Stability of osteopontin in plasma and serum 1

Clin Chem Lab Med 2012;50(11):1979–1984 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/cclm-2012-0177 Stability of osteopontin in plasma and serum1) Patrizia Lanteri1,*, Giovanni Lombardi1, Alessandra Colombini1, Dalila Grasso1 and Giuseppe Banfi1,2 Keywords: immunodetection; osteopontin; pre-analytical phase; stability. 1 Experimental Biochemistry and Molecular Biology Laboratory, I.R.C.C.S. Istituto Ortopedico Galeazzi, Milan, Italy 2 Chair of Clinical Biochemistry, School of Medicine, University of Milano, Milan, Italy Abstract Background: Osteopontin is a glycoprotein widely expressed in many tissues and in different physiological conditions. Osteopontin concentrations are usually measured through immunological methods; however, little is known about the pre-analytical management of the sample. We evaluated the effects of different times and temperatures storage conditions on serum and plasma concentrations of osteopontin. Methods: Serum and plasma aliquots were frozen at –80°C, following storage at 4°C or room temperature for 0, 2, 4, 8, 12, 24 and 48 h. Osteopontin concentrations were determined by enzymoimmunometric assay. Serum samples obtained from tubes with or without gel separator were compared to verify the effect of gel. Western blotting analysis was performed to characterize the antibody. Results: Osteopontin concentrations were stable over time in all conditions in both serum and plasma. Plasma showed 3.8–4.8-fold higher concentrations than serum. Comparable levels were found between serum tubes with or without gel separator and always lower than those in plasma, demonstrating no effect of gel in serum tubes. Western blotting analysis showed various osteopontin bands, indicating that the antibody recognizes the entire panel of different osteopontin forms. Conclusions: We demonstrated the stability across 48 h of osteopontin in serum and plasma at either room temperature or 4°C, when the evaluation is carried out by an immunebased method. The minimal variations observed over time were always lower than the calculated intra- and inter-assay coefficients of variation. Plasma specimens should be preferred when osteopontin concentration are assayed by immunological methods. 1) This work was partially presented at the 1st EFCC-BD European Conference on Preanalytical Phase, Parma, April 2011. *Corresponding author: Dr. Patrizia Lanteri, PhD, I.R.C.C.S. Istituto Ortopedico Galeazzi, via R. Galeazzi, 4 – 20161, Milan, Italy Phone: +39 0266214068, Fax: +39 0266214060, E-mail: [email protected] Received March 21, 2012; accepted May 14, 2012; previously published online June 3, 2012 Introduction Osteopontin (OPN) is a phosphorylated acidic, integrin-binding, glycoprotein expressed by various cell types, including osteoblasts, osteoclasts, T-cells, macrophages and hematopoietic cells (1). This multifunctional molecule has been detected in many tissues and in all body fluids, and it is expressed in a series of physiological conditions (e.g., tissue remodeling, inhibition of biomineralization and chemotaxis) (2). Clinically, OPN, which acts as a potential pro-inflammatory molecule, has been associated with several diseases, including osteoarthritis, ischemia, rheumatic diseases and mesenchymal-derived tumors, and in response to injury or infection (1, 3). Expression of OPN is increased by steroids, retinoic acid and glucocorticoids as well as pro-inflammatory cytokines (4, 5). OPN modulates osteoclast differentiation and bone resorption (6) and it is involved itself in the inflammatory reactions by inducing adhesion, migration and survival of different cell types (2, 7, 8). The reported size of secreted protein varies from 44 kDa to 75 kDa (5, 9). The translated protein undergoes to a series of substantial post-translational modifications, namely glycosylation, transglutamination, sulfatation, serine/threonine phosphorylation, Ca2+-dependent polymerization by transglutaminase II and proteolytic cleavage, which generate different functional isoforms of OPN that can be found in the same or in different tissues (3, 5, 10, 11). Among these modifications, thrombin-cleavage enhances interactions with integrins allowing OPN to mediate cell-matrix and, possibly, cell-cell interactions (12, 13). The presence of a conserved thrombin cleavage site, within six amino acids downstream to the RGD cell-binding domain (the Arg-Gly-Asp sequence that binds integrins), suggests that OPN activity is dependent on thrombin cleavage (13–15). The interaction of OPN with its own receptors, RGD or not RGD-mediated, could depend on thrombin cleavage (16). Indeed, the proteolytic cut allows the exposure of cryptic binding sites which were previously hidden by the interactions between upstream and downstream located domains (17). In bloodstream, OPN is cleaved by thrombin wherever the blood coagulation pathway is activated, such as at sites of inflammation (13, 14, 18). The rapid cleavage of OPN in biological fluids determines a reduction of detectable levels of the intact molecule (19). Immunodetection represents the most used method to measure OPN concentration; however, antibodies