[1st Edition] 9780128124277, 9780128119198

Table of contents :
Content:
CopyrightPage iv
ContributorsPage vii
PrefacePage ixGregory S. Makowski
Chapter One - Advances in Cardiac Biomarkers of Acute Coronary SyndromePages 1-58A.K. Saenger, N. Korpi-Steiner
Chapter Two - Vitamin D Testing—Where Are We and What Is on the Horizon?Pages 59-101N. Heureux
Chapter Three - Urine Exosomes: An Emerging Trove of BiomarkersPages 103-122J.M. Street, E.H. Koritzinsky, D.M. Glispie, R.A. Star, P.S.T. Yuen
Chapter Four - Identification of Genomic Somatic Variants in Cancer: From Discovery to ActionabilityPages 123-162G.L. Fawcett, A. Karina Eterovic
Chapter Five - Challenges in Laboratory Detection of Unusual Substance Abuse: Issues with Magic Mushroom, Peyote Cactus, Khat, and Solvent AbusePages 163-186A. Dasgupta
Chapter Six - Hydrogen Sulfide as a “Double-Faced” Compound: One with Pro- and Antioxidant EffectPages 187-196B. Olas
IndexPages 197-202

Citation preview

Academic Press is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 125 London Wall, London, EC2Y 5AS, United Kingdom First edition 2017 © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-811919-8 ISSN: 0065-2423 For information on all Academic Press publications visit our website at https://www.elsevier.com/

Publisher: Zoe Kruze Acquisition Editor: Poppy Garraway Editorial Project Manager: Shellie Bryant Production Project Manager: Vignesh Tamil Cover Designer: Greg Harris Typeset by SPi Global, India

CONTRIBUTORS A. Dasgupta University of Texas–Houston Medical School, Houston, TX, United States G.L. Fawcett Institute for Personalized Cancer Therapy (IPCT) at University of Texas M.D. Anderson Cancer Center, Houston, TX, United States D.M. Glispie Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States N. Heureux DIAsource Immunoassays, Louvain-la-Neuve, Belgium A. Karina Eterovic Institute for Personalized Cancer Therapy (IPCT) at University of Texas M.D. Anderson Cancer Center, Houston, TX, United States E.H. Koritzinsky Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States N. Korpi-Steiner University of North Carolina, Chapel Hill, NC, United States B. Olas Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland A.K. Saenger University of Minnesota, Minneapolis, MN, United States R.A. Star Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States J.M. Street Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States P.S.T. Yuen Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States

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PREFACE The first volume of the Advances in Clinical Chemistry series for 2017 is presented. In Chapter 1, advances in biomarkers for acute coronary syndromes are presented. This syndrome represents a broad spectrum of clinical presentations resulting from coronary artery disease causing a sudden depression in cardiac perfusion. In Chapter 2, vitamin D is reviewed. This biomarker has undergone a significant transformation in the clinical laboratory due to its expanding role beyond traditional bone disease. Assays involving the multiple forms of vitamin D are highlighted. In Chapter 3, the use of biofluid exosomes as diagnostic tools is reviewed. These small vesicles, formed as part of the endosomal pathway, contain cellular material traceable to the cell of origin. Their role as urinary biomarkers is explored. In Chapter 4, progress in discovery and validation or actionable somatic variants is presented. Although advances in throughput and efficiency of sequencing technologies have facilitated their identification, the role of bioinformatics plays an increasingly important role in their assessment for clinical relevance. In Chapter 5, the role of the clinical laboratory in elucidating less commonly abused drugs/agents is reviewed. These rare agents elude detection by traditional drugs-of-abuse testing via immunoassay typically requiring more sophisticated methods of analysis including LC/MS/MS. In Chapter 6, the role of hydrogen sulfide as a gasotransmitter is reviewed. This unique molecule acts to regulate oxidative stress via interaction with reactive oxygen species and as such is an important physiological antioxidant. I thank each contributor of Volume 78 and colleagues for their thoughtful peer review. I thank Shellie Bryant and Vignesh Tamil for expert editorial support. I hope the first volume for 2017 will be enjoyed. Comments and feedback from the readership are always appreciated. I would like to dedicate Volume 78 to MV 2016. We’re back. GREGORY S. MAKOWSKI

ix

CHAPTER ONE

Advances in Cardiac Biomarkers of Acute Coronary Syndrome A.K. Saenger*, N. Korpi-Steiner†,1 *University of Minnesota, Minneapolis, MN, United States † University of North Carolina, Chapel Hill, NC, United States 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 1.1 Acute Coronary Syndrome 1.2 Myocardial Infarction 2. Evolution of the Criteria to Define Myocardial Infarction 3. Troponin Structure, Function, and Circulating Isoforms 4. Standardization of Troponin Assays 5. Analytical Performance Characteristics and Terminology Related to Troponin Assays 5.1 Limit of Blank, Limit of Detection, and Limit of Quantitation 5.2 Analytical Sensitivity and Imprecision at the 99th Percentile URL 5.3 Classification of cTn Assays 6. Serial “Delta” Troponin Changes 7. Point of Care Troponin Assays 7.1 Analytical Recommendations for POC cTn Assays 8. Preanalytical Factors Influencing cTn Results 8.1 Specimen Type 8.2 Hemolysis 8.3 Endogenous Interferences 9. Defining Normality and the 99th Percentile URL 9.1 Sex 9.2 Age 9.3 Surrogate Biomarkers 9.4 Population Size 9.5 Recommendations for Defining Normality 10. High-Sensitivity Troponin and Advancements in the Diagnosis of AMI 10.1 Accelerated Serial Sampling Protocols for Rapid Rule-In/Rule-Out 10.2 Frequency of AMI Diagnosis 11. High-Sensitivity Troponin Rule-Out Strategies 11.1 Undetectable Baseline Troponin Concentrations 11.2 Accelerated Diagnostic Protocols 12. Cardiac Troponin: Risk Stratification and Prognosis in Various Populations

Advances in Clinical Chemistry, Volume 78 ISSN 0065-2423 http://dx.doi.org/10.1016/bs.acc.2016.07.001

#

2017 Elsevier Inc. All rights reserved.

2 2 3 3 7 9 14 14 15 17 20 21 23 24 24 25 27 28 28 29 30 30 31 32 33 34 34 34 36 37

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A.K. Saenger and N. Korpi-Steiner

12.1 Renal Disease 12.2 Perioperative Setting 13. Other Biomarkers in ACS 13.1 Natriuretic Peptides 13.2 Copeptin 13.3 Soluble ST2 14. Conclusions References

38 43 43 43 45 46 48 48

Abstract Acute coronary syndrome (ACS) encompasses a pathophysiological spectrum of cardiovascular diseases, all of which have significant morbidity and mortality. ACS was once considered an acute condition; however, new treatment strategies and improvements in biomarker assays have led to ACS being an acute and chronic disease. Cardiac troponin is the preferred biomarker for the diagnosis of myocardial infarction, and there is considerable interest and efforts toward development and implementation of highsensitivity cardiac troponin (hs-cTn) assays worldwide. Analytical and clinical performance characteristics of hs-cTn assays as well as testing limitations are important for laboratorians and clinicians to understand in order to utilize testing appropriately. Furthermore, expanding the clinical utility of hs-cTn into other cohorts such as asymptomatic community dwelling populations, heart failure, and chronic kidney disease populations supports novel opportunities for improved short- and long-term prognosis.

