Stability of Fibroblast Growth Factor 23 in Human Plasma

Transcription

1 Stability of Fibroblast Growth Factor 23 in Human Plasma Salena Cui, 1 Sucheta M. Vaingankar, 2 Anneke Stenger, 3 Sushrut S. Waikar, 1 and David E. Leaf 1 * Background: Given the important emerging field of fibroblast growth factor 23 (FGF23) biology, there is a growing need for reliable data on FGF23 assay characteristics. We therefore evaluated the effects of different processing and storage conditions on FGF23 stability, as well as the assay precision of FGF23 measurements using 2 commercially available FGF23 ELISA kits. Methods: We measured plasma concentrations of intact FGF23 (ifgf23) and C-terminal FGF23 (cfgf23) in duplicate in 12 patients with a wide range of kidney function. We used blinded replicate samples to calculate the interassay CV for both assays. We processed the samples immediately after collection, after6hat22 C,orafter 24 h at 4 C. We also exposed samples to 0, 1, 2, or 3 freeze-thaw Results: The interassay CVs for ifgf23 and cfgf23 were 5.2% and 7.2%, respectively. Delayed processing for either6hat22 Cor24hat4 Chadnosignificant effect on either ifgf23 or cfgf23, although a nonsignificant trend toward decreased ifgf23 concentrations was observed compared with immediate processing (23% relative decline in concentrations under both delayed processing conditions). Three freeze-thaw cycles had no effect on either ifgf23 or cfgf23 concentrations. Conclusions: FGF23 measurements in human plasma are stable with delayed processing or after undergoing multiple freeze-thaw IMPACT STATEMENT Increased plasma fibroblast growth factor 23 (FGF23) concentrations are associated with and possibly directly related to adverse outcomes in patients with chronic kidney disease. Few studies have comprehensively evaluated the effects of different preanalytical processing and storage conditions on FGF23 stability. The findings in the current study suggest that FGF23 is stable in human plasma under a variety of different conditions, including delayed processing and multiple freezethaw 1 Division of Renal Medicine, Brigham and Women's Hospital, Boston, MA; 2 School of Medicine and 3 Division of Nephrology, University of California at San Diego, San Diego, CA. *Address correspondence to this author at: Renal Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA Fax ; deleaf@partners.org. DOI: /jalm American Association for Clinical Chemistry 4 Nonstandard abbreviations: FGF23, fibroblast growth factor 23; CKD, chronic kidney disease; ifgf23, intact fibroblast growth factor 23; cfgf23, C-terminal fibroblast growth factor 23; egfr, estimated glomerular filtration rate; RU, relative units. May : JALM 729

2 Fibroblast growth factor 23 (FGF23) 4 is an osteocyte-derived phosphaturic hormone that plays an important role in vitamin D metabolism (1). Over the past decade, FGF23 has emerged as a powerful biomarker of death (2, 3) and other adverse outcomes (4 6) in patients with chronic kidney disease (CKD). Moreover, recent studies in animal models suggest that increased plasma FGF23 concentrations in CKD patients may be directly implicated in the pathogenesis of left ventricular hypertrophy (7), immune dysfunction (8), and inflammation (9). Given the important emerging field of FGF23 biology, there is a growing need for reliable data on FGF23 assay characteristics. We therefore sought to determine the precision of 2 commonly used, commercially available FGF23 ELISA kits, and to test the effect of different preanalytical processing and storage conditions on the stability of FGF23. Specifically, we measured plasma intact FGF23 (ifgf23) and C-terminal FGF23 (cfgf23) concentrations in samples obtained from patients with a wide range of kidney function. METHODS 80 C until batched analysis. Samples remained at 80 C storage for up to 9 months before batched analysis. To investigate the effect of freeze-thawing on FGF23 stability, samples underwent 0, 1, 2, or 3 freeze-thaw For each freeze-thaw cycle, samples were thawed on ice for 2 h and then returned to 80 C storage. Samples remained at 80 C storage for up to 3 months before batched analysis. Assays FGF23 circulates predominantly in 2 forms: ifgf23, which is biologically active, and cfgf23, which is inactive. We measured both ifgf23 and cfgf23 in duplicate using second-generation ELISA kits (Immutopics) designed for research use only. The intact assay uses 2 polyclonal antibodies that bind to epitopes on N- and C-terminal fragments, while the C-terminal assay uses 2 polyclonal antibodies that recognize epitopes within the C-terminal portion only. Plates consisted of 12 eight-well strips for a total of 96 wells. Preparation and storage of assay kit components were according to the manufacturer's package insert, and all assays were performed by manual technique. Sample collection and processing All patients provided written informed consent, and all protocols were approved by our hospital's Institutional Review Board. We collected venous blood samples in EDTA-containing vacutainers from patients with varying concentrations of kidney function as follows: estimated glomerular filtration rate (egfr) >60 ml/min/1.73 m 2 (n = 3), egfr ml/min/1.73 m 2 (n = 3), egfr <30 ml/min/1.73 m 2 (n = 3), and end-stage kidney disease receiving hemodialysis (n = 3). To investigate the effect of delayed processing, samples were centrifuged immediately after collection, after 6 h at room temperature (22 C), or after 24 h at 4 C. Samples were centrifuged for 15 min at 1700g, and the plasma was extracted, aliquoted, and stored at Assay precision We used blinded replicate samples from patients with a broad range of kidney function to calculate the interassay CV for ifgf23 and cfgf23. Blinded replicate samples consisted of patient samples that were assigned random numerical identifiers and tested in duplicate across plates on different days. Specifically, ifgf23 and cfgf23 concentrations were measured in triplicate in 8 patients with a wide range of kidney function. For these analyses, 2 plates were used for ifgf23 and cfgf23 each, and measurements were conducted over 2 days. A single lot of reagents was used. In 2 additional patients, both of whom were receiving hemodialysis, we also measured ifgf23 concentrations 11 times across 11 plates on 11 days, and 730 JALM :06 May 2017