recognizing different parts or molecule cleavage products are used. Brought to you by | Università degli Studi di Milano Authenticated | 159.149.103.9 Download Date | 10/3/13 3:11 PM 1980 Lanteri et al.: Pre-analytical phase of osteopontin For this purpose, it is necessary to obtain from the manufacturer the technical indications about the antibodies specificity. To our knowledge, very few studies reported variation of OPN levels in plasma and serum when different pre-analytical settings are applied (1, 19). Indeed, in 2007, Sennels and colleagues analyzed only the effect of freeze-thaw cycles on plasma samples (1), while, in 2010, Cristaudo and colleagues evaluated the effects of different storage conditions of whole blood (19). The study of the pre-analytical precautions that must be carried out for the correct measurement of a specific parameter, represents one of the most important concerns in clinical chemistry. Indeed, most detectable errors occur in the pre-analytical phase (20) and mainly during sample collection (21, 22), transport, sample treatment and storage, all of which will affect measurement accuracy, ultimately leading to uncertainty about results (23). Although unquestionably valuable clinical tools, little is known about needed pre-analytical cautions in the measurement of bone derived molecules, and particularly, in the pre-analytical phase of OPN (24). We aimed at evaluating the effects of different times and temperatures storage conditions on serum and plasma concentrations of OPN. Moreover, to better characterize the specificity of anti-OPN antibody used in diagnostic immunoenzymatic assay, Western blotting analysis was performed using the same antibody in order to detect the panel of OPN-isoforms recognized by the antibody. Materials and methods Blood drawings Blood samples were collected in the morning (08:00–10:00), following overnight fasting, from 11 resting healthy volunteers by standard antecubital venipuncture in plain (serum tubes without gel BD Plus Serum with clot activator 7 mL and thixotropic gel separation tubes BD SST™ II Advance 7 mL tubes) and K2EDTA (BD K2EDTA 3.5 mL tubes) tubes (BD Vacutainer Systems, Becton-Dickinson, Franklin Lakes, NJ, USA) of the same lot for each type. Immediately after drawing, tubes were gently inverted 10 times, plain tubes were kept at rest for 30 min prior to be centrifuged for 10 min, 1200 × g (2500 rpm), at 4°C, in order to separate the soluble fraction and the curpuscular one. Pre-analytical warnings, for what concern the blood drawings, were strictly followed to avoid any possible affecting factor on analytical phase (25). The study was approved by the reference Ethical Committee and an informed consent was signed by each subject. Pre-analytical settings Aliquots of serum samples obtained from tubes with gel separator (in the rest of the article named “gel serum”) and plasma were frozen at –80°C immediately, or following storage at 4°C or room temperature (RT) over different times from drawing (2, 4, 8, 12, 24 and 48 h). To verify differences between serum and plasma OPN content possibly due to the tube gel, aliquots of serum obtained from tubes without gel with clot activator (in the rest of the article named “clot activated serum”) were frozen at –80°C immediately after the blood drawing. ELISA test Serum and plasma OPN levels were assayed through an enzymelinked immune-sorbent assay (ELISA) using a capture monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA). Briefly, according to manufacturer specifications, microplates were coated for 18 h with the anti-hOPN capture antibody (2.0 µg/mL), washed three times with 0.05% Tween 20 in phosphate buffered saline (PBS), pH 7.3, and blocked for 1 h with 1% bovine serum albumin (BSA) in PBS, pH 7.3. Following three washes, diluted samples (1:25) in 1% BSA in PBS, pH 7.3 were added in duplicates and incubated for 2 h. The HRP-conjugated polyclonal detection antibody (0.2 µg/mL) (R&D Systems Inc.) was added and incubated for 2 h. Following three washes, a solution of 1:1 H2O2-TMB was added for 20 min and then the reaction was stopped with 1N H2SO4. All steps were performed at RT. The optical density was read at λ = 450 nm (VICTORTM X3, Perkin Elmer, Waltham, MA, USA) while reference reading was set at λ = 620 nm. The OPN concentration in samples was calculated against a standard curve built with reconstituted lyophilized OPN (R&D Systems Inc.). Samples and standards were assayed in duplicate. Intra- (n = 20 replicates) and inter-assay (n = 10 samples in 4 different assays) variability (CV%) were also determined. Western blotting analysis Gel serum and plasma OPN levels were analyzed in samples frozen at –80°C until assay or kept for 48 h at RT and then frozen at –80°C until assay, in order to investigate the specificity of the antibody used in the ELISA test. Equal amounts of proteins (corresponding to 15 µg) deriving from plasma or serum were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) under non-denaturating conditions. After separation, proteins were transferred to polyvinylidene difluoride membranes (PVDF) and immunoblotted using specific anti-OPN and