1. MonashPathology 17-HYDROXYPROGESTERONE ASSAYS – TIME FOR HARMONISATION
MonashHealth
17-HYDROXYPROGESTERONE ASSAYS – TIME FOR HARMONISATION
R Tudball1, KL Wan1, KW Choy1, N Wijeratne1,2,3, J Montalto2, C Tran4, L Jolly5, JCG Doery1,3
1Monash Pathology, Monash Health, 2Dorevich Pathology, Heidelberg, 3Department of Medicine, Monash University, Clayton, Vic.,
4Pathology Department, Royal Children’s Hospital, Parkville, Vic.3053; 5RCPA-QAP 257 Gilbert St, Adelaide, SA 5000, Australia
17-OH progesterone assays are poorly harmonised
INTRODUCTION
The adoption of LC-MS/MS methods for measurement of steroids has highlighted the need for improved harmonisation of these assays. Following implementation of a LC-MS/MS method for 17-OHP, we realised that adoption of appropriate age and gender related reference ranges needed to take account of the biases in existing assays seen in QA programmes and sample exchanges which we have investigated.
METHODS
Using clinical patient samples we measured 17-OHP by isotope dilution LC-MS/MS (Shimadzu Nexeria LC and AB Sciex 5500 MS/MS; functional sensitivity <0.14nmol/L) using Isosciences CertimassTM Reference Standard calibrator material. Immunoassays were performed by MP Biomedicals RIA and Immunotech RIA following serum extraction.
RCPA QAP data is from Endocrine Cycle 43.
RESULTS
Using commutable patient samples the Immunotech extraction immunoassay (n=50) showed a +50% positive bias while MP Biomedicals (n=33) demonstrated a -23% negative bias to LC-MS/MS. (Fig 1&2). Thus Immunotech read 94% higher than MP Biomedicals. Review of the RCPAQAP Endocrinology Cycle 43 reflected poor correlation of immunoassays generally compared to LC-MS/MS group (n=6) (Fig 3). Best precision and accuracy are seen with the LC-MS group.
A sampling of adult reference range comparisons demonstrate lack of harmonisation as expected !! (Fig 4)
DISCUSSION
As more laboratories move to the more precise and accurate LC-MSMS assay of specialised steroids such as 17-OHP, there is a clear potential to use harmonised reference intervals which will necessarily differ from the less specific immunoassay reference ranges.
CONCLUSION
The current lack of harmonisation of assays, reference ranges and diagnostic cut-offs is highly unsatisfactory as they confuse clinical endocrinologists and degrade the use of the published literature as well as determination of cut-offs for dynamic endocrine tests.
In view of the difficulty of developing age and gender specific reference ranges and diagnostic cut-offs in children and adults, it is critical for the IVD industry to realign their assays to LC-MS/MS reference methods and seek to further improve antibody specificity until LC-MS becomes the universal norm.
Fig 1. Immunotech Extraction Immunoassay
Fig 2. MP Biomedicals Immunoassay
LAB
AGE
MALE
FEMALE
Lab X -Immunotech
Immunoassay
 > 10 y
1.8 – 10.3 
0.5 – 3.3 (Follicular)
2.1 – 9.4 (Luteal)
(post menopause)
Lab Y - MP Biomedicals
>11 y
Nichols Institute
Extraction RIA
Adult
MAYO LC-MSMS
<6.7
<2.4 (Follicular)
<8.6 (luteal)
<1.54 (post menopause)
ARUP Laboratories
LC-MSMS
 > 18y
 <4.2
<6.3
0.45 – 2.1 (Follicular) 
1.1 – 8.8 (Luteal)
MMC
 16 (post pub)-45
>45
<5.0
<3.0
REFERENCES
1 A 17-year audit of testing for 21-hydroxylase deficiency
Wan KL, Choy KW, Wijeratne N, Doery JCG
Clin Biochem Revs 2015; Poster 60ting fo
r 21-hydroxylase deficiency
RCPAQAP data used with permission
Fig 3. RCPA QAP demonstrating poor correlation of immunoassays (black) with LC-MS (yellow) &
MMC LC-MS (red)
Fig 4.
Sample of adult 17-OHP reference ranges(nmol/L) in
current use.
Monash Medical Centre, Clayton & Monash Children’s Hospital (under construction). Future site of Monash Heart Hospital.