3 we measured cfgf23 concentrations 22 times across 22 plates on 22 days. A single lot of reagents was used for these measurements. Statistical analysis We used R version for all statistical analyses. We used paired 2-sample t-tests to compare FGF23 concentrations in samples obtained under different preanalytical storage conditions, i.e., immediate processing vs delayed processing for 6 h at 22 C, and immediate processing vs delayed processing for 24 h at 4 C. We used one-way ANOVA with repeated measures to test the effect of multiple freeze-thaw cycles on FGF23 concentrations (overall and stratified by egfr 30 vs <30 ml/min/1.73 m 2 ). We expressed concentrations as a percentage of their original value obtained under ideal conditions of immediate processing and without additional freeze-thaw All comparisons are 2-tailed, with P <0.05 considered significant. RESULTS FGF23 (% of original) P = 0.26 P = 0.35 ifgf23 (n = 4) P = 0.94 P = 0.87 cfgf23 (n = 4) Immediate 1439 (2407) 2439 (3498) 6 h at 22 C 1410 (2566) 2457 (3539) 24 h at 4 C 1343 (2435) 2405 (3444) Processing Immediate 6 h at 22 C 24 h at 4 C Fig. 1. Effect of delayed processing on FGF23 stability. FGF23 concentration as a percentage of the concentration measured under the reference condition (immediate processing). The bars represent mean (SE) relative concentration. Mean (SD) raw data are included at the bottom of the image. ifgf23 units are expressed in pg/ml. cfgf23 units are expressed in RU/mL. Time and temperature effect on FGF23 The effect of delayed vs immediate processing on ifgf23 and cfgf23 concentrations is shown in Fig. 1. On average, ifgf23 concentrations showed a trend toward decreased concentrations when processing was delayed by 6 h at 22 C[23% decrease (SD ±33.3%), P = 0.26] and when processing was delayed by 24 h at 4 C [23% decrease (SD ±42.1%), P = 0.35]; however, these changes were not statistically significant. In contrast, cfgf23 concentrations showed virtually no change when processing was delayed for 6 h at 22 C [0.03% decrease (SD ±0.9%), P = 0.94] or for 24 h at 4 C [0.2% decrease (SD ±2.4%), P = 0.87]. Freeze-thaw stability Next we investigated the effect of freeze-thawing on ifgf23 and cfgf23 concentrations. As shown in Fig. 2, up to 3 freeze-thaw cycles had no appreciable effect on ifgf23 concentrations. After 3 freeze-thaw cycles, ifgf23 concentration decreased by 4.6% from baseline (SD ±11.9%, P = 0.64). Among patients with an egfr 30 ml/min/ 1.73 m 2, ifgf23 concentrations decreased by 0.6% from baseline (SD ±7.5%, P = 0.23), and among patients with an egfr <30 ml/min/1.73 m 2, ifgf23 concentrations decreased by 13.4% from baseline (SD ±14.0%, P = 0.89) with 3 freezethaw We observed similar findings with cfgf23. After 3 freeze-thaw cycles, cfgf23 concentrations decreased by 1.1% from baseline (SD ±15.6%, P = 0.35). Among patients with an egfr 30 ml/min/ 1.73 m 2, cfgf23 concentrations increased by 0.9% from baseline (SD ±3.8%, P = 0.26), and among patients with an egfr <30 ml/min/1.73 m 2, May : JALM 731