anti-transferrin (as loading control) antibodies. Western blotting to OPN The non-specific binding sites were blocked with 5% non-fat dry milk in TBS-T buffer (10 mM Tris base, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) at RT for 1 h. The membrane was incubated with mouse monoclonal to osteopontin antibody (R&D Systems Inc.) in 5% milk TBS-T overnight at 4°C, followed by reaction with secondary horseradish peroxidase (HRP)-conjugated antibody (R&D Systems Inc.) for 45 min and enhanced chemiluminescence detection with Supersignal® West Dura Chemioluminescent Substrate (Thermo Scientific, Rockford, IL, USA). The image acquisition was performed using a Kodak Gel Logic 2200 imaging system (Eastman Kodak Company, Rochester, NY, USA). Film bands were quantified using Image J software (US National Institutes of Health, Bethesda, MD, USA) and measured values were normalized towards transferrin. Western blotting to transferrin After blocking with 5% non-fat dry milk in TBS-T buffer (10 mM Tris base, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) overnight at 4°C, the membrane was incubated with mouse monoclonal to transferrin antibody (R&D Systems Inc.) in 5% milk TBS-T at RT for 1 h. The PVDF was subsequently, incubated with secondary horseradish peroxidase (HRP)-conjugated antibody (R&D Systems Inc.) for 45 min and enhanced chemiluminescence detection with Supersignal® West Dura Chemioluminescent Substrate (Thermo Scientific). The image acquisition was performed using Kodak Gel Logic 2200 imaging system (Eastman Kodak Company). Film bands were quantified using Image J software (US National Institutes of Health). Brought to you by | Università degli Studi di Milano Authenticated | 159.149.103.9 Download Date | 10/3/13 3:11 PM Lanteri et al.: Pre-analytical phase of osteopontin Statistical analysis Statistical analyses were performed with GraphPad Prism v5.00 software (GraphPad software, La Jolla, CA, USA). For the ELISA tests, all values in the descriptive analysis were expressed as mean ± SD. Normal distribution of values was assayed by Kolmogorov-Smirnov normality test. One-way Analysis of Variance (ANOVA) for repeated measures, using Kruskal-Wallis test with Bonferroni’s correction, was performed to compare data over time; paired comparisons, for each timepoint, were performed by two-tailed paired t-test, between: i) same sample matrix and different storage conditions; and ii) different sample matrixes and same storage condition. Data obtained from the quantification of the Western blotting bands were compared by Friedman’s test with Dunn’s post-hoc test, in order to compare corresponding bands from samples stored at different conditions. Results In freshly separated plasma and gel serum, we found mean OPN concentrations of 43.96 ng/mL ± 9.10 and 10.98 ng/mL ± 6.20, respectively. The behavior of OPN concentrations over time in plasma and in gel serum was identical. No differences were found comparing samples stored at either 4°C or RT and frozen at different times for either plasma or gel serum. At 4°C, in both plasma and gel serum, OPN showed a slightly decreasing trend, but without reaching the statistical significance (Figure 1). On the contrary, when stored at RT, OPN concentrations in plasma and gel serum samples showed a faint, but not significant, increase (Figure 2). Interestingly, plasma concentrations were found to be always significantly higher (3.8–4.8-fold) than those of gel serum. OPN concentrations, for each timepoint, compared 1981 among the two different sample matrixes, were always significantly different (p < 0.0001). Differences between plasma and gel serum OPN concentrations were always higher than the calculated intra- and inter-assay variations (Table 1). Considering the same timepoint at different temperatures of storage, significantly high concentrations of OPN were found in gel serum when stored at RT compared to 4°C for 24 (p < 0.05) and 48 h (p < 0.001). However, in plasma samples higher OPN concentrations were found following storage at RT for 4, 8 (p < 0.001), 12 and 24 h (p < 0.05). These differences were always lower than the calculated analytical variability. In order to verify if the differences in OPN concentration between plasma and gel serum could be due to the gel, acting as a capture matrix, additional ELISA tests were performed on freshly separated soluble fractions of whole blood (plasma EDTA, gel serum and clot activated serum). Interestingly, other than the already observed difference between plasma and gel serum, we found comparable values of OPN in clot activated serum (8.15 ng/mL ± 5.74) with those of gel serum and, thus, significantly lower OPN concentrations than plasma (p < 0.0001). To further characterize the antibody used in the ELISA test, Western blotting analysis was performed by using the same antibody both in samples kept for 48 h at RT and then frozen at –80°C until assay (48 h) and in freshly separated samples (0 h). In both plasma and gel serum we observed different bands (Figure 3A): two between 150 and 100 kDa (a), one, darker, at about 70 kDa (b) and an additional one at 25 kDa (c). Western blotting analysis to transferrin was carried