an experimental demonstration of the matrix effect ...

Vol. 74 | No. 5/1 | May 2018 DOI: 10.21506/j.ponte.2018.5.4

International Journal of Sciences and Research

AN EXPERIMENTAL DEMONSTRATION OF THE MATRIX EFFECT ASSOCIATED WITH THE USE OF HEPATITIS B IMMUNOGLOBULIN ON IMMUNOSUPPRESSANT LEVELS MEASURED WITH THE LC-MS/MS METHOD Ataman Gonel (Corresponding Author) Department of Biochemistry/Harran University/Turkey/[email protected] Ismail Koyuncu Department of Biochemistry/ Harran University/Turkey/[email protected]

Abstract Some of the liver transplant patients need to use hepatitis B immunglobulin (HBIG) preparations due to hepatitis B reinfections. Blood level measurement of immunosuppressants used to prevent rejection is performed by immunoassay methods and LC-MS/MS reference method. It is known that immunoassay methods are influenced by immunoglobulins. Therefore, the LC-MS/MS method should be preferred in order to minimize the risk of organ rejection. Although LC-MS/MS is the reference method, some molecules in the serum may cause incorrect measurement of the analyte through matrix effect that is changing ionization efficiency. The aim of this article is to investigate the exposure of immunosuppressant blood level to the matrix effect with HBIG drug used in liver transplant patients Experimentally, it was observed that low and medium blood concentrations of tacrolimus, everolimus, sirolimus and cyclosporin were significantly increased in the interference study by adding HBIG (Hepatect CP 50 IU) to the control materials. Amount of increase is between 15,38% - 35,61%. Incorrect laboratory results may lead clinicians to adjust the wrong blood drug dose. It is vital to keep the immunosuppressive drug levels in the therapeutic index in organ transplant patients. Immunosuppressants should be measured from blood sample that is taken prior to HBIG administration, since incorrect measurements caused by the matrix effect would increase the risk of liver rejection.

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Keywords: immunosuppressant, LC-MS/MS, matrix effect, HBIG, interference

Introduction In some liver transplant patients, there is a need for hepatitis B immunoglobulin preparates related to previously contracted hepatitis B infection [1]. These types of preparates are effective both in protecting against infection and in preventing organ rejection [2]. Serum levels of immunosuppressants must be kept at a specific therapeutic dose in these patients to prevent rejection. To ensure this, medication levels must be checked periodically and oral doses should be adjusted accordingly. Therapeutic drug monitoring (TDM) of immunosuppressants is applied with immunoassay (IA) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods [3]. The levels of cyclosporin A, tacrolimus, sirolimus and everolimus in the blood from immunosuppressant drugs are generally measured with IA methods. Many IA methods have been developed for TDM [4]. In the majority of IA methods there is exposure to interference originating from metabolites of the main drug [5]. At the same time, immunoglobulins can cause incorrect measurements of immunosuppressant levels found in the serum by creating an immunocomplex or by competing with enzyme-marked immunoglobulins found in the IA reactives (Figure 1). These kinds of interferences have a negative effect on the follow-up of immunosuppression [6, 7]. It is not known how many patients to date have been exposed to organ rejection because of incorrect immunosuppressant measurement. When the factors causing incorrect immunosuppressant measurement are not prevented, there is a serious risk of morbidity and mortality. It cannot be predicted which component will create the interference effect in the blood. When a result with interference from the laboratory is accepted as true by the clinician, the immunosuppression treatment will continue. Due to these types of disadvantages of the IA method,

chromatographic

methods

are

recommended

for

the

measurement

of

immunosuppressives. LC-MS/MS is the accepted gold standard, although despite acceptance as such, incorrect results may be given in this method because of the matrix effect [6-8]. The matrix effect is the incorrect measurement of an analyte because of the change in ionisation productivity of some molecules found in the matrix. This event was first proven by Tang and Kebarle (1993) with the demonstration that as organic-based concentrations increased, the 38