4 120 P = 0.64 P = 0.23 P = 0.89 P = 0.34 P = 0.26 P = FGF23 (% of original ) Overall (n = 12) egfr 30 egfr <30 Overall (n = 12) egfr 30 egfr <30 ifgf23 #Freeze-thaw cycles cfgf23 #Freeze-thaw cycles (788) 65 (10) 861 (993) 382 (688) 74 (13) 752 (932) (780) 64 (12) 858 (979) 393 (720) 74 (11) 776 (980) (789) 70 (12) 868 (994) 395 (698) 76 (15) 778 (939) (778) 65 (12) 863 (974) 400 (766) 75 (14) 790 (1057) Fig. 2. Effect of freeze-thawing on FGF23 stability. FGF23 concentration as a percentage of the concentration measured under the reference condition (0 freeze-thaw cycles). The bars represent mean (SE) relative concentration. Mean (SD) raw data are included at the bottom of the image. ifgf23 units are expressed in pg/ml. cfgf23 units are expressed in RU/mL. cfgf23 concentrations decreased by 2.0% from baseline (SD ±24.0%, P = 0.58) with 3 freeze-thaw Assay precision The interassay CV for ifgf23, tested across a total of n = 46samples [mean (SD) ifgf23 concentration 696 (742) pg/ml], was 5.2%. When stratified by egfr 30 ml/min/1.73 m 2 [n = 12, mean (SD) ifgf23 concentration, 59 (8) pg/ml] vs <30 ml/min/1.73 m 2 [n = 34, mean (SD) ifgf23 concentration, 921 (742) pg/ml], the interassay CVs for ifgf23 were 1.9% and 6.3%, respectively. The interassay CV for cfgf23, tested across a total of n = 68 samples [mean (SD) cfgf23 concentration 6783 (12852) relative units (RU)/mL], was 7.2%. When stratified by egfr 30 ml/min/1.73 m 2 [n = 12, mean (SD) cfgf23 concentration 75 (13) RU/ ml] vs <30 ml/min/1.73 m 2 [n = 56, mean (SD) cfgf23 concentration 8571 (13952) RU/mL], the interassay CVs for cfgf23 were 0.9% and 7.8%, respectively. DISCUSSION In the current study, we evaluated 2 commercially available ELISAs commonly used for the measurement of plasma FGF23 concentrations in humans. We found the interassay CVs to be <10% for both ifgf23 and cfgf23. We found that de- 732 JALM :06 May 2017

5 layed processing for 6 h at 22 C or 24 h at 4 C had little effect on ifgf23 or cfgf23 concentrations. Additionally, we found that up to 3 freeze-thaw cycles had little effect on ifgf23 or cfgf23 concentrations. Notably, these findings were consistent across a wide range of kidney function levels. Prior studies have evaluated the stability of FGF23 in EDTA-containing vacutainers after delayed processing under a variety of conditions and have reported conflicting results. Using plasma samples from 15 healthy volunteers and 15 patients with mild-to-moderate CKD, Smith et al. (10) reported that delayed processing for 4 h at 22 C resulted in a significant decline in ifgf23 concentrations and a significant increase in cfgf23 concentrations compared to samples processed immediately. In a more recent study conducted in hemodialysis patients, Smith et al. (11) reported that delayed processing for 8hat22 C resulted in a significant average decline of 23% in ifgf23 concentrations but no change in cfgf23 concentrations. In contrast, using plasma samples from both healthy volunteers and dialysis patients, Fassbender et al. (12) reported that delayed processing of samples for 6 days at 4 C resulted in no significant difference in ifgf23 or cfgf23 concentrations compared to samples stored at 20 C. Similarly, using plasma samples from a single patient with tumor-induced osteomalacia, El- Maouche et al. (13) recently reported that delayed processing for up to 48 h at 4 C or 22 C had little effect on ifgf23 or cfgf23 concentrations compared to immediate processing, although delayed processing at 37 C decreased both ifgf23 and cfgf23 in a time-dependent fashion. Our findings are consistent with the studies by Fassbender et al. and El-Maouche et al. We speculate that the discrepancies between these studies vs the studies by Smith et al. could be due to method-specific differences in assays (e.g., first- vs second-generation ifgf23 assays) or differences in patient populations. Prior studies on the effect of freeze-thaw cycles on FGF23 stability are sparse. One study conducted in a single patient with tumor-induced osteomalacia found both ifgf23 and cfgf23 concentrations to be stable after 5 freeze-thaw cycles (samples frozen in dry ice) (13). In contrast, another study found that 3 freeze-thaw cycles ( 20 C to 24 C) caused a 37% decline in plasma ifgf23 concentrations, although the described results were unpublished (14). In the current study, we evaluated the effects of repeated freeze-thawing in 12 patients with a wide range of kidney function and found both ifgf23 and cfgf23 concentrations to be stable with up to 3 freezethaw We acknowledge several limitations including the small sample size. We limited our analyses to EDTA plasma samples, which is the recommended sample type for both of the FGF23 assays that were studied. We did not assess linearity-ofdilution; however, these investigations have been previously reported by others (11). We did not evaluate the effects of long-term storage of plasma at 80 C on FGF23 stability, although others have evaluated this and found only minor degradation of both ifgf23 and cfgf23 after frozen storage for up to 40 months (13). We did not evaluate the effects of storage of plasma at other temperature conditions, such as 20 C. Finally, other FGF23 ELISAs such as the Kainos assay for ifgf23 were not evaluated. In conclusion, we found that delayed processing had no significant effect on ifgf23 or cfgf23 concentrations, although we did observe a trend toward decreased ifgf23 concentrations. Multiple freeze-thaw cycles had no effect on ifgf23 or cfgf23 concentrations across a wide range of kidney function levels. May : JALM 733