out as loading control. A specific band, in both plasma and serum, was detected at approximately 90 kDa (Figure 3B). Film bands were quantified by densitometry, and values were normalized towards transferrin. No significant differences Storage 4°C 60 50 OPN, ng/mL 40 30 20 10 0 0 2 4 8 12 24 Time, h 48 Figure 1 Time course of OPN concentrations (ng/mL) in plasma ( ) and gel serum () samples following storage at 4°C. Values are presented as mean ± SD. ANOVA for repeated measures was performed to compare data over time. No significant differences in OPN concentrations, for each matrix, among different timepoints, have been reported. OPN concentrations, for each timepoint, between different sample matrixes, are always significantly different (two-tailed paired t-test), p < 0.0001. Brought to you by | Università degli Studi di Milano Authenticated | 159.149.103.9 Download Date | 10/3/13 3:11 PM 1982 Lanteri et al.: Pre-analytical phase of osteopontin Storage RT 60 50 OPN, ng/mL 40 30 20 10 0 0 2 4 8 12 24 Time, h 48 Figure 2 Time course of OPN concentrations (ng/mL) in plasma ( ) and gel serum () samples following storage at RT. Values are presented as mean ± SD. ANOVA for repeated measures was performed to compare data over time. No significant differences in OPN concentrations, for each matrix, among different timepoints, have been reported. OPN concentrations, for each timepoint, between different sample matrixes, are always significantly different (two-tailed paired t-test), p < 0.0001. were observed among densities of identical bands in samples stored at different conditions. Discussion A number of papers reported a wide in vitro instability of OPN (1, 13, 14, 26). However, these studies did not consider the possible variation of OPN concentration during a time course and in different storage conditions. Recently, efforts have been spent in defining the tissue-specific expression pattern of this molecule and, thus, its putative role as marker in a variety of pathological conditions (2, 5, 15). For example, blood levels of OPN have been reported to correlate with numerous pathological situations, including osteoarthritis, ischemia, rheumatoid arthritis and mesenchymal-derived tumors, and in response to injury or infections (1, 3, 17). Even though many studies have been performed, the usefulness of OPN as diagnostic and/or prognostic marker for this wide variety of inflammatory diseases was not definitely assessed. Pre-analytical modifications of protein concentration represented a source of differences affecting the clinical Table 1 Intra- and inter-assay variations of OPN ELISA test. OPN, ng/mL Within-run CV% Between-run CV% 10.00 44.00 3.00 3.35 2.31 2.26 Within-run precision was determined for 20 replicates of four samples on four plates. Between-run precision was determined for 10 samples in four separates evaluations. validity of data and their interpretation. However, it must be considered that the experimental conditions and the kind of sample and assay, often not so well specified, could play a pivotal role on these differences and the similarities among these studies could be explained. In 2007 Sennels and colleagues reported the biological variations and the reference interval for circulating OPN in plasma samples obtained from 300 Danish healthy blood donors (1); however, the authors did not report the epitope-specificity of the antibody used in the ELISA test. In our study we analyzed the OPN concentrations in plasma and serum by ELISA test using that same monoclonal antibody: the OPN concentrations we measured were similar to the previously described ones (median of 51 µg/L) (1), but it was not possible to define neither the specific epitope recognized by the antibody nor the detected isoforms (intact or modified molecule). The manufacturer of the antibody, indeed, did not indicate the antibody specificity reporting a general OPN identification. However, since we did not observe any significant variation in plasma and gel serum OPN concentrations, in samples stored at RT or 4°C, among the different timepoints and, since it was observed that the molecule is unstable in blood due to proteolytic activities (1, 13, 19), we can argue that our ELISA test was able to detect a panel of OPN isoforms currently present in the bloodstream. Probably, different assays use antibodies which recognize specific more sensible forms. Real and accurate specificity of the antibodies definition by producers is acknowledged. In the absence of that, we demonstrated that the use of an antibody which recognizes all the protein forms could assure an effective stability of OPN across a 48-h long period, yielding the assay reliable and clinically valid. Moreover, considering different temperatures of storage, significant higher concentrations of OPN, Brought to you by | Università degli Studi di Milano Authenticated | 159.149.103.9 Download Date | 10/3/13 3:11 PM Lanteri et al.: Pre-analytical phase of osteopontin A OPN 48 h P S B 0h P Transferrin 48 h P S S 0h P S kDa kDa 250 150 100 75 50 250 150 100 75 50 37 37 25 25 20 20 15 15 a a b c 1983 Figure 3 Western blotting analysis performed in both plasma (P) and gel serum (S) using an anti-OPN (A) and anti-transferrin (B) antibodies both in samples kept for 48 h at RT and then frozen at –80°C until assay (48 h) and in freshly separated samples (0 h). (a) Two OPN bands detected at apparent molecular masses between 150 and 100 kDa. (b) OPN band detected at about 70 kDa. (c) OPN band detected at molecular mass of 25 kDa. both in plasma and in serum, were observed at each timepoint when samples were stored at RT compared to 4°C. Moreover, higher OPN levels were observed in plasma rather than in gel serum (3.8–4.8-fold). We hypothesize that it could be due to the different intrinsic characteristics of plasma and serum. Our findings confirmed this hypothesis: OPN levels in both kinds of serum tubes (with or without gel) were comparable and always lower than those measured in plasma, demonstrating that this is not an effect due to the presence of the gel into the serum tubes; the minimal variations observed over the time were always lower than the calculated intra- and inter-assay ones. Possible explanations to this phenomenon are: i) OPN could be sequestered into the clot; ii) Ca2+-dependent thrombin activation, taking place in serum during the clot formation, could result in the proteolytic cleavage of the molecule and in loss of immunoreactivity; and iii) Ca2+-dependent transglutaminase II activity, more pronounced in serum rather than in plasma, since the higher free Ca2+ concentration could modify OPN immunoreactivity. In plasma, lower Ca2+ concentration would result in a lower degree of transglutaminase II activation possibly resulting in higher number of monomeric forms of OPN in the bloodstream. On the contrary, in serum, the higher degree of transglutaminase II activation would result in higher level of polymeric OPN, which is likely to be more easily sequestered in clot. Polymerization would hide the traditional integrin binding site, probably generating a new one (11), inducing a reduction of OPN immunoreactivity proportional to the amount of protein polymerized. However, current attempts are focused in clarifying all these aspects. To further characterize the antibody used in the ELISA test, Western blotting analysis was performed using the same antibody: in both freshly separated plasma and serum different bands were observed. The same protein pattern was observed in plasma/serum following storage for 48 h at RT before freezing. According to the literature, we can define that the upper bands represent polymerized forms of OPN (9, 12, 27), that differ in multimer size, the lower band is the thrombin-cleaved OPN fragment (9, 27) and the middle band is the intact OPN (10). The stability of OPN concentrations could be explained by the ability of this monoclonal antibody to recognize the thrombin-cleaved products, which rises after exposure to proteolytic cleavage, as well as the intact isoforms (native and post-translationally modified). The statistical analysis on Western blotting data demonstrated that thrombin-cleaved OPN fragment form did not increase over the time, confirming the data obtained by the ELISA test. However, while plasma concentrations were found to be always significantly higher than those of serum when assayed by ELISA test (differences were always higher than the calculated intra- and inter-assay variations), as previously observed by Cristaudo and colleagues (19), no differences were observed between OPN bands in plasma and serum on Western blotting analysis. Anyway, some differences between Cristaudo’s work and ours have to be pointed out: in their study, they evaluated the effects of storage length, at RT or at 4°C, of whole blood before plasma/serum separation. Consequently, unlike what we did, Cristaudo and colleagues observed that serum was less stable than plasma over time, but this may be due to the different method they used for samples treatment. In conclusion, the stability of OPN in serum and plasma for 48 h at either RT or 4°C was demonstrated. Plasma specimens should be preferred when immunoassay methods and used for measuring the protein. A warning about the interpretation of previously published data concerning OPN, when preanalytical and analytical conditions are not properly detailed, should be outlined, considering that these conditions widely affect measurements. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this Brought to you by | Università degli Studi di Milano Authenticated | 159.149.103.9 Download Date | 10/3/13 3:11 PM 1984 Lanteri et al.: Pre-analytical phase of osteopontin article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: This work was funded by the Italian Ministry of Health Employment or leadership: None declared. Honorarium: None declared. References 1. Sennels HP, Jacobsen S, Jensen T, Hansen MS, Ostergaard M, Nielsen HJ, et al. Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high-sensitivity C-reactive protein. 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Transglutaminase-catalyzed cross-linking of osteopontin is inhibited by osteocalcin. J Biol Chem 1997;272:22736–41. Brought to you by | Università degli Studi di Milano Authenticated | 159.149.103.9 Download Date | 10/3/13 3:11 PM