electrospray reactions of analytes decreased. Although the mechanism of the matrix effect is not known, it is thought to most probably originate from matrix components that cannot be determined or associated with an analyte [9]. As the matrix effect has a negative effect on the accuracy and sensitivity of analyte data, this seems to be the most important problem in LC-MS/MS methods that are accepted as reference methods [10]. The agents causing the matrix effect in the LC-MS/MS method are shown in Table 1. Table1: Factors causing the matrix effect in the LC-MS/MS method [11]. S. No Agent Explanation Carbohydrates, lipids, phospholipids, 1 Endogenous components in the matrix proteins, bile salts etc. Formulation assistive substances, components which may leak from 2 Exogenous components in the matrix laboratory equipment, anticoagulants, analyte balancers, sample preparation reactives. Fragmented components and prodrug 3 Fragmented products originating from the analyte sensitive to pressure, pH, temperature or light. Found in analytes and standard compound 4 Impurities and salts content Binding to biological matrix or sample 5 Poor recovery of analytes dishes 6 Solvents and additives Used in LC Other drugs present in the blood sample of 7 Xenobiotics and their metabolites the patient

Literature Review The aim of this study was to raise awareness of the causes of interference created during the measurement of immunosuppressants with the immunoassay method, and to test the exposure of immunosuppressants to the matrix effect with HBIG, which is used in liver transplant patients in particular, in the LC-MS/MS method, although it is a reference method.

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Methodology Materials: A Jasem brand, 6-level calibrator (Lot: CL-3000420150616) and a 3-level control solution (Lot: CL-3000620150616) were used (Tables 2, 3). For the interference assay, a Hepatect CP 50 IU flacon HBIG preparate was used. All solvents and reagents were of HPLC or analytical grade and were purchased from JASEM Laboratory Systems and Solutions A.S. Nitrogen (99.999%) was purchased from Harran University Central Laboratory. Table 2: Levels of calibrators S. No

Drug

1 2 3 4

Cylosporine A Tacrolimus Sirolimus Everolimus

Table 3: Levels of controls S. No Drug 1 Cylosporine A 2 Tacrolimus 3 Sirolimus 4 Everolimus

Level-0 ug/L 0 0 0 0

Level-1 ug/L 25.2 1.36 1.52 1.35

Level-1 ug/L 53.15±7.27 3.61±0.46 3.71±0.46 3.65±0.45

Level-2 ug/L 47.8 2.80 2.98 2.79

Level-2 ug/L 118±12 7.47±0.74 11.25±1.12 11.33±1.13

Level-3 ug/L 91 5.50 5.92 5.64

Level-4 ug/L 180 11.3 12.5 11.5

Level-5 ug/L 446 22.8 24.3 23.8

Level-6 ug/L 1258 46.3 49.5 46.5

Level-3 ug/L 227.5±22.75 14.6±1.45 18.25±1.82 18.75±1.87

Instrumentation: Shimadzu Nexera X2 ultra high performance liquid chromatograph (UHPLC) coupled with a Shimadzu 8045 triple quadrupole mass spectrometer (MS/MS) (Shimadzu, Japan) was used. All data collected were reprocessed using Shimadzu Software, which automatically calculated the concentration of each compound. Sample Preparation: 500µL of control solution was placed in a centrifuge tube, and after the addition of 25µL internal standard, this was mixed with a vortex for 5 seconds. Then 975µL of precipitation property reagent 1 was added and it was mixed again with a vortex for 15 secs. The mixture obtained was centrifuged for 5 mins at 3000 rpm. The supernatant was withdrawn into a vial and read on the Shimadzu 8045 LC-MS/MS device. Similar procedures were repeated for level 1, level 2, and level 3 control solutions and readings were taken on the device. The first stage and the reading procedures were repeated for the interference assay by adding 50µL distilled water to each control level, For each control level, 50µL was taken from the Hepatect CP 50 IU 10mL flacon and the measurements were repeated. Ten separate repetitions were made for each level of control solution of control +50µL distilled water and

40



control + 50µL Hepatect CP 50 IU solutions and the mean values were taken for the calculation of the bias percentage according to the bias formula. To discount interference that could occur associated with volume expansion at each control level, the results obtained by adding 50µL distilled water were accepted as the target value.