1. INTRODUCTION 1.1 Acute Coronary Syndrome Acute coronary syndrome (ACS) is a broad term used to describe a spectrum of clinical presentations which result from coronary artery disease and cause a sudden reduction in myocardial perfusion. The pathophysiology of ACS involves thickening of the coronary artery vessel(s) by lipid-filled plaques. Initially the plaques are often small, and the patient may be asymptomatic. Patients with stable angina exhibit chest pain upon exertion or exercise because the presence of plaques cause a reduction in coronary flow which is insufficient to maintain the increased oxygen demand and required output. Stable angina can progress to ACS which is a comprised of unstable angina (UA) and myocardial infarction (MI). Rupture of a coronary arterial plaque causes platelet aggregation and intraluminal thrombosis resulting in reduced blood flow in the involved vessel. UA arises when nonocclusive thrombi cause ischemia and chest pain at rest. The continuum of ACS

Biomarkers in ACS

3

disease further progresses to myocardial infarction in the presence of concurrent intraluminal thrombosis, ischemia, and myocardial injury. The diagnosis of ACS is based upon electrocardiogram (ECG) changes, i.e., ST-elevation myocardial infarction (STEMI), and those without ECG changes in non-ST elevation myocardial infarction (NSTEMI) and UA. For STEMI patients the ECG is diagnostic for acute myocardial infarction (AMI), and cardiac biomarkers are of little utility. However, the sensitivity of the ECG alone is poor. A rise and/or fall in cardiac biomarkers is reflective of acute myocardial injury facilitating differentiation of NSTEMI from UA (Fig. 1). Cardiac troponin (cTn) is endorsed by the Universal Definition of Myocardial Infarction as the cardiac biomarker of choice (Table 1; [1]).

1.2 Myocardial Infarction Globally, MI is a major cause of morbidity and mortality; annually, an estimated 660,000 new individuals in the Unites States will have an MI, and approximately every 90 s someone will succumb to one [2,3]. During an AMI, injury to myocardial tissue results in rapid release of cTn into circulation. A rise in cTn typically occurs within a few hours of symptom onset and can remain elevated above the upper reference limit (URL) for the reference population for several days until it is cleared [4]. Rapid diagnosis of AMI is critical due to the risk of mortality, and early medical intervention (e.g. revascularization) has the greatest benefit within the first few hours [5–7]. A delay in ruling out a diagnosis of AMI contributes to patient overcrowding in the emergency department and associated costs [8]. Inappropriate hospital admissions associated with attempts to rule in an AMI are also problematic.

2. EVOLUTION OF THE CRITERIA TO DEFINE MYOCARDIAL INFARCTION The diagnosis of myocardial infarction has evolved substantially over the past few decades with important implications to clinical outcomes as well as epidemiological and translational research. Troponin assays were first introduced into clinical practice in the late 1990s and in the first Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC) Universal Definition clinical guidelines in 2000 [9]. The original cTn assays were relatively insensitive, but highly specific, and cutpoints were optimized to yield equivalent clinical performance compared to the old World Health Organization (WHO) definition of AMI. Therefore, very high cutoffs were

Fig. 1 Myocardial injury—cardiac troponin >99th percentile URLa.

Biomarkers in ACS

5

Table 1 Criteria for Diagnosis of Acute Myocardial Infraction Requires evidence of myocardial injury and clinical evidence of myocardial ischemia

• Evidence of myocardial injury


Detection of a rise and/or fall of cardiac biomarker values (preferably cardiac troponin) with at least one value >99th percentile upper reference limit • Clinical evidence of myocardial ischemia, with at least one of the following:
Clinical symptoms of ischemia
New or presumed new significant ST-segment-T wave (ST-T) changes or new left bundle branch block
Development of pathological Q waves in the ECG
Imaging evidence of new loss of viable myocardium or new regional wall motion abnormality
Identification of an intracoronary thrombus by angiography or autopsy Adapted from Thygesen et al. [1].

used and clearly differentiated the diagnosis of AMI into “yes/no” categories. Since that time there has been considerable improvement in the analytical sensitivity of cTn assays and a clinical awareness that even low concentrations of cTn yield both diagnostic and prognostic significance in ACS. Cardiac troponin was, and remains, the recommended biomarker of choice due to its superior clinical sensitivity and specificity compared to creatine kinase-MB (CK-MB) [10]. The prevalence of MI using CK-MB was 30% and increased to 80% with the inclusion of cTn in the 2000 guidelines. The 2000 guidelines also recommended serial cTn sampling and required one of the cTn values to be above the 99th percentile URL of the assay if used to diagnose AMI. The 99th percentile URL was recommended because it was approximately three standard deviations from the mean and minimized potential false-positive values. In 2007, the second Universal Definition of Myocardial Infarction was introduced and defined five different MI subtypes that were endorsed by the major cardiology societies [11]. The 2012 recommendations refined these subtypes and also incorporated coronary endothelial dysfunction as one of the variables to consider when evaluating supply/demand ischemia (Fig. 1; [11,12]). Several etiologies of myocardial injury spanning ischemic and nonischemic pathologies can give rise to detectable cTn values and is not limited to AMI caused by ACS. Progressive improvements in cTn assay sensitivity have also broadened the number of clinical conditions which have cTn elevations which are detectable (Fig. 1). Thus, an elevated cTn result by itself is insufficient for the diagnosis of AMI and must be interpreted in the clinical