6 Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: D.E. Leaf, National Institute of Diabetes and Digestive Kidney Diseases (K23DK106448), UAB-UCSD O Brien Center for Acute Kidney Injury Research (NIH P30-DK079337) to the institution; S.M. Vaingankar, National Institute of Diabetes and Digestive Kidney Diseases (R01DK094894), and the National Heart, Lung, and Blood Institute (R01HL108629); S.S. Waikar, National Institute of Diabetes and Digestive Kidney Diseases (R01DK093574, R01DK103784, U01DK104308, and U01DK085660). Expert Testimony: None declared. Patents: None declared. Role of Sponsor: No sponsor was declared. REFERENCES 1. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, Takeuchi Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 2004;19: Gutierrez OM, Mannstadt M, Isakova T, Rauh-Hain JA, Tamez H, Shah A, et al. Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. N Engl J Med 2008;359: Isakova T, Xie H, Yang W, Xie D, Anderson AH, Scialla J, et al. Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease. JAMA 2011;305: Wright CB, Shah NH, Mendez AJ, DeRosa JT, Yoshita M, Elkind MS, et al. Fibroblast growth factor 23 is associated with subclinical cerebrovascular damage: the Northern Manhattan Study. Stroke 2016;47: Mehta R, Cai X, Lee J, Scialla JJ, Bansal N, Sondheimer JH, et al. Association of fibroblast growth factor 23 with atrial fibrillation in chronic kidney disease, from the Chronic Renal Insufficiency Cohort Study. JAMA Cardiol 2016;1: Gutierrez OM, Januzzi JL, Isakova T, Laliberte K, Smith K, Collerone G, et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. Circulation 2009;119: Faul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, et al. FGF23 induces left ventricular hypertrophy. J Clin Invest 2011;121: Rossaint J, Oehmichen J, Van Aken H, Reuter S, Pavenstadt HJ, Meersch M, et al. FGF23 signaling impairs neutrophil recruitment and host defense during CKD. J Clin Invest 2016;126: Singh S, Grabner A, Yanucil C, Schramm K, Czaya B, Krick S, et al. Fibroblast growth factor 23 directly targets hepatocytes to promote inflammation in chronic kidney disease. Kidney Int 2016;90: Smith ER, Ford ML, Tomlinson LA, Weaving G, Rocks BF, Rajkumar C, et al. Instability of fibroblast growth factor- 23 (FGF-23): implications for clinical studies. Clin Chim Acta 2011;412: Smith ER, McMahon LP, Holt SG. Method-specific differences in plasma fibroblast growth factor 23 measurement using four commercial ELISAs. Clin Chem Lab Med 2013;51: Fassbender WJ, Brandenburg V, Schmitz S, Sandig D, Simon SA, Windolf J, et al. Evaluation of human fibroblast growth factor 23 (FGF-23) C-terminal and intact enzymelinked immunosorbent-assays in end-stage renal disease patients. Clin Lab 2009;55: El-Maouche D, Dumitrescu CE, Andreopoulou P, Gafni RI, Brillante BA, Bhattacharyya N, et al. Stability and degradation of fibroblast growth factor 23 (FGF23): the effect of time and temperature and assay type. Osteoporos Int 2016;27: Smith ER, McMahon LP, Holt SG. Fibroblast growth factor 23. Ann Clin Biochem 2014;51: JALM :06 May 2017