MS) total testosterone (T) assay into into routine clinical practice. This method is highly sensitive and specifi and is superior to immunoassay for measuring T
Analysis of sex hormone binding globulin (SHBG), and either determining the free androgen index (FAI) in females or the calculated free T (cFT) in males is used to support the clinical interpretation of equivalent T levels
Our aim was to derive gender-specific SHBG, LC-MS/MS FAI and cFT reference intervals (RI) M
Reference interval distribution plots
Females
Males
Testosterone
Method
Seventy five females and 38 males with an age range spanning 20 to 61 years were recruited. Exclusion criteria included hormonal/steroid therapy (including OCP), drugs (including thyroxine and anti-convulsants), albumin <35 g/L, and known Polycystic Ovary Syndrome
T was measured by an automated reverse phase/ion-exchange extraction procedure and analyzed by chromatography using an Acquity UPLC linked to a Waters Xevo tandem mass spectrometer. SHBG was measured using immunoassay (IA) on an Abbott Architect i2000sr
The FAI was used to determine the free T in females1. The Vermuelen equation was used to determine the cFT in males2
The female FAI RI was validated by:
Comparing T and FAI determined by IA at MMUH and by LC-MS/MS at a referral lab over a two year period, n=186
Auditing the clinical presentation and imaging, where available, of all FAI’s since the introduction of the new LC-MS/MS assay at MMUH, n=66
Relationship between FAI and CFT
Validation of LC-MS/MS Derived FAI
Results
The SHBG RI’s for females and males were 20.9–226.5 nmol/L and 5.9–68.5 nmol/L respectively. The RI’s for the female FAI and for the male cFT were 0.29–5.26% and nmol/L respectively
The FAI correlated well with cFT in females (r=0.945). In males, the FAI was shown to correlate poorly with cFT (r=0.184) and over estimate cFT at low SHBG concentrations
Our new LC-MS/MS FAI RI is lower than the previous upper limit of the MMUH (8.5%) which was determined using IA. We validated our new LC-MS/MS FAI RI by confirming an observed positive bias over a 2 year period for IA compared to LC- MS/MS values. In addition, we found that by applying the new LC-MS/MS FAI, the clinical sensitivity increases without compromising the clinical specificity
Method Comparison
Clinical Performance
Conclusion
We have derived gender specific SHBG RI’s and also LC-MS/MS derived female FAI and male cFT RI’s in a healthy population. The female SHBG RI differed significantly from the manufacture’s quoted interval ( nmol/L). Our new RI may benefit from pre- and post-menopausal partitioning
Our LC-MS/MS FAI and cFT will improve the diagnostic efficiency of interpreting equivalent T levels when introduced into routine clinical practice.
References
GD Carter et al. Investigation of hirutism: testosterone is not enough. Ann Clin Biochem 1983;
Vermeulen et al. A critical evaluation of simple methods for the estimations of free testosterone in serum. J Clin Endocrinol 1999;