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A.K. Saenger and N. Korpi-Steiner

scenario. Type I MI arises from a primary coronary event and spontaneous atherosclerotic plaque rupture in patients with ACS; it is managed acutely with aggressive antithrombotic therapy, angiography, and revascularization as appropriate. Spontaneous Type I MI with intraluminal thrombosis and ensuing myocyte necrosis may also arise with ulceration, fissuring, erosion, or dissection of one or more coronary arteries in the presence and occasionally in the absence of CAD. Type 2 MI evolves secondary to an imbalance in myocardial oxygen supply and/or demand which has several potential etiologies, such as endothelial dysfunction, vasospasm, severe tachycardia, hypo- or hypertension, sepsis, anemia, respiratory failure, and others. The treatments are heterogeneous for type 2 MI and may include treating the underlying cause [12]. While an acute elevation in cTn with a rise and/ or fall pattern with at least one value 99th percentile reference limit is necessary for AMI diagnosis, it currently does not help identify etiology. The differentiation between type I and type 2 MI relies on a careful clinical assessment and judgment to identify the cause of the cardiomyocyte injury because type 2 MI patients most often have a slight elevation in cTn, and the rise/fall pattern may be more subtle than in type 1 MI [12,13]. Type 3 MI diagnosis is independent of cTn test results due to unavailability at the time of cardiac death caused by an AMI [1]. Patients are classified as having a fatal AMI based on clinical symptoms of myocardial ischemia or presumed new ischemic ECG changes or new left bundle branch block resulting in cardiac death. In this clinical setting, the patient suffered a cardiac death before blood collection for cTn testing or prior to identification of elevated cTn results. Types 4 and 5 MI are associated with periprocedural myocardial injury or infarction that is associated with revascularization procedures and include cardiac biomarker criteria. The criteria for diagnosing periprocedural myocardial infarction (PMI) have changed over the past decade. In the 2000 and 2007 Universal Definition of Myocardial Infarction guidelines [9,11], increased concentrations of cardiac biomarkers (preferably cTn) postpercutaneous coronary intervention (PCI) was sufficient to diagnose MI. However, this changed with the 2012 Universal Definition guideline which requires other criteria beyond elevated cTn, including ischemic chest pain for greater than 20 min, ischemic ECG changes and abnormal findings on imaging [1]. There are patients who have elevated cTn concentrations postPCI but meet no other criteria and vice versa [14–16]. A study by Idris et al. determined the impact of the 2012 guidelines on the frequency of type 4 MI [17]. They found a reduction in the number of PMI by approximately

Biomarkers in ACS

7

60%, largely due to exclusion of cardiovascular events which did not have prognostic significance. Type 5 MI is related to coronary artery bypass surgery (CABG) procedures and was redefined in the 2012 Task Force guidelines using a defined arbitrary cardiac biomarker cutoff concentration >10  99th percentile URL during the first 48 h following CABG. Type 5 MI must also satisfy one of the additional clinical evidence criteria in the clinical practice guideline.

3. TROPONIN STRUCTURE, FUNCTION, AND CIRCULATING ISOFORMS Troponin is comprised of three proteins (Troponin C, I, and T) that collectively form a contractile apparatus. The troponin complex, together with tropomyosin and Ca2+ ions, plays a central role in the regulation of thin myofilament contraction and relaxation in striated cardiac and skeletal muscle tissues. Within the thin filament, tropomyosin dimers coil around actin filaments and function to modulate myosin interaction with myosin-binding sites on the actin filaments. Troponin T (TnT; 37 kDa) binds to tropomyosin and positions the troponin–tropomyosin complex on the actin filament. Troponin I (TnI; 24 kDa) inhibits the actinomyosin ATPase enzymatic hydrolysis of adenosine triphosphate (ATP) that provides energy for myofilament contraction. Troponin C (TnC; 18 kDa) is the calcium-binding component of the contractile apparatus and upon binding intracellular Ca2+ ions induces a conformational change in the troponin–tropomyosin complex, reducing TnI inhibition of the actinomysin ATPase and allowing muscle contraction to occur [18]. As Ca2+ ions are transported back into the sarcoplasmic reticulum, the troponin–tropomyosin complex returns to its previous conformation, inhibiting actinomysin ATPase activity resulting in muscle relaxation. The troponin contractile apparatus is expressed in striated skeletal and cardiac muscle tissue, although tissue-specific isoforms exist for each troponin protein. Two different isoforms of human TnC encoded by individual genes have been described, including an isoform exclusively expressed in fast-twitch skeletal tissue and an isoform expressed in both cardiac and slow-twitch skeletal muscle tissue [19]. There is significant homology between the cardiac and slow-twitch skeletal TnC isoforms which reduces cardiac specificity; consequently, detection of TnC has limited diagnostic utility in ACS.

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Human TnI exhibits heterogeneity due to tissue-specific isoform expression, posttranslational modifications, and proteolytic degradation. Three human TnI isoforms have been identified, including slow twitch skeletal TnI (slow sTnI), fast-twitch skeletal TnI (fast sTnI), and cardiac muscle TnI (cTnI) [18]. Interestingly, during fetal heart development slow sTnI is the dominant isoform expressed in the neonatal period which transitions to cTnI isoform expression by the first 9 months of life [20]. The amino acid sequences of cTnI, slow and fast sTnI isoforms show 40% dissimilarity supporting use of immunoassays that are specific for cTnI [21,22]. The cTnI isoform is comprised of 209 amino acid residues and is approximately 23–24 kDa in size [23]. Posttranslational modifications including phosphorylation, oxidation, reduction, and proteolytic degradation contribute to cTnI molecular heterogeneity (Fig. 2; [18,23,24]). The cTnI molecule is highly susceptible to proteolysis with cleavage of the N- and C-terminal regions, whereas the central region located between residues 30 and 110 exhibits higher stability possibly due to protection by TnC [25]. During myocardial tissue necrosis, cTnI is rapidly released into circulation which is accompanied by liberation of lysosomal proteolytic enzymes that can degrade cTnI. The majority of circulating cTnI after an AMI appears to be in the complexed form, whereas only a small amount of free cTnI has been detected [24,26]. Adult TnT has three tissue-specific isoforms encoded by individual genes. These are expressed in cardiac muscle tissue (cTnT) as well

Fig. 2 Cardiac troponin I epitopes that are prone to interference. Reproduced with permission from Apple and Collinson [23].

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slow- and fast-twitch skeletal muscle tissues (sTnT). Sequence homology exists between the 3 isoforms consisting of 125 amino acid residue differences between the adult slow sTnT and cTnT isoforms (56.6% homology) and 120 amino acid residue differences between the adult slow sTnT and cTnT (58.3% homology) [27]. Unlike cTnI, alternate splicing of mRNA leads to additional cTnT isoforms transiently expressed in fetal human skeletal muscle [28]. As fetal development progresses, cTnT is downregulated and sTnT expression is upregulated, and cTnT is undetectable in nondiseased adult skeletal muscle [29]. Reexpression of cTnT isoforms has been demonstrated in patients with heterogeneous skeletal muscle diseases [30,31], though the prevalence of detectable cTnT due to skeletal muscle disease has not yet been elucidated. The majority of cTn is bound to thin filaments as part of the structural pool and only a small amount is free in the cytoplasm as the cytoplasmic pool, estimated at 3–8% of the total amount of cellular cTn [32,33]. During myocardial injury, the cTn cytoplasmic pool is thought to be rapidly released into circulation, followed by the remaining degraded structural cTn pool. The major forms of cTn released into circulation are cTnT and the cTnI–TnC complex. Additional circulating forms include cTnT–cTnI– TnC ternary complex and free cTnI [33,34]. The detailed mechanism by which cTn is released from myocytes in the absence of acute injury or necrosis is not fully elucidated. Following cTn release the heterogeneous forms are subsequently degraded, fragmented, and cleared. Many of these mechanisms are also not yet fully understood, which likely contributes to the ongoing analytical and clinical challenges associated with cTn assays.

