TOP-DOWN DETERMINATION OF SERUM INSULIN-LIKE GROWTH FACTORS BY LC-MS Richard KT Kam, Ph.D. Scientific Officer, Department of Chemical Pathology Prince of Wales Hospital
Prince of Wales Hospital, Hong Kong Established 1984 Teaching hospital of The Chinese University of Hong Kong Serving population ~1.3 million Specimen throughput: ~3500/day Centralized laboratory for IGF-I analysis
Background GHRH + Somatostatin Hypothalamus Pituitary Insulin-like Growth Factors 1 (IGF-I) is the mediator of pituitary growth hormone (GH). GH level is pulsatile and fluctuating, while IGF-I is released in a more stable manner. Determination of circulating IGF-I level is more useful than GH in the diagnosis of GH disorders, e.g. Acromegaly GH deficiency GH therapy Growth Hormone (GH) + Liver - IGF-I ALS Target tissue Growth and differentiation IGFBP-3
Positive shift in patient median over 5 years Algeciras-Schimnich A, Bruns DE, Boyd JC, Bryant SC, La Fortune KA, Grebe SK. Failure of current laboratory protocols to detect lot-to-lot reagent differences: findings and possible solutions. Clin Chem. 2013 Aug;59(8):1187-94.
de Kock SS, Rodgers JP, Swanepoel BC. Rapid Commun Mass Spectrom. 2001;15(14):1191-7. Bobin S, Popot MA, Bonnaire Y, Tabet JC. Analyst. 2001 Nov;126(11):1996-2001.
Determination of IGF-I by top-down approach for clinical application Bystrom CE, Sheng S, Clarke NJ. Narrow mass extraction of time-of-flight data for quantitative analysis of proteins: determination of insulin-like growth factor-1. Anal Chem. 2011 Dec 1;83(23):9005-10.w
Methodology Acid ethanol extraction of serum Analysis by UPLC-TOF MS (Waters G2XS) Resolution > 30000 Calibrator traceable to WHO 02/254 in artificial matrix (4% BSA in 0.1% saline) QC pooled from patient serum
Methodology Dissociate from acid labile subunit + internal standard Precipitation of large protein, neutralize buffer Concentrate IGF for sensitivity + 1.5 M Tris Vacuum dry + Ethanol / HCl Reconstitution 150 ul serum IGF extract Discard acid labile proteins Salt-out large protein IGF extract High resolution LC-TOF analysis
Full-scan spectrum of IGF-I, IGF-II and IS 7+ cluster IGF-1 IGF-2 8+ cluster N-15 IGF-1 9+ cluster
Peak detection of IGF-I IGF-2 IGF-1 N-15 IGF-1 Chromatographic peak area for quantitation 30 ug/l S/N: 44.4 Monitor m/z 1093.5240
Reference Interval Age-specific: 28 ranges Male + Female Total 56 age- and genderspecific reference ranges (not including Tanner stage) Red: Quest Diagnostics LC-TOF MS Blue: Immunoassay (Nichols Advantage), 2003 Bystrom C, Sheng S, Zhang K, Caulfield M, Clarke NJ, et al. (2012) Clinical Utility of Insulin-Like Growth Factor 1 and 2; Determination by High Resolution Mass Spectrometry. PLoS ONE 7(9): e43457. doi:10.1371/journal.pone.0043457 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0043457
Overview of IGF-I LC-MS Assay Reference interval from the overseas laboratory was adopted. Precision (CV%) Linear range (mg/l) Within batch Between batch 4 Hrs room temperature 3 freeze-thaw cycles IGF-I (PWH) 10 2000 3.3% 5.3% -0.9% -0.3% IGF-II (PWH) 30 2000 9.3% 6.5% -0.9% 2.6% 15.6 2000 5.0% 4.4% N/A* N/A IGF-I (Overseas) 800 Slope = 0.97 Y-intercept = -2.8 Pearson R2 = 0.82 N = 150 G2XS 150 700 600 G2XS 200 100 1200 Slope = 0.93 Y-intercept = -8.3 Pearson R2 = 0.90 N = 85 500 800 400 50 200 200 100 0 50 100 150 QD 1 *N/A: not available 200 250 600 400 300 0 Slope = 0.94 Y-intercept = 2.42 Pearson R2 = 0.98 1000 PWH 250 Accuracy assessment High-end (> 200 mg/l) Low-end (0 200 mg/l) PWH IGF-1 assay Instability (% deviation) 0 100 200 300 Overseas laboratory assay 400 500 QD 2 600 700 800 0 200 400 RCPA 600 RCPA EQA+ 800 1000 1200 median +: Royal College of Pathologist, Australia. External Quality Assurance.
IGF-I service since April 2016 Sample throughput: ~3000/year Reference interval adopted from the overseas laboratory What we have learnt thus far... 1. Collection tube interference; 2. Oxidized protein in EQA materials;
Interlaboratory comparison Comparison to an overseas laboratory using a similar method Y = 0.865X + 6.6 R2 = 0.81 N = 669 1000 CurrentPWH assay 800 600 400 > 90% samples from certain hospitals were undetectable... 200 0 0 200 400 600 Quest Overseas laboratory 800 1000
Pilot study Brand B serum tube IGF-I + IS Brand G serum tube Brand G serum microtube
m/z: 55.5 (double charge) -> mass difference = 111 Da Polyvinylpyrrolidone (PVP) polymer MW: 111.12 Da Drake SK, Bowen RA, Remaley AT, Hortin GL. Potential interferences from blood collection tubes in mass spectrometric analyses of serum polypeptides. Clin Chem. 2004; 50 (12): 2398-401.
