Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone

Consortium and methods

The Bologna ethics committee approved the HarmoSter study (n° 141/2017/U/Tess). Laboratory A coordinated the study, recruited patients and collected samples. Laboratories B to L were measuring centers: all measured cortisol and 17OH-progesterone; five also measured aldosterone (Supplemental Table 1 and Table 1). Eleven LDTs (Laboratories B to I) and two panel MassChrom^® kit (Chromsystems; Munich, Germany; https://chromsystems.com) (Laboratory L) were involved. Among LDTs, 6PLUS1^® Multilevel Serum Calibrator set (Chromsystems) was used for in-house calibration for cortisol and aldosterone by Laboratories D and E, and for 17OH-progesterone by Laboratories D, E and F. Technical details and in-house measurement ranges are shown in Table 1 and Table 2.

Study samples

The sample set included authentic samples, EQA materials and calibrators. We recruited 26 volunteers (women/men: 13/13; age: 20–69 years) following informed consent. Nine subjects were healthy and medication-free. Six women had hyperandrogenism and one had an estroprogestogen patch; two men had hypogonadism and one inhaled budesonide; others were taking one or more medications (cholecalciferol, thyroxine, folic acid, insulin sensitizers, cholesterol-lowering, anti-hypertensive, expectorants and antibiotics). Blood was taken at 7:30–8:30 am after overnight fasting. For each subject, three types of tubes were randomly alternated, containing gel separator (Vacutainer™ SST™ II Advance, cat.366468, Becton Dickinson, Milan, Italy), lithium-heparin (Vacutainer™, 102 IU, cat.368886, Becton Dickinson) or beads clot activator (Vacutest, cat.10636, KIMA S.R.L., Azergrande, Italy). After 30 min settling, samples were centrifuged (2000 rcf, 10 min, room temperature). Supernatants of each specimen were mixed, aliquoted and stored at −80 °C. Seventy-eight samples were obtained.

EQA materials were sent to Laboratory A and stored according to manufacturers’ specifications. The Reference Institute for Bioanalytics (RfB; Bonn, Germany; www.rfb.bio) donated four materials (HM40121, HM40122, HM40123 and HM40124; lyophilized human recalcified plasma spiked with steroids and no preservatives), and assigned with cortisol, 17OH-progesterone and aldosterone RMP target values. The United Kingdom National External Quality Assessment Service (UKNEQAS; Birmingham, UK; https://ukneqas.org.uk) donated eight liquid materials (off-the-clot minimally manipulated human serum), four with cortisol (C568 and C532), 17OH-progesterone (H408, spiked with 17OH-progesterone) and aldosterone (L125, spiked with aldosterone) target values determined as means of all MS-based methods in the survey. Instand e.V (Düsseldorf, Germany; https://www.instand-ev.de). donated three low/high concentration paired materials (human serum spiked with steroids and no additives), two with target values assigned by RMP for cortisol (N°302, liquid), and as means of all methods in the survey for aldosterone (N°304, lyophilic). None of the EQA materials were directly tested for commutability.

The Biological Sales Network (BSN Srl, Castelleone, Italy; https://www.bsn-srl.it) donated seven level serum-based liquid calibrators for a ten-steroid panel (EUM01041, lot.M01411808VEQ) traceable to the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP). Target values of RCPAQAP materials were determined by LC-MS/MS by the National Measurement Institute Australia (traceable to CRM-6007a) for cortisol, and by all laboratory medians for 17OH-progesterone and aldosterone. Freshly prepared calibrators were delivered to Laboratory A, immediately aliquoted and stored at −80 °C. Chromsystems donated serum-based lyophilic calibrators for a fifteen-steroid panel (6PLUS1^® Multilevel Serum Calibrator set, lot.5016, different from lots used for in-house calibration by Laboratories D, E, F and L). Cortisol was traceable to NIST SRM-971 – frozen human serum; 17OH-progesterone and aldosterone were traceable to CRMs – methanol – from ISO 17025 and 17034 certified supplier. At Laboratory A, calibrators of each level were reconstituted according to manufacturer’s instructions, mixed together, aliquoted and stored at −80 °C. BSN and Chromsystems measurement ranges were 11.92–3,182 and 25.6–806 nmol/L for cortisol, 0.61–166.2 and 0.27–43.9 nmol/L for 17OH-progesterone, and 0.11–25.68 and 0.075–12.5 nmol/L for aldosterone, respectively.

Running scheme and quantitation

Two aliquots from 110 samples were shipped to measuring centers on the same day and stored at −80 °C. EQA materials were stored and handled according to manufacturers’ indications. All were measured within 4 months by two identical runs, each including 110 singlets and an independent in-house calibration set, according to protocols ordinarily used by each laboratory. Results by in-house calibration (CAL1) were sent to Laboratory A before measuring centers received external calibrators’ nominal values. They were then asked to use BSN (CAL2) and Chromsystems (CAL3) sets for re-quantification of results. External calibrators were included in the curve calculation if their nominal concentration was within each method’s range. Calibration curves displayed R²>0.98.