4. STANDARDIZATION OF TROPONIN ASSAYS Standardization and harmonization efforts have focused on cTnI assays, which are available from a wide variety of manufacturers and analytical platforms. Standardization is generally not an issue for cTnT because only one diagnostic company (Roche Diagnostics, Penzberg, Germany) holds the patent and antibodies for cTnT. Clinical diagnostic assays for cTn are primarily sandwich-type immunometric methods with capture and detection antibodies, though antigen epitope specificities and detection antibody tags vary. There are several cTnI diagnostic assays commercially available with variable analytical and clinical performance characteristics and different target antibodies (Table 2) [23]. The International Federation of Clinical Chemistry (IFCC) website (http://www.ifcc.org/) maintains a

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Table 2 Analytical Characteristics of Contemporary Sensitive Cardiac Troponin Assay Systems Cardiac Troponin Concentrations at: Amino Acid Residues of 99th 10% CV Epitopes Recognized by Concentration Capture (C) and Company/ LODa percentile Platform/Assay (ng/L) (ng/L) (CV)b (ng/L) Detection (D) mAbs Laboratory test systems

Abbott 20 AxSYM ADV

40 (14%)

160

C: 87–91, 41–49; D: 24–40

Abbott 9 ARCHITECT

28 (14%)

32

C: 87–91, 24–40; D: 41–49

Beckman Access AccuTnl

10

40 (14%)

60

C: 41–49; D: 24–40

bioMerieux Vidas Ultra

10

10 (27.7%)

110

C: 41–49, 22–29; D: 87–91, MAb 7B9

Ortho Vitros ECi ES

12

34 (10%)

34

C: 24–40, 41–49; D: 87–91

Roche Elecsys 10 TnT Gen 4

14 ng/L

0.535

Continued

Table 7 Performance of High-Sensitivity Troponin for Diagnosis of AMI in CKD Patients—cont’d Diagnosis Evaluated hs-cTnT and hs-cTn Study Assay Study Patients (#) Threshold AUC Sensitivity (%) Specificity (%) PPV (%)

Reichlin et al. [59]

Roche Elecsys 2010

836 125 with CKD

NPV (%)

AMI

1 h: Absolute change

0.88

Relative change

0.62

2 h:

a

AUC, area under the receiver operator characteristic curve. PPV, positive predictive value. NPV, negative predictive value. Adapted from Parikh et al. [143].

b c

Absolute change

0.94

Relative change

0.70

N/A

N/A

N/A

N/A

Biomarkers in ACS

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populations who are often asymptomatic at baseline [147]. A summary of studies evaluating the prognostic value of hs-cTn (both hs-cTnT and hs-cTnI) in CKD populations is presented in Table 8 [143,148–151]. A prospective study by Jacobs et al. compared the performance of hs-cTnT with contemporary cTn assays to detect elevated cTn in 32 ESRD patients on hemodialysis (HD) [148]. Troponin concentrations were assessed every 2 months for 6 months and follow-up occurred for 6 months. In the evaluated population, 100% of patients had elevated hs-cTnT compared to 81% and 28% using cTnT and cTnI assays, respectively. The median hs-cTnT concentration was 17 ng/L (IQR 11–29 ng/L). Another prospective study of 143 dialysis patients demonstrated superior predictive power for all-cause mortality using hs-cTnT compared to NT-proBNP, with a median follow-up of 46.7 months [149]. These findings were confirmed in another cross-sectional study evaluating baseline hs-cTnT concentrations in 239 dialysis patients followed for 6 months; however, multivariate analysis did not factor NT-proBNP into the analysis [150]. A study published by Hassan et al. followed stable HD and peritoneal dialysis (PD) patients for a year and demonstrated stable hs-cTnT concentrations independent of cardiovascular events [151]. The incidence of AMI increased significantly across hs-cTnT quartiles in both HD and PD patients; however, only mortality increased in HD patients (p ¼ 0.015). To date there have been few studies evaluating the utility of hs-cTnI as a prognostic marker in patients with ESRD. As a caveat there are reports of hs-cTnI studies in renal patients but the studies actually used contemporary assays. None have evaluated sex-specific cutoffs in renal patients. Elevated hs-cTn (both cTnI and cTnT) in any patient with CKD or ESRD is associated with an increased presence of underlying structural heart disease and cardiovascular risk factors with a greater probability to progress to symptomatic heart disease, particularly heart failure (HF) and death [152,153]. The use of hs-cTn assays in the CKD population, although associated with more persistently elevated values, appears to be able to accelerate the time to diagnose AMI in a similar fashion compared to patients with normal renal function [154]. In order to optimize clinical sensitivity and specificity for the diagnosis of AMI, it is crucial to evaluate the change in cTn values even more so than in the healthy population without renal disease. Further longitudinal studies are required to evaluate the potential role of hs-cTn in clinical management of asymptomatic CKD patients. Ideally hs-cTn could be used as a biomarker to guide therapeutic interventions in patients with CKD or ESRD which may ultimately prevent or slow further progression to cardiovascular disease.

