Methodological considerations in determining sex steroids in children

[madman]

Abstract

Objectives: In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays.

Methods: The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/ MS assays (n=3).

Results: For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9 ± 1.2 and 33.9 ± 1.6 pmol/L (group mean ± standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3 ± 18.1 and 45.7 ± 19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06 ± 0.00 to 1.00 ± 0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12 ± 0.11 and 0.85 ± 0.23 nmol/L.

Conclusions: For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometrybased methods is recommended.




Conclusions

This study enabled the following general conclusions to be drawn from pediatric samples.

(1) Commercially available estradiol immunoassays are not suitable for diagnosis in children.

(2) Commercially available testosterone immunoassays have an uncertainty of reproducibility in the low range that each individual user should consider.

(3) For the safe diagnosis and determination of sex steroids in children, analysis with MS-based methods is recommended.

(4) Every pediatric endocrinologist or laboratory scientist should be familiar with the principles and pitfalls of the sex steroid methods they use.

(5) To increase conformity of methods used for diagnostics in children, participation in an EQA scheme is highly recommended.

(6) We recommend that manufacturers of sex steroid CLIA tests and laboratories that analyze them advertise these tests as recommended for adults, not children.

(7) Since reliable sex steroid quantitation in children requires extremely high expertise, collaboration between biomedical laboratory scientists and clinicians is highly advantageous.

 

[madman]

Table 1A: Summary of performance characteristics for the routine immunoassays used for estradiol analysis. Pediatric reference intervals were downloaded from the attended laboratories’ website, if present; otherwise, they were taken from published data. Puberty-specific intervals were given priority over age-specific intervals. For Siemens Advia Centaur there were no published data available

 

[madman]

Table 1B: Summary of performance characteristics for the routine immunoassays used for testosterone analysis. Pediatric reference intervals were downloaded from the attended laboratories’ website, if present; otherwise, they were taken from published data. Puberty-specific intervals were given priority over age-specific intervals. For Siemens Advia Centaur there were no published data available.

 

[madman]

Table 2: Summary of estradiol and testosterone liquid chromatography-mass spectrometry (LC-MS/MS) validation data provided by the users.

 

[madman]

Table 3: Number of users and their reporting limits for the immunoassays used. (participants in EQA round 2020:01 [article number: KSP 014], Equalis AB, Uppsala, Sweden)

 

[madman]

Figure 1: Method comparison of three different liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS) methods for estradiol (A) and testosterone (B) determinations in child samples. Each symbol and connected line represent one laboratory’s results plotted against the mean value of the three LC-MS/MS method results. Sample E defines the lowest mean concentration of estradiol: 4.9 pmol/L, sample A: 8.5 pmol/L, sample B: 9.4 pmol/L, sample D: 16.6 pmol/L and the highest concentration in sample C: 33.9 pmol/L. The corresponding mean concentration of testosterone was 0.06 nmol/L in sample E, 0.19 nmol/L in sample A, 0.62 nmol/L in sample B, 0.99 nmol/L in sample D and 1.00 in sample C

 

[madman]

Table 4: Serum estradiol and testosterone results from participants in EQA round 2020:01 (article number: KSP 014), Equalis AB, Uppsala, Sweden. Samples A and E were derived from prepubertal children and samples B-D from children in early pubertal development.

 

[madman]

Figure 2: Graphical representation of differences in estradiol quantitation, in order of evaluated concentration, in five child samples labelled A–E, between various immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. Upper panel: Six immunoassays’ analytical biases (A) and relative biases (B) estimated using the mean value of three LC-MS/MS. Each symbol represents the estradiol mean value of each method’s results with standard deviations as error bars. ■ = Abbot Alinity I (n=1), ● = Beckman UniCel DxI 800 (n=2),;= Cobas Roche e601/e801 analyzer series and e411 analyzer (n=10),:= Ortho Vitros 3,600 (n=1),A= Siemens Avida Centaur (n=2),== Cisbio CT RIA + in-house extraction (n=1). Two results from Abbot Alinity, were designated as outliers (1,591 % relative bias at 4.9 and 1,303 % at 8.5 pmol/L) and therefore omitted in the graphic presentation. Lower panel: Detailed plot of analytical bias (C) and relative bias (D) for ten Cobas Roche e601/e801 analyzer series and e411 analyzer, estimated using the mean value of three LC-MS/MS. Each connected line represents one laboratory’s estradiol results

 

[madman]

Figure 3: Graphical representation, in evaluated concentration order, of differences between testosterone liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS) methods and the various immunoassays for five serum samples labeled A–E. Upper panel: Six immunoassay’s analytical bias (A) and relative bias (B) estimated using the mean value of three LC-MS/MS. Each symbol depicts the testosterone mean value of each method’s results with standard deviation error bars. ■ = Abbot Alinity I (n=1), ● = Beckman UniCel DxI 800 (n=2), ; = Cobas Roche e601/e801 analyzer series and e411 analyzer (n=11), : = Ortho Vitros 3,600 (n=1),A= Siemens Avida Centaur (n=1), = = Cisbio CT RIA (n=1). One result from Ortho Vitros and one from Siemens Avida Centaur, were designated as outliers (reported values <0.49 nmol/L and <0.24 nmol/L at 0.06 nmol/L) and therefore omitted in the graphic presentation, panel B. Lower panel: detailed plot of analytical bias (C) and relative bias (D) for the eleven Cobas Roche e601/e801 analyzer series and e411 analyzer, estimated using the mean value of three LC-MS/MS. Each connected line represents one laboratory’s testosterone results. One laboratory result was designated as an outlier (217 % relative bias at 0.06 nmol/L) and therefore omitted in the graphic presentation.