MS N N+1 N+2 N+3 111 Da Δ m/z 85 N Polyvinylpyrrolidone (PVP) polymer MSMS 85 Da N C4H7NO + N
Δ m/z 44 MSMS Polyethylene glycol (PEG) polymer Δ m/z 44 N 44 Da O HO OH 44 Da O Δ m/z 44 CH3CH=O + H OH
Slope = 0.870 Y-int = 6.6 R2 = 0.93 N = 494 1000 Without interference CurrentPWH assay 800 600 400 Rate of detection = 100.0% 200 0 0 200 400 600 800 1000 Quest Overseas laboratory Slope = 0.807 Y-int = 11.2 R2 = 0.55 N = 175 1000 With interference CurrentPWH assay 800 600 Rate of detection = 73.1% 400 200 0 0 200 400 600 800 Quest Overseas laboratory 1000
Standardization of Collection Tubes Blood collection tubes were standardized to ensure the quality of service. Certain brands of blood collection tube were unsuitable for use due to interference. Brand B tube as the standardized collection tube for IGF-I service A pilot study on collection tube should be done when introducing HRMSbased assay for clinical service
Case M/70, adrenal insufficient History of impaired fasting glucose, poor vision IGF-I: 119 µg/l Mass spectrum Patient Quality control IGF-I EIC BPC
IGF-I and Internal standard Patient 12+ 11+ 10+ 9+ IGF-I IGF-I not reported Unknown interferent included for monitoring
Qualification of IGF-I quantitation Traditionally done by qualifying MRM channel in LCQQQ instrument RT + parent mass + fragment mass (ion ratio) How about HRMS? RT + parent mass +??? Application of isotope ratio?
M M-1 M+2 M-2 M/M-2 2500 Concentration (mg/l) M/M+2 M/M-1 2000 1500 1000 500 0 1.00 2.00 0.87 1.37 Isotope ratio 0.70 1.20
35 30 IGF-I, isotope ratio (TOF) Mean = 1.56 RSD = 7.8% N = 430 25 Conc All patient isotope ratio vs concentration Androstenedione, ion ratio (QQQ) 20 15 10 1000 Mean = 1.25 RSD = 14.5% N = 534 800 0 0.5 1.0 1.5 2.0 Ratio 2.5 3.0 3.5 600 Ang-I, ion ratio (QQQ) 400 70 60 200 Mean = 0.96 RSD = 15.5% N = 574 50 0 0.5 1.0 1.5 M/ 2.0 2.5 Ratio ratio M-1 3.0 3.5 Conc Conc (µg/l) Concentration 5 40 30 20 10 0 0.4 0.9 1.4 1.9 Ratio 2.4 2.9 3.4
IGF-I and Internal standard Patient 12+ 11+ 10+ 9+ IGF-I IGF-I not reported Unknown interferent included for monitoring
2. Oxidized protein in EQA material
Oxidized material in EQA samples IGF-I contains exactly 1 methionine residue Wild-type IGF-I (7+) = m/z 1093.5240 oxmet IGF-I (7+) = m/z 1095.79 Found in commercial QC pool and RCPA EQA material Concentration dependents? Methionine Methionine sulphoxide (+ 16 Da) Bystrom C, Sheng S, Zhang K, Caulfield M, Clarke NJ, Reitz R. PLoS One. 2012;7(9):e43457.
Mass spectrum IGF-I chromatogram oxmet IGF-I chromatogram QC 469 ng/ml ND Patient samples 487 ng/ml ND RCPA EQA 669 ng/ml 105 ng/ml oxmet IGF-I IGF-I
Observed concentration (µg/l) RCPA EQA performance 1200 Total IGF-I y = 0.9392x + 7.5577 R² = 0.9777 800 Native IGF-I y = 0.769x + 32.14 R² = 0.9838 400 oxmet IGF-I y = 0.162x - 10.836 R² = 0.8933 0 0 400 800 1200 RCPA median (µg/l) RCPA materials (N = 18) Patient samples (N = 20) 462.5 108 891 99.1 26 160 426.5 103 749 21.6 NA Samples with detectable level of oxmet IGF-I (%) 83.3 5.0 Relative abundance of oxmet IGF-I in detectable samples (%) 17.6 5.1 IGF-I (mg/l) oxmet IGF-I (mg/l) Median Range Median Range
Summary Serum collection tube as a critical pre-analytical factors; Commutability of EQA material; Always check raw data.
Acknowledgement Funding support from Hospital Authority Resource Allocation Exercise 2014-15 (15-011PAT) Logistic support from all Hospital Authority Pathology Laboratories in Hong Kong Technical support from Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong Data from RCPA Quality Assurance Program
Thank you Faculty of Medicine The Chinese University of Hong Kong
3. How to interpret the results?
Case #1 F/8, precocious puberty, body height > 97th percentile IGF-I: undetectable Examination of raw data Internal standard IGF-I IGF-I chromatographic peak
Case #1 Instrument lock mass failure Mass tolerance < 20 ppm 1st injection Reinjection ND 43.3 ppm 162 mg/l 0.3 ppm IGF-I chromatographic peak
Case #2 F/11, short stature Body weight and height < 3rd percentile IGF-I: undetectable Mass spectrum Patient Quality control IGF-I EIC BPC
Mass spectrum Case #2 Collection tube interference PVP polymer Patient IGF-I not reported IGF-I and Internal standard Quality control