Table 8 Prognostic Performance of hs-cTn in CKD Patients Type of Study (C-S, Study n cTn Assay longitudinal, Both) Key Cross-Sectional Findings Key Longitudinal Findings

Jacobs et al. [148]

32

hs-cTnT

Follow-Up

Both

Significant association with history of cardiac disease

All patients had elevated cTn at least once during follow-up

McGill et al. [149] 143 hs-cTnT

Longitudinal

N/A

Independent predictive variables of Median 46.7 months all-cause mortality: age, log hs-cTnT, log CRP, albumin. After 46.7 months hs-cTnT replaced NT-proBNP as the most powerful predictor of mortality (risk increased 1.4-fold for every 2.72 ng/L increase in hs-cTnT)

Wolley et al. [150] 239 hs-cTnT

Cross-sectional

Increase in baseline troponin N/A by 100 units increased odds of CV death (OR 1.5, Cl 1.2–1.9). Multivariate analysis after adjustment for CRP and LVEF: OR 1.3 (Cl 1.1–1.7)

6 months

Hassan et al. [151]

Both

HD patients had significantly more DM, viral hepatitis, PVD when compared to PD patients

12 months

393 hs-cTnT

Adapted from Parikh et al. [143]

Increasing MI and mortality across hs-cTnT quartiles for HD patients but only increasing MI across hs-cTnT quartiles for PD patients

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12.2 Perioperative Setting Myocardial ischemia and/or infarction early following noncardiac surgery is major determinants of short- and long-term cardiovascular outcomes. There is an influx of outpatient noncardiac surgeries occurring largely in elderly populations, with an average overall complication rate as high as 17% and a mortality rate that can exceed 2% [155–157]. Perioperative management of this population represents a significant opportunity for potential reduction of these risks. Closer examination of the morbidity and mortality associated with noncardiac surgery reveals a substantial percentage of precipitating events are cardiovascular in origin [158]. Perioperative myocardial infarction represents the most common cardiovascular complication following noncardiac surgery, and therefore, there has been significant interest and emphasis placed on identifying such events. Perioperative ischemic events often lack the typical clinical signs and symptoms associated with ACS or AMI, which has led some to advocate further assessment of elevated cTn in the perioperative setting. However, there are clinical challenges associated with interpretation of frequent elevations of cTn (and hs-cTn) in this setting. Serial troponin measurements in the absence of clinical symptoms have been suggested for perioperative care but are not currently recommended by clinical guidelines. Hypotheses around the mechanism of perioperative myocardial infarction are similar to type 1 and type 2 MI defined in the Universal Definition of Myocardial Infarction. Type 2 MI, however, is thought to be a much more common cause of perioperative myocardial infarction, representing up to 65% of MIs in the perioperative setting, and presenting as silent ST-segment depression on ECG [159,160]. Since a majority of these patients will not experience ischemic symptoms, detection of these events is often late and beyond the timeframe of meaningful intervention [159]. As such, the utility of cardiac biomarker surveillance is of particular interest. Cardiac troponin demonstrates great utility in diagnosis of perioperative myocardial infarction, particularly in noncardiac (primarily vascular) surgery [161]. The same cTn diagnostic cutoff used to diagnose MI should be used for perioperative MI (i.e., the 99th percentile multiplied by 5 or 10 for type 4 and 5, respectively).

13. OTHER BIOMARKERS IN ACS 13.1 Natriuretic Peptides B-type natriuretic peptide (BNP) and the N-terminal portion of the proBNP peptide (NT-proBNP) are secreted by the ventricles in response to

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cardiomyocyte stress. BNP binds and activates receptors causing reduction in systemic vascular resistance, central venous pressure, and natriuresis. BNP and NT-proBNP are utilized primarily for diagnosis of acute decompensated HF and monitoring therapeutic efficacy in patients with HF. However, the natriuretic peptides (NPs) represent a different but complimentary mechanistic modality compared with cardiac troponins and have prognostic utility postmyocardial infarction. Several large clinical trials have measured BNP or NT-proBNP in patients presenting with ACS (both STEMI and NSTEMI) and consistently demonstrated elevated NPs yield important prognostic information [162,163]. Both BNP and NT-proBNP have been shown to be predictive of future adverse outcomes independent of other biomarkers, including cardiac troponin. Furthermore, elevated NPs typically predict future onset of HF or death rather than ischemic events, whereas cardiac troponin typically predicts recurrent ischemic events. NT-proBNP and BNP provide incremental information regarding cardiovascular morbidity and mortality at 1 year beyond the GRACE score [164]. It is similar to the GRACE score when used alone to predict in-hospital morality following MI. In particular NT-proBNP/ BNP is useful in NSTE-ACS patients and predicts in-hospital and 180-day mortality and development of HF. The evidence for use of NPs and prognosis in ACS is comprehensive and included in the clinical guidelines [165]. The therapeutic implications for elevated NP values in the ACS setting are still undefined due to inconsistent results between clinical trials. Studies have assessed whether circulating NPs may provide utility to identify subpopulations of patients for whom prescription of angiotensin-converting enzyme inhibitor (ACEI) treatment confers benefit. After adjustment for important clinical covariates ACEI treatment was associated with survival benefits only for those ACS patients with marked elevation of plasma NT-proBNP, after a minimum follow-up time of 1 year [166]. The Metabolic Efficiency with Ranolazine for Less Ischemia in Non-ST Elevation ACS-thrombolysis in myocardial infarction (MERLIN-TIMI 36) investigated the relationship between BNP and cardiovascular outcomes and the effect of ranolazine, which exerts antiischemic effects by reducing myocardial sodium and calcium overload and consequently ventricular wall stress [167]. Conclusions from this trial suggest use of ranolazine reduces the risk of cardiovascular death or MI in NST-ACS patients with elevated BNP. In a substudy of the Fragmin and fast revascularization during InStability in Coronary artery disease trial [168], 2-year mortality was reduced by 7.3% in patients with elevated NT-proBNP and interleukin-6 levels at admission

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who underwent an early invasive strategy (RR: 0.46, 95% CI: 0.21–1.00). However, only elevated cTnT was independently associated with a reduction of MI by means of an invasive strategy. Another study did not find any significant interaction between NT-proBNP concentrations and clinical benefits of tirofiban treatment [163]. Unifying conclusions regarding the role of NPs in guiding ACS management are difficult to make as these studies are limited by the heterogeneity of NP cutoff points used for the trials. Despite intense interest in identifying treatments that might modify the risk associated with elevated concentration of NPs, further studies are required to assess the utility of NPs as a biomarker which can predict who will benefit from specific interventions or therapeutic modalities in patients with ACS.

13.2 Copeptin Despite the exquisite clinical sensitivity and specificity of cTn for diagnosis of AMI there remains potential for other biomarkers to detect very early, small ischemic events. In addition, if a biomarker is additive to cTn such that early rule-out strategies could be adopted without requiring serial measurements would represent an ideal situation. The first single center study reported in 2009 that copeptin analysis combined with cTn at presentation allowed for an early rule-out strategy and discharge [169]. Copeptin is the C-terminal portion of provasopressin and is secreted in a 1:1 fashion with arginine vasopressin (AVP), a biomarker used to assess osmoregulation and cardiovascular homeostasis. Copeptin is a stable molecule, unlike AVP, and can be measured using electrochemiluminescent immunoassay (Kryptor, Thermo Fisher, Waltham, MA, USA). After myocardial necrosis copeptin is hypothesized to increase peripheral vasoconstrictor activity as well as increase protein synthesis in myocytes which leads to hypertrophy and vasoconstriction of coronary arteries. Copeptin concentrations increase 0–4 h following onset of signs and symptoms of MI, although it is not specific for myocardial necrosis and can be increased in HF, stroke, traumatic brain injury, and hemorrhagic or septic shock [170]. A meta-analysis of 168 studies which evaluated copeptin and cTn (contemporary and high-sensitivity assays) demonstrated significant improvement in diagnostic sensitivity [171]. Clinical sensitivity at baseline increased from 91% using hs-cTn alone compared with 98% when copeptin was added; similarly with the Siemens Centaur TnI-Ultra contemporary assay the clinical sensitivity increased from 0.79 to 0.93 [171]. Although