Data analysis and statistics

Hormone values are reported in nmol/L. To convert cortisol, 17OH-progesterone and aldosterone to ng/mL, multiply by 0.362, 0.33 and 0.36, respectively. Results were excluded if below the lower limit of quantification (LLOQ) or above the upper LOQ (ULOQ) of CAL1. Moreover, results were excluded from CAL2 or CAL3 datasets when out of the respective measurement range. Duplicate means and CVs were calculated (Supplemental Tables 2–4). Within-subject (CVi) and between-subjects (CVg) biological variabilities [27] were used to assess the maximum allowable imprecision (MAI; 0.5 × CVi) and bias (MAB; 0.25 × (CVi² + CVg²)^0.5) and total allowable error (TAE; 0.25 × (CVi² + CVg²)^0.5 + 1.65 × (0.5 × CVi)) (cortisol: 11.6, 13.5 and 32.5%; 17OH-progesterone: 14.2, 12.0 and 35.3%; aldosterone 18.3, 12.6 and 42.8%; respectively) [28].

Cortisol

Authentic sample results ranged from 221 to 994 nmol/L (median of all laboratories by CAL1). Three to nine samples were above CAL1 ULOQ in three laboratories. The highest CAL2 calibrator was above the in-house measurement range of all laboratories. Conversely, three samples were above the CAL3 measurement range (Supplemental Table 2). The intra-laboratory CVs ranged from 2.8 to 7.4%, all below the MAI (Table 2). Calibration influenced the measures within all laboratories (p<0.001). Laboratory D results were largely lowered when using CAL2 (−40.2%) and CAL3 (−31.8%) compared to CAL1. For other laboratories, when compared with CAL1, CAL2 determined −18.8 to −9.7% deviation, while CAL3 yielded modest deviations (Supplemental Table 5). Comparing CAL1 vs. CAL3 by Passing-Bablok within laboratories using Chromsystems’ as in-house calibration, confirmed the large proportional overestimation of the former in Laboratory D and the consistency of results in Laboratories E and L (Supplemental Figure 1)

EQA material target values ranged from 265.0 to 948.5 nmol/L; two were above CAL1 and CAL3 measurement ranges in Laboratories D, E and L (Supplemental Table 2). Trueness and bias by CAL1 and CAL3 were mostly within MAB, except Laboratory D, displaying a large positive bias with CAL1. CAL2 determined a negative deviation, with several cases exceeding the MAB, especially for Laboratories D and F (Figure 1). Commutability was demonstrated except for some high level EQA materials slightly outside the interval for three laboratories (Supplemental Figure 2).

Figure 1:

Trueness and bias of steroid measurement in external quality assessment materials as function of the calibration system. % Difference = ((laboratory value – target value)/target value) × 100. Trueness vs. target values determined by reference measurement procedure was evaluated in materials HM40121, HM40122, HM40123, HM40124 for all analytes and in material N°302 for cortisol. Bias vs. target values determined as mean/median of the EQA survey was evaluated in materials C568 and C532 for cortisol, H408 for 17OH-progesterone, N°304 and L125 for aldosterone. Lines: zero ± maximum allowable bias (cortisol: 13.5%, 17OH-progesterone: 12.0% and aldosterone: 12.6%). Black dots: in-house calibration (CAL1); gray dots: BSN calibration (CAL2); white dots: Chromsystems calibration (CAL3).

Given the large deviation shown by Laboratory D, inter-laboratory analyses were performed both with and without data from that laboratory, the latter reported as follows. Median inter-laboratory CV by CAL1 was 4.9%, and it was significantly reduced to 3.6% with CAL3 (p<0.001). No cases were detected with CV >MAI (Figure 2 and Supplemental Table 6).

Figure 2:

Inter-laboratory coefficient of variation according to the calibration system.

Black dots: in-house calibration (CAL1); white dots: Chromsystems calibration (CAL3). Horizontal lines: maximal allowable imprecision (cortisol: 11.6%; 17OH-progesterone: 14.2%; aldosterone: 18.3%).

At Passing-Bablok analysis, slopes were similar to 1 in two laboratories with CAL1 and in six with CAL3 (Table 3).

Median bias ranged from −6.6 to 6.9% for CAL1 and −2.3 to 5.5% for CAL3, with all results within the TAE (Figure 3). Compared to CAL1, CAL3 significantly reduced the bias median within six and variance within three laboratories (Supplemental Table 7). Bland-Altman analyses per laboratory are shown in Supplemental Figures 3 and 4.

Figure 3:

Laboratories % bias vs. median of all laboratories as function of the calibration system. % Bias = ((laboratory value – median of all laboratories)/median of all laboratories) × 100. Segments, median; error bars, 2.5 and 97.5 centiles; horizontal lines, zero ± total allowable error (cortisol: 32.5%; 17OH-progesterone: 35.3%; aldosterone: 42.8%). CAL1, in-house calibration; CAL3, Chromsystems calibration.