Results In the results obtained by adding level 1 control solution, there was observed to be a significant deviation from the target value of 15.47%-35.61%. In the results obtained by adding the level 2 control solution, the deviation from the target value was 15.38%-21.20%. The deviation percentages with the addition of the level 3 control solution were not significant. At level 3, minimal reductions were observed of 1.59% in everolimus level and 0.79% in tacrolimus level. Sirolimus and Cyclosporin A levels increased by 2.34% and 1.32% respectively (table 4).

Control Sample +50 uL water (V1) level1 2.65 1 level2 6.37 Everolimus 3-8 level3 11.29 level1 3.96 2 level2 10.98 Sirolimus 3-18 level3 19.19 level1 2.86 3 level2 5.00 Tacrolimus 1.78-19.2 level3 11.35 level1 47.76 4 level2 86.39 Cyclosporin A 50-400 level3 178.79 Table 4: Measurement results from HBIG added samples S. No Drug

Therapeutic index (ng/dL)

Control Levels

41

Control Sample +50uL Hepatect CP 50 IU (V2) 3.06 7.35 11.11 5.37 13.27 19.64 3.47 6.06 11.26 63.08 101.57 181.15

Bias

Bias (%)

0.41 0.98 -0.18 1.41 2.29 0.45 0.61 1.06 -0.09 15.32 15.18 2.36

15.47 15.38 -1.59 35.61 20.86 2.34 21.33 21.20 -0.79 32.08 17.57 1.32



Discussion HBIG is extremely successful in the preventionof hepatitis B re-infection following liver transplantation (LT). Furthermore, immunoglobulins are known to affect the measurements of immunosuppressants used by patients. This interaction is between the antibodies in the HBIG drugs and the immunoglobulins used in the immunoassay kits (Figure 1). As a result of competitive inhibition in the interaction, low or high immunosuppressant levels determined are misleading for clinicians [12]. These types of interferences are a significant risk factor for organ rejection. In previous studies, mean cyclosporin concentrations have been shown to have been measured 12%-40% higher with ACMIA (Automated Antibody Conjugated Magnetic Immunoassay), EMIT (Enzyme multiplied immunoassay Technique), CEDIA (Cloned Enzyme Donor Immunoassay), and FPIA (Fluorescence polarization immunoassay), compared with chromatographic methods [13-17]. Soldin et al (2010) reported that elevated blood cyclosporin levels were related to the presence of endogenous antibodies in the ACMIA cyclosporin test assayed on the Dimension RXL analyser. De Jonge et al reported that the cyclosporin level of 492 ng/mL in a 77-year old patient was incorrect as when the measurement was made using LC-MS/MS, cyclosporin was not determined [18]. Although the main reason for interference in the measurement of tacrolimus is the metabolites of the drug, low hematocrit values and incorrect tacrolimus concentrations have been reported in the MEIA (microparticle enzyme-linked immunoassay) method of the AXSYM device [5]. Westley et al compared the CEDIA and MEIA methods with LC-MS/MS in respect of kidney transplants and determined a 33.1% bias in the CEDIA method and a 20.1% bias in the MEIA method [19]. Bazin et al evaluated the CMIA (Chemiluminescent Microparticle Immunoassay) architect tacrolimus test and observed a mean 20% bias between the values determined with LC-MS/MS and the CMIA test [20]. The ACMIA tacrolimus test is affected by rheumatoid factors and endogenous heterophilic antibodies. Altinier et al described heterophilic antibody interference in the ACMIA tacrolimus method. In a case study it was reported that after termination of the treatment, the therapeutic level of tacrolimus determined in the blood was probably due to heterophilic antibodies [21]. Sirolimus is also exposed to interference of metabolites in immunoassay measurements. Morris et al determined bias at 49.2% in measurements made with the MEIA method