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other individual cTn assays have been studied in this context, there were a limited number of studies that could be adequately combined for statistical significance in the meta-analysis. Copeptin will not be useful as the sole biomarker for AMI because the overall sensitivity (45–84%), specificity (41–77%), and clinical performance are not superior to cTn. In addition, use of this multimarker strategy further decreases the clinical specificity for AMI, indicating false-positive results could be more prevalent than true positives. Routine adoption of copeptin into clinical practice and laboratories is hindered by lack of available platforms and therefore is currently not a financially feasible or viable option. In addition, copeptin is a Research Use Only (RUO) test in the United States and does not have FDA approval. Additional studies are warranted to prospectively analyze the utility of copeptin in different emergency department settings, i.e., where patients present early following signs and symptoms and those where patients often present late.

13.3 Soluble ST2 ST2 is a member of the interleukin-1 receptor family and has two isoforms that are directly implicated in progression of cardiac disease: soluble ST2 (sST2) and a transmembrane-bound form, ST2 ligand (ST2L). Interleukin33 (IL-33) is a cytokine which can be secreted by most cells in response to damage. IL-33 is also the hormone that interacts with ST2L, protecting against left ventricular hypertrophy and myocardial fibrosis to effectively preserve cardiac function. When sST2 concentrations are high, it will bind preferentially to IL-33, making IL-33 unavailable for cardioprotective signaling and the heart vulnerable to the effects of sST2. High concentrations of sST2 result in cellular death, tissue fibrosis, reduced cardiac function, and an increase in the rate of disease progression. sST2 has been largely studied in the realm of HF and is a biomarker which has strong prognostic utility for cardiovascular and all-cause morbidity and mortality. The Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA) is the only assay which is FDA approved for use as an aid in assessing prognosis in chronic HF patients. There are other research use only assays but the analytical and clinical performance cannot be assumed to be comparable to the Presage ST2 assay. sST2 can be quantitated in serum or plasma and currently available as a 96-well ELISA plate. There are sex-specific differences in reference intervals in normal healthy individuals, with higher values in males (52 ng/mL) compared to females (38.8 ng/mL). sST2 is unaffected by

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renal function, body mass index, or age. The clinical cutpoint is more relevant than the reference interval and a value of >35 ng/mL is the threshold consistently associated with a worse prognosis. The biological variability of sST2 is low, with a reference change value of 30% and much lower than NPs which have a reference change value of 92% [172]. Therefore, sST2 may have more utility as a serial marker of response to treatment compared to NPs, simply due to lower biological variability. Elevated concentrations of circulating sST2 are associated with relevant pathophysiological processes in cardiovascular diseases. A link between sST2 and myocardial stretch, fibrosis, adverse remodeling, inflammation, impaired hemodynamics, and vascular diseases has been described in experimental and human studies. In patients diagnosed with AMI, circulating sST2 concentrations were elevated early and correlated with adverse cardiac remodeling as measured with magnetic resonance [173]. There was an inverse correlation of sST2 with left ventricle (LV) ejection fraction and a positive correlation with infarct volume index at baseline and 24 weeks postAMI. The relationship between sST2 and adverse remodeling after AMI is also supported by the results in an experimental model of AMI, where myocardial expression of sST2 was rapidly upregulated during the first 4 weeks and correlated with the ongoing processes of fibrosis and inflammation [174]. Further evidence in the progression of adverse remodeling to overt HF was notable among ACS patients in the MERLIN-TIMI 36 trial. Individuals with higher circulating sST2 concentrations had a higher rate of development of HF, in both short- (HR 2.56, 95% CI 1.77–3.71) and longterm follow-up (HR 1.67, 95% CI 1.32–2.11) [175]. Higher sST2 concentrations are associated with an adverse remodeling phenotype which is prone to development of HF, which may be particularly relevant postmyocardial infarction. In the setting of AMI, early concentrations of sST2 in the first 24-h post-AMI predict mortality, cardiovascular mortality, and HF at 30 days [176]. The predictive value of sST2 was independent and complementary to NPs; the combination of sST2 and NT-proBNP significantly improved risk stratification [177]. In patients with non-ST elevation ACS, sST2 was also predictive of cardiovascular death and HF at 30 days and 1 year [175]. In stable coronary disease, sST2 is also predictive of mortality and cardiovascular death independent of clinical variables and biomarkers [178]. In contrast to the prognostic value of sST2, in patients presenting to the emergency department with chest pain, the reported AUC for diagnosis of AMI or ACS was 0.579, and there is clearly not a diagnostic role for sST2 above and beyond troponin [179,180].

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Additional studies are warranted to evaluate the utility of increased sST2 postmyocardial infarction, which is suggestive of a maladaptive response and predictive of an adverse phenotype susceptible to development of overt HF through myocardial fibrosis and remodeling.

14. CONCLUSIONS Cardiac troponin is the preferred biomarker for the diagnosis of myocardial infarction. Continuous advancements in the analytical performance of cTn testing methods will improve rapid rule-in/rule-out diagnosis of MI and expand the clinical utilities to include risk stratification and prognostic values in other patient cohorts. The ongoing search and refinement for new biomarkers in ACS and approaches to measurement represent a dynamic field.