17OH-Progesterone

Authentic sample results ranged from 0.34 to 6.63 nmol/L (median of all laboratories by CAL1). Values were below the CAL2 measurement range in 33 authentic samples (Supplemental Table 3). The intra-laboratory CV ranged from 4.4 to 18.0%, exceeding the MAI in Laboratory H and L (Table 2). Duplicate-CV increased at values <1.0 nmol/L. Calibration influenced results within all laboratories (p<0.001). Compared to CAL1, CAL2 determined −20.3 to −5.7% lower values, while CAL3 determined modest variations (Supplemental Table 5). CAL1 vs. CAL3 Passing-Bablok analysis within laboratories using Chromsystems’ as in-house calibration detected small deviations in Laboratory D and E, but substantial consistency in Laboratories F and L (Supplemental Figure 1).

EQA materials target values ranged from 2.41 to 12.73 nmol/L. Trueness and bias by CAL1 and CAL3 were mostly within the MAB, except for Laboratory G (−22.2 to −12.3%), and Laboratory H, showing higher values at lower levels (−25.8 to 109.1%). CAL2 increased the negative biases, with several cases exceeding the MAB (Figure 1). A slight deviation from the commutability interval was observed in high level EQA materials in two laboratories. Moreover, HM materials lacked commutability in Laboratory H (Supplemental Figure 2).

The 11.8% median inter-laboratory CVs observed with CAL1 reduced to 10.3% with CAL3 (p<0.001), while cases with inter-laboratory CV>MAI were 33.3 and 7.7%, respectively. Notably, CAL1 CVs increased at levels <1 nmol/L (Figure 2 and Supplemental Table 6).

Passing-Bablok slopes were similar to 1 in two laboratories with both CAL1 and CAL3. Laboratory G and H displayed the lowest slope coefficients (95CI) when using CAL1 (0.844 (0.789–0.896) and 0.777 (0.738–0.809), respectively). Laboratory H also showed a large intercept (95CI) (0.318 (0.274–0.363)). CAL3 improved Laboratory H (slope: 0.922 (0.868–0.954); intercept: 0.066 (0.014–0.112)) but not Laboratory G regression (Table 3).

Median bias vs. median of all laboratories ranged from −17.2 to 7.8% for CAL1 and -20.9 to 10.3% for CAL3 (Figure 3). With both calibrations, Laboratory G showed the largest negative median and Laboratory H the largest variance of bias. Compared to CAL1, CAL3 significantly reduced the bias median within four and variance within three laboratories (Supplemental Table 7). Moreover, CAL3 reduced the opposite systematic bias shown by Laboratory D and H at lowering concentrations with CAL1 (Supplemental Figure 5), almost eliminating cases exceeding the TAE (Figure 3).

Aldosterone

Authentic sample results ranged from 0.07 to 1.22 nmol/L (median of all laboratories by CAL1). Values were below the CAL2 measurement range in 32 authentic samples for most of the laboratories (Supplemental Table 4).

The intra-laboratory CV ranged from 5.2 to 22.2%, with Laboratory L exceeding the MAI (Table 2). Duplicate-CV increased at values <0.2 nmol/L. Calibration influenced results within all laboratories (p<0.001). Compared to CAL1 values, the deviation was −13.1 to 25.2% for CAL2 and −10.0 to 22.1% for CAL3 (Supplemental Table 5). CAL1 vs. CAL3 Passing-Bablok analysis within laboratories using Chromsystems’ as in-house calibrators detected deviations in Laboratory D and E, but consistent results in Laboratory L (Supplemental Figure 1).

EQA material target values ranged from 0.21 to 1.33 nmol/L. A large negative bias was shown for N°304 low (0.21 nmol/L) by all laboratories and calibration sets (−48.6 to −31.6%, or below measurement range). Trueness and bias by CAL1 mostly exceeded the MAB in Laboratory D, G and H. External calibrations improved trueness and bias in Laboratory G only (Figure 1). A slight deviation from the 95% commutability interval was noted in high level EQA materials in three laboratories (Supplemental Figure 2).

Median inter-laboratory CVs with CAL1 was 13.8% and reduced to 8.6% with CAL3 (p<0.001), while cases with inter-laboratory CVs>MAI were 15.4 and 2.6%, respectively (Figure 2 and Supplemental Table 6).

Passing-Bablok slopes in CAL1 and CAL3 were similar to 1 in three and two laboratories, respectively. Compared to CAL1, CAL3 improved the slope (95CI) in Laboratory G (CAL1: 0.846 (0.821–0.870); CAL3: 1.000 (1.000–1.000)) but worsened it in Laboratory H (CAL1: 0.969 (0.909–1.000); CAL3: 0.875 (0.829–0.933)) (Table 3).

Using CAL1, median bias vs. median of all methods ranged from −12.0 to 16.8%, with almost all cases within the TAE. CAL3 significantly reduced bias median, ranging from −8.8 to 3.4%, in three laboratories and variance in one (Figure 3, Supplemental Table 7 and Supplemental Figure 6).