42



compared to LC-MS/MS [22]. Schmidt et al evaluated CMIA sirolimus analysis on an architect analyser and determined that sirolimus had entered a cross-reaction with metabolites. In another study, CMIA was compared with the LC-MS/MS method and deviation between the mean values was observed from 14% up to 39%. Higher results were obtained from the CMIA method than in LC-MS/MS [23]. Holt et al reported positive bias of 21.9% in the CMIA method compared to LC-MS/MS [24]. In a study that evaluated the everolimus level with the Quantitative Microsphere System (QMS) immunoassay method in 90 samples, the everolimus values determined with the QMS everolimus test were found to be mean 11% higher than the values obtained with the LCMS/MS method [25]. In a study by Hoffer et al of 169 patient samples, the mean everolimus concentration in the QMS everolimus test was determined to be 31.2% higher than that determined with the LC-MS/MS method. As a result of analysis with the QMS everolimus test, 69% of the samples were determined at a supratherapeutic concentration, and the everolimus values determined with the LC-MS/MS method were in the therapeutic range [26]. Sallusto et al observed deviation of 30% between the measurements of everolimus with FPIA and LC-MS/MS [27]. Although many studies have shown interference from exposure to immunoglobulins in immunoassay methods, there has been no previous study that has examined how the matrix effect in the LC-MS/MS method is affected by the HBIG drug that has to be used for liver transplant patients in particular. In LC-MS/MS measurement, no interference is expected associated with competing antibodies as in the IA method. However, the matrix effect, which is observed as a change in ionization activity in the presence of flammable substances, changes the analyte results [28]. It must not be forgotten that even if measurements of tests with some critical importance made with this reference method provide the clinician with confidence, there is a possibility of deviations associated with interactions during the measurement.

Therefore,

by

affecting

the

measurement

concentrations

of

immunosuppressants, the presence in the blood of large molecule drugs such as HBIG, which are used in liver transplant patients in particular, can cause misinterpretation by clinicians. In this study, by adding HBIG (Hepatect CP 50 IU) to the control materials, for which the mean values were fully known, in the interference study, the low and moderate blood concentrations of tacrolimus, everolimus, sirolimus and cyclosporin A were observed to have 43



significantly increased. The increases were in the range of 15.38%-35.61%. If clinicians accept false high values positively affected at this level by drugs with a narrow therapeutic index (TI) and reduce the drug dose, there is an increased risk of rejection. The change in drug level at higher concentrations is at a minimal level such as -1.59%- +2.34%. In a series of experiments, King et al showed that the matrix effects resulted in competition between nonvolatile matrix components and between analyte ions and analyte ions in the transition to the gas phase [29]. The finding of the current study that the matrix effect was minimalised as the immunosuppressant concentrations increased confirmed the competition determined by King et al at the ionisation stage. The effectiveness of the formation of analyte ions depends on the matrix density entering the electrospray ion source. Bonfiglio et al reported that the chemical nature of a component had a significant effect on the degree of the matrix effect. In a study of 4 components of different polarities under the same mass spectrometric conditions, the most polarised was determined to have the largest ion suppression rate and the least polarised was less affected by ion suppression [30]. In some studies it has been shown that signal suppression in the emergence of the matrix effect is complex and includes many factors. It is believed that gas phase proton transfer reactions and competition in high viscosity are major factors in the formation of the matrix effect [31].

Figure 1: Interference examples of endogenous antibodies in the immunoassay method.

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(A) Sandwich immunoassay; EA may be connected to both capture and detection antibodies. The parasite signal formed causes a falsely elevated result. (B) Competitive immunoassay; the EA is seen to be connected to both a capture antibody and a labelled analogue (not an analyte). In this situation the parasite gives a false low result [32].

Figure2: The matrix effect creating an incorrect measurement in the LC-MS / MS method

Conclusions Although the LC-MS/MS method is a reference method with high specificity, excellent sensitivity and accuracy in the measurement of immunosuppressant drugs, the matrix effect must be carefully evaluated. The matrix effect can cause incorrect test results. Especially in liver transplant patients, the measurement of blood immunosuppressants must be made before the injection of immunoglobulins such as Hepatect CP, which is used to prevent hepatitis B re-infection. It is extremely important to maintain the immunosuppressant drug levels within the therapeutic index in organ transplant patients. Moreover, it must not be forgotten that analytical errors occurring in the immunosuppressant measurement have life-threatening organ rejection risks. Until the development of methods that will eliminate these types of effects, the administration of drugs that will increase the molecule density in the matrix in the pre-analytical phase should be avoided.