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[87] R. Sodi, S.M. Darn, A.S. Davison, et al., Mechanism of interference by haemolysis in the cardiac troponin T immunoassay, Ann. Clin. Biochem. 43 (2006) 49–56. [88] O. Sonntag, Haemolysis as an interference factor in clinical chemistry, J. Clin. Chem. Clin. Biochem. 24 (1986) 127–139. [89] G. Lippi, R. Aloe, T. Meschi, et al., Interference from heterophilic antibodies in troponin testing. Case report and systematic review of the literature, Clin. Chim. Acta 426 (2013) 79–84. [90] K.D. Onuska, S.A. Hill, Effect of rheumatoid factor on cardiac troponin I measurement using two commercial measurement systems, Clin. Chem. 46 (2000) 307–308. [91] J. Krahn, D.M. Parry, M. Leroux, J. Dalton, High percentage of false positive cardiac troponin I results in patients with rheumatoid factor, Clin. Biochem. 32 (1999) 477–480. [92] S. Eriksson, H. Halenius, K. Pulkki, et al., Negative interference in cardiac troponin I immunoassays by circulating troponin autoantibodies, Clin. Chem. 51 (2005) 839–847. [93] M. Adamczyk, R.J. Brashear, P.G. Mattingly, Coprevalence of autoantibodies to cardiac troponin I and T in normal blood donors, Clin. Chem. 56 (2010) 676–677. [94] N. Bolstad, D.J. Warren, K. Nustad, Heterophilic antibody interference in immunometric assays, Best Pract. Res. Clin. Endocrinol. Metab. 27 (2013) 647–661. [95] P.O. Collinson, Y.M. Heung, D. Gaze, et al., Influence of population selection on the 99th percentile reference value for cardiac troponin assays, Clin. Chem. 58 (2012) 219–225. [96] P.M. McKie, D.M. Heublein, C.G. Scott, et al., Defining high-sensitivity cardiac troponin concentrations in the community, Clin. Chem. 59 (2013) 1099–1107. [97] T. Keller, F. Ojeda, T. Zeller, et al., Defining a reference population to determine the 99th percentile of a contemporary sensitive cardiac troponin I assay, Int. J. Cardiol. 167 (2013) 1423–1429. [98] C. Prontera, A. Fortunato, S. Storti, et al., Evaluation of analytical performance of the Siemens ADVIA TnI ultra immunoassay, Clin. Chem. 53 (2007) 1722–1723. [99] P.A. Cain, R. Ahl, E. Hedstrom, et al., Age and gender specific normal values of left ventricular mass, volume and function for gradient echo magnetic resonance imaging: a cross sectional study, BMC Med. Imaging 9 (2009) 2. [100] M. Franzini, V. Lorenzoni, S. Masotti, et al., The calculation of the cardiac troponin T 99th percentile of the reference population is affected by age, gender, and population selection: a multicenter study in Italy, Clin. Chim. Acta 438 (2015) 376–381. [101] O. Bergmann, R.D. Bhardwaj, S. Bernard, et al., Evidence for cardiomyocyte renewal in humans, Science 324 (2009) 98–102. [102] R. Matyal, Newly appreciated pathophysiology of ischemic heart disease in women mandates changes in perioperative management: a core review, Anesth. Analg. 107 (2008) 37–50. [103] A.S. Shah, M. Griffiths, K.K. Lee, et al., High sensitivity cardiac troponin and the under-diagnosis of myocardial infarction in women: prospective cohort study, BMJ 350 (2015) g7873. [104] A. Bagai, B.R. Chaitman, G. Gosselin, et al., Substantial variability between laboratories in troponin decision level for diagnosis of myocardial infarction and assay 99th percentile: findings from the international study of comparative health effectiveness with medical and invasive approaches (ISCHEMIA) trial, J. Am. Coll. Cardiol. 63 (2014) A1878. [105] P. Venge, N. Johnston, B. Lindahl, S. James, Normal plasma levels of cardiac troponin I measured by the high-sensitivity cardiac troponin I access prototype assay and the impact on the diagnosis of myocardial ischemia, J. Am. Coll. Cardiol. 54 (2009) 1165–1172.

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[106] T.C. Aw, S.K. Phua, S.P. Tan, Measurement of cardiac troponin I in serum with a new high-sensitivity assay in a large multi-ethnic Asian cohort and the impact of gender, Clin. Chim. Acta 422 (2013) 26–28. [107] M. Krintus, M. Kozinski, P. Boudry, et al., Defining normality in a European multinational cohort: critical factors influencing the 99th percentile upper reference limit for high sensitivity cardiac troponin I, Int. J. Cardiol. 187 (2015) 256–263. [108] T. Zeller, F. Ojeda, F.J. Brunner, et al., High-sensitivity cardiac troponin I in the general population—defining reference populations for the determination of the 99th percentile in the Gutenberg Health Study, Clin. Chem. Lab. Med. 53 (2015) 699–706. [109] Y. Sandoval, F.S. Apple, The global need to define normality: the 99th percentile value of cardiac troponin, Clin. Chem. 60 (2014) 455–462. [110] P.E. Hickman, T. Badrick, S.R. Wilson, D. McGill, Reporting of cardiac troponin— problems with the 99th population percentile, Clin. Chim. Acta 381 (2007) 182–183. [111] CLSI, Defining, establishing, and verifying reference intervals in the clinical laboratory, approved guideline-third ed, CLSI document EP28-A3c. Clinical Laboratory Standards Institute, Wayne, PA, 2010. 28: 59. [112] J.R. Tate, W. Ferguson, R. Bais, et al., The determination of the 99th centile level for troponin assays in an Australian reference population, Ann. Clin. Biochem. 45 (2008) 275–288. [113] J. Todd, B. Freese, A. Lu, et al., Ultrasensitive flow-based immunoassays using singlemolecule counting, Clin. Chem. 53 (2007) 1990–1995. [114] AACC, Biomarkers of acute cardiovascular disease division news, American Association for Clinical Chemistry 2015. 1 (1) (2015). https://www.aacc.org//media/files/ divisions/bacd/bacd_division_newsletter_2515.pdf?la¼en. [115] A.S. Jaffe, The 10 commandments of troponin, with special reference to high sensitivity assays, Heart 97 (2011) 940–946. [116] F.S. Apple, L.A. Pearce, S.W. Smith, et al., Role of monitoring changes in sensitive cardiac troponin I assay results for early diagnosis of myocardial infarction and prediction of risk of adverse events, Clin. Chem. 55 (2009) 930–937. [117] F.S. Apple, S.W. Smith, L.A. Pearce, et al., Use of the Centaur TnI-Ultra assay for detection of myocardial infarction and adverse events in patients presenting with symptoms suggestive of acute coronary syndrome, Clin. Chem. 54 (2008) 723–728. [118] T. Reichlin, W. Hochholzer, S. Bassetti, et al., Early diagnosis of myocardial infarction with sensitive cardiac troponin assays, N. Engl. J. Med. 361 (2009) 858–867. [119] T. Keller, T. Zeller, D. Peetz, et al., Sensitive troponin I assay in early diagnosis of acute myocardial infarction, N. Engl. J. Med. 361 (2009) 868–877. [120] C.W. Hamm, J.P. Bassand, S. Agewall, et al., ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: the task force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC), Eur. Heart J. 32 (2011) 2999–3054. [121] M. Roffi, C. Patrono, J.P. Collet, et al., 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC), Eur. Heart J. 37 (2016) 267–315. [122] E. Moy, M. Barrett, R. Coffey, et al., Missed diagnoses of acute myocardial infarction in the emergency department: variation by patient and facility characteristics, Diagnosis 2 (2015) 29–40. [123] B.D. McCarthy, J.R. Beshansky, R.B. D’Agostino, H.P. Selker, Missed diagnoses of acute myocardial infarction in the emergency department: results from a multicenter study, Ann. Emerg. Med. 22 (1993) 579–582.

