TOP-DOWN DETERMINATION OF SERUM INSULIN-LIKE GROWTH FACTORS BY LC-MS

Transcription

1 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

2 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

3 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

4 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 Aug;59(8):

5 de Kock SS, Rodgers JP, Swanepoel BC. Rapid Commun Mass Spectrom. 2001;15(14): Bobin S, Popot MA, Bonnaire Y, Tabet JC. Analyst Nov;126(11):

6 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 Dec 1;83(23): w

7 Methodology Acid ethanol extraction of serum Analysis by UPLC-TOF MS (Waters G2XS) Resolution > Calibrator traceable to WHO 02/254 in artificial matrix (4% BSA in 0.1% saline) QC pooled from patient serum

8 Methodology Dissociate from acid labile subunit + internal standard Precipitation of large protein, neutralize buffer Concentrate IGF for sensitivity 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

9 Full-scan spectrum of IGF-I, IGF-II and IS 7+ cluster IGF-1 IGF-2 8+ cluster N-15 IGF-1 9+ cluster

10 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

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13 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): e doi: /journal.pone

14 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) % 5.3% -0.9% -0.3% IGF-II (PWH) % 6.5% -0.9% 2.6% % 4.4% N/A* N/A IGF-I (Overseas) 800 Slope = 0.97 Y-intercept = -2.8 Pearson R2 = 0.82 N = 150 G2XS G2XS Slope = 0.93 Y-intercept = -8.3 Pearson R2 = 0.90 N = QD 1 *N/A: not available Slope = 0.94 Y-intercept = 2.42 Pearson R2 = PWH 250 Accuracy assessment High-end (> 200 mg/l) Low-end (0 200 mg/l) PWH IGF-1 assay Instability (% deviation) Overseas laboratory assay QD RCPA 600 RCPA EQA median +: Royal College of Pathologist, Australia. External Quality Assurance.

15 IGF-I service since April 2016 Sample throughput: ~3000/year Reference interval adopted from the overseas laboratory What we have learnt thus far Collection tube interference; 2. Oxidized protein in EQA materials;

16 Interlaboratory comparison Comparison to an overseas laboratory using a similar method Y = 0.865X R2 = 0.81 N = CurrentPWH assay > 90% samples from certain hospitals were undetectable Quest Overseas laboratory

17 Pilot study Brand B serum tube IGF-I + IS Brand G serum tube Brand G serum microtube

18 m/z: 55.5 (double charge) -> mass difference = 111 Da Polyvinylpyrrolidone (PVP) polymer MW: 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):

19 MS N N+1 N+2 N Da Δ m/z 85 N Polyvinylpyrrolidone (PVP) polymer MSMS 85 Da N C4H7NO + N

20 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

21 Slope = Y-int = 6.6 R2 = 0.93 N = Without interference CurrentPWH assay Rate of detection = 100.0% Quest Overseas laboratory Slope = Y-int = 11.2 R2 = 0.55 N = With interference CurrentPWH assay Rate of detection = 73.1% Quest Overseas laboratory 1000

22 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

23 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

24 IGF-I and Internal standard Patient IGF-I IGF-I not reported Unknown interferent included for monitoring

25 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?

26 M M-1 M+2 M-2 M/M Concentration (mg/l) M/M+2 M/M Isotope ratio

27 35 30 IGF-I, isotope ratio (TOF) Mean = 1.56 RSD = 7.8% N = Conc All patient isotope ratio vs concentration Androstenedione, ion ratio (QQQ) Mean = 1.25 RSD = 14.5% N = Ratio Ang-I, ion ratio (QQQ) Mean = 0.96 RSD = 15.5% N = M/ Ratio ratio M Conc Conc (µg/l) Concentration Ratio

28 IGF-I and Internal standard Patient IGF-I IGF-I not reported Unknown interferent included for monitoring

29 2. Oxidized protein in EQA material

30 Oxidized material in EQA samples IGF-I contains exactly 1 methionine residue Wild-type IGF-I (7+) = m/z oxmet IGF-I (7+) = m/z 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.

31 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

32 Observed concentration (µg/l) RCPA EQA performance 1200 Total IGF-I y = x R² = Native IGF-I y = 0.769x R² = oxmet IGF-I y = 0.162x R² = RCPA median (µg/l) RCPA materials (N = 18) Patient samples (N = 20) NA Samples with detectable level of oxmet IGF-I (%) Relative abundance of oxmet IGF-I in detectable samples (%) IGF-I (mg/l) oxmet IGF-I (mg/l) Median Range Median Range

33 Summary Serum collection tube as a critical pre-analytical factors; Commutability of EQA material; Always check raw data.

34 Acknowledgement Funding support from Hospital Authority Resource Allocation Exercise (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

35 Thank you Faculty of Medicine The Chinese University of Hong Kong

36 3. How to interpret the results?

37 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

38 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

39 Case #2 F/11, short stature Body weight and height < 3rd percentile IGF-I: undetectable Mass spectrum Patient Quality control IGF-I EIC BPC

40 Mass spectrum Case #2 Collection tube interference PVP polymer Patient IGF-I not reported IGF-I and Internal standard Quality control