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References [1] R. Adler, R. Safadi, Y. Caraco, M. Rowe, A. Etzioni, Y. Ashur, D. Shouval, Comparison of immune reactivity and pharmacokinetics of two hepatitis B immune globulins in patients after liver transplantation, Hepatology 29(4) (1999) 1299-1305. [2] S.Y. Wong, J. Levitsky, Chronic rejection related to hepatitis B immunoglobulin discontinuation in a liver transplant recipient, Transplant International 24(12) (2011). [3] A.J. McShane, D.R. Bunch, S. Wang, Therapeutic drug monitoring of immunosuppressants by liquid chromatography–mass spectrometry, Clinica Chimica Acta 454 (2016) 1-5. [4] Y. Zhang, R. Zhang, Recent Advances in Analytical Methods for the Therapeutic Drug Monitoring of Immunosuppressive Drugs, Drug testing and analysis (2017). [5] Y. Armendáriz, S. García, R.M. Lopez, L. Pou, Hematocrit influences immunoassay performance for the measurement of tacrolimus in whole blood, Therapeutic drug monitoring 27(6) (2005) 766-769. [6] E. Tudela, G. Muñoz, J.A. Muñoz-Guerra, Matrix effect marker for multianalyte analysis by LC– MS/MS in biological samples, Journal of Chromatography B 901 (2012) 98-106. [7] C. Côté, A. Bergeron, J.-N. Mess, M. Furtado, F. Garofolo, Matrix effect elimination during LC– MS/MS bioanalytical method development, Bioanalysis 1(7) (2009) 1243-1257. [8] P. Kaczyoski, Clean-up and matrix effect in LC-MS/MS analysis of food of plant origin for high polar herbicides, Food chemistry 230 (2017) 524-531. [9] L. Tang, P. Kebarle, Dependence of ion intensity in electrospray mass spectrometry on the concentration of the analytes in the electrosprayed solution, Analytical chemistry 65(24) (1993) 3654-3668. [10] Y. Huang, R. Shi, W. Gee, R. Bonderud, Matrix effect and recovery terminology issues in regulated drug bioanalysis, Bioanalysis 4(3) (2012) 271-279. [11] H.-J. Kim, J.-S. Kang, Matrix effects: Hurdle for development and validation of bioanalytical LC– MS methods in biological samples analyses, Biodesign 4 (2016) 46-58. [12] S.H.B. Han, J. Ofman, C. Holt, K. King, G. Kunder, P. Chen, S. Dawson, L. Goldstein, H. Yersiz, D.G. Farmer, An efficacy and cost‐effectiveness analysis of combination hepatitis B immune globulin and lamivudine to prevent recurrent hepatitis B after orthotopic liver transplantation compared with hepatitis B immune globulin monotherapy, Liver Transplantation 6(6) (2000) 741-748. [13] W. Steimer, Performance and specificity of monoclonal immunoassays for cyclosporine monitoring: how specific is specific?, Clinical chemistry 45(3) (1999) 371-381. [14] E. Schütz, D. Svinarov, M. Shipkova, P.-D. Niedmann, V.W. Armstrong, E. Wieland, M. Oellerich, Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and Emit): a critical overview of performance characteristics and comparison with HPLC, Clinical chemistry 44(10) (1998) 2158-2164. [15] A. Hamwi, M. Veitl, G. Männer, K. Ruzicka, C. Schweiger, T. Szekeres, Evaluation of four automated methods for determination of whole blood cyclosporine concentrations, American journal of clinical pathology 112(3) (1999) 358-365. [16] A.R. Terrell, T.M. Daly, K.G. Hock, D.C. Kilgore, T.Q. Wei, S. Hernandez, D. Weibe, L. Fields, L.M. Shaw, M.G. Scott, Evaluation of a no-pretreatment cyclosporin A assay on the Dade Behring dimension RxL clinical chemistry analyzer, Clinical chemistry 48(7) (2002) 1059-1065. [17] A.W. Butch, A.M. Fukuchi, Analytical performance of the CEDIA® cyclosporine PLUS whole blood immunoassay, Journal of analytical toxicology 28(3) (2004) 204-210. [18] H. de Jonge, I. Geerts, P. Declercq, H. de Loor, K. Claes, K. Desmet, D.R. Kuypers, Apparent elevation of cyclosporine whole blood concentrations in a renal allograft recipient, Therapeutic drug monitoring 32(5) (2010) 529-531. [19] I.S. Westley, P.J. Taylor, P. Salm, R.G. Morris, Cloned enzyme donor immunoassay tacrolimus assay compared with high-performance liquid chromatography-tandem mass spectrometry and microparticle enzyme immunoassay in liver and renal transplant recipients, Therapeutic drug monitoring 29(5) (2007) 584-591.