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CHAPTER TWO

Vitamin D Testing—Where Are We and What Is on the Horizon? N. Heureux1 DIAsource Immunoassays, Louvain-la-Neuve, Belgium 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Measurement of Vitamin D Metabolites 2.1 Measurement of 25OH Vitamin D 2.2 Measurement of 1,25(OH)2 Vitamin D 2.3 Free and Bioavailable 25OH Vitamin D 2.4 24,25(OH)2 Vitamin D 2.5 C3-epi-25OH Vitamin D 3. Future Directions 3.1 Improvement of Existing Methods 3.2 Novel Assays 4. Conclusion References

59 60 62 76 83 87 88 90 90 92 93 94

Abstract Vitamin D testing is part of laboratory practice since more than 30 years but has become a routine parameter only recently, due to a highly increasing amount of research in the field resulting in new clinical applications. Vitamin D actually represents a family of molecules of which 25OH Vitamin D and 1,25(OH)2 Vitamin D, under their D3 and D2 forms, are the most important to date. Physical detection methods and immunoassays exist for both molecules and are being reviewed and discussed. New developments in the measurement of C3-epi-25OH Vitamin D, 24,25(OH)2 Vitamin D, and free/bioavailable 25OH Vitamin D are also presented. The future of Vitamin D testing is considered based on the evolution of laboratories and based on the scientific research that is currently performed.

1. INTRODUCTION Vitamin D is part of the laboratories tests menu since the 1980s. It has been used during three decades in the diagnostic and monitoring of Vitamin D deficiency linked to bone diseases such as rickets, osteoporosis, Advances in Clinical Chemistry, Volume 78 ISSN 0065-2423 http://dx.doi.org/10.1016/bs.acc.2016.07.002

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and osteomalacia [1–3]. Although bone-related issues remain the main field of interest for the measurement of Vitamin D metabolites, the massive amount of research that has been performed during the last decade has led to a number of novel clinical applications, with a large portion still being to be fully proven by large random clinical trials [4–6]. This continuously increasing interest for the action of Vitamin D and for the implications of its deficit has led over time to a real explosion of the number of tests performed by the laboratories. Clinicians must understand the fundamental difference between 25OH Vitamin D and 1,25(OH)2 Vitamin D, which is not always the case as 1,25 (OH)2 Vitamin D is still largely misordered [7]. In addition, several immunoassay types and physical detection methods exist for both parameters, and professionals must understand the performance and the limitations of each technique in order to correctly interpret the results of the analyses, together with all the other clinical and diagnostic tools that they have. Finally, the physiology and metabolism of Vitamin D is not fully understood yet and novel assays are currently being developed with the objective of closing this gap. This chapter aims at clarifying the different aspects of Vitamin D testing, from a scientific and from an operational perspective. Existing methods are being reviewed and compared, and research assays are also being discussed.

2. MEASUREMENT OF VITAMIN D METABOLITES Vitamin D is the parent compound of the Vitamin D family (Fig. 1) [8]. As for other Vitamin D metabolites, they exist under two forms, the D2 and D3 forms. Vitamin D3 is synthesized in the skin from 7-dehydrocholesterol under the action of the sun’s UVB radiation [9]. This pathway represents around 90% of the natural sources of Vitamin D. The remaining 10% come from food such as fat fishes, e.g., salmon, sardines, tuna, and eggs. Nowadays it is also common to find in fortified food such as cow’s milk, tofu, cereals, and orange juice. The third source of Vitamin D is the supplementation that is classically prescribed to Vitamin D deficient individuals. Vitamin D2, on the other hand, is less ubiquitous and is not produced in the human body. It is found in very low quantities in some mushrooms and is mostly limited to high-dose supplements in some countries (Fig. 2) [10]. Once present in the human body, Vitamin D is rapidly metabolized into 25-hydroxyvitamin D (25OH Vitamin D) in the liver [11]. This

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H3C

H3C

H3C

H3C

CH2

O

O

Synonym: Cholecalciferol Chemical formula: C27H44O MW: 384.6 g/mol

Synonym: Ergocalciferol Chemical formula: C28H44O MW: 396.7 g/mol O

H

H3C

H3C

O

H3C

H

H3C

H 3C CH3

CH3

O

H3C

H

O

H

H

O

OH CH3

CH3

CH2

H O

H3C

OH

CH3

CH2

CH2

O

Synonym: 25OH Ergocalciferol Chemical formula: C28H44O2 MW: 412.7 g/mol

Synonym: Calcifediol Chemical formula: C27H44O2 MW: 400.6 g/mol CH3

CH3

H

25OH Vitamin D2

25OH Vitamin D3

CH3

H

O

O

Vitamin D2

O

H

H

Vitamin D3

CH3

CH2

CH2

H

H3C

H3C CH3

CH3

CH2

H

H CH3

H3C CH3

CH3

O

CH3

H3C

CH3

H3C

H

O

CH3

CH3

CH2

H

H O

H O

O

1,25(OH)2 Vitamin D3

1,25(OH)2 Vitamin D2

24,25(OH)2 Vitamin D3

24,25(OH)2 Vitamin D2

Synonym: Calcitriol Chemical formula: C27H44O3 MW: 416.6 g/mol

Synonym: NA Chemical formula: C28H44O3 MW: 428.7 g/mol

Synonym: Secalciferol Chemical formula: C27H44O3 MW: 416.6 g/mol

Synonym: NA Chemical formula: C28H44O3 MW: 428.7 g/mol

Fig. 1 Vitamin D ID: Vitamin D metabolites identity sheet.

Sources of

Synthesis in the skin

Vitamin D3

Vitamin D2

7-Dehydrocholesterol + UVB (sun)

–

Nonfortified food

Fortified food

–

Supplements

Fig. 2 Sources of Vitamin D: Natural and synthetic sources of Vitamin D3 and Vitamin D2.

transformation is executed by an enzyme from the cytochrome P family named CYP2R1. 25OH Vitamin D has a long half-life of 2–3 weeks, compared to the other Vitamin D metabolites, and is so far the best biomarker to evaluate the Vitamin D status of an individual [12].

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25OH Vitamin D is further metabolized into the biologically active 1,25 (OH)2 Vitamin D through the action of the cytochrome P family enzyme CYP27B1. Historically, the kidney was thought to be the only organ where this oxidation takes place. Recent studies have identified the presence and the role of CYP27B1 in many other