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[20] C. Bazin, A. Guinedor, C. Barau, C. Gozalo, P. Grimbert, C. Duvoux, V. Furlan, L. Massias, A. Hulin, Evaluation of the Architect® tacrolimus assay in kidney, liver, and heart transplant recipients, Journal of pharmaceutical and biomedical analysis 53(4) (2010) 997-1002. [21] S. Altinier, M. Varagnolo, M. Zaninotto, P. Boccagni, M. Plebani, Heterophilic antibody interference in a non-endogenous molecule assay: an apparent elevation in the tacrolimus concentration, Clinica Chimica Acta 402(1-2) (2009) 193-195. [22] R.G. Morris, P. Salm, P.J. Taylor, F.A. Wicks, A. Theodossi, Comparison of the reintroduced MEIA® assay with HPLC-MS/MS for the determination of whole-blood sirolimus from transplant recipients, Therapeutic drug monitoring 28(2) (2006) 164-168. [23] R.W. Schmid, J. Lotz, R. Schweigert, K. Lackner, G. Aimo, J. Friese, T. Rosiere, D. Dickson, D. Kenney, G.T. Maine, Multi-site analytical evaluation of a chemiluminescent magnetic microparticle immunoassay (CMIA) for sirolimus on the Abbott ARCHITECT analyzer, Clinical biochemistry 42(15) (2009) 1543-1548. [24] D.W. Holt, D.A. Mandelbrot, M.A. Tortorici, J.M. Korth‐Bradley, D. Sierka, D.I. Levy, S. See Tai, G.L. Horowitz, Long‐term evaluation of analytical methods used in sirolimus therapeutic drug monitoring, Clinical transplantation 28(2) (2014) 243-251. [25] A. Dasgupta, B. Davis, L. Chow, Evaluation of QMS everolimus assay using Hitachi 917 Analyzer: comparison with liquid chromatography/mass spectrometry, Therapeutic drug monitoring 33(2) (2011) 149-154. [26] E. Hoffer, D. Kurnik, E. Efrati, I. Scherb, M. Karasik, G. Ring, Y. Bentur, Comparison of everolimus QMS immunoassay on architect ci4100 and liquid chromatography/mass spectrometry: lack of agreement in organ-transplanted patients, Therapeutic drug monitoring 37(2) (2015) 214-219. [27] B.C. Sallustio, B.D. Noll, R.G. Morris, Comparison of blood sirolimus, tacrolimus and everolimus concentrations measured by LC-MS/MS, HPLC-UV and immunoassay methods, Clinical biochemistry 44(2-3) (2011) 231-236. [28] P.J. Taylor, Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–electrospray–tandem mass spectrometry, Clinical biochemistry 38(4) (2005) 328334. [29] R. King, R. Bonfiglio, C. Fernandez-Metzler, C. Miller-Stein, T. Olah, Mechanistic investigation of ionization suppression in electrospray ionization, Journal of the American Society for Mass Spectrometry 11(11) (2000) 942-950. [30] R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds, Rapid Communications in Mass Spectrometry 13(12) (1999) 1175-1185. [31] X.S. Tong, J. Wang, S. Zheng, J.V. Pivnichny, P.R. Griffin, X. Shen, M. Donnelly, K. Vakerich, C. Nunes, J. Fenyk-Melody, Effect of signal interference from dosing excipients on pharmacokinetic screening of drug candidates by liquid chromatography/mass spectrometry, Analytical chemistry 74(24) (2002) 6305-6313. [32] E. García-González, M. Aramendía, D. Álvarez-Ballano, P. Trincado, L. Rello, Serum sample containing endogenous antibodies interfering with multiple hormone immunoassays. Laboratory strategies to detect interference, Practical laboratory medicine 4 (2016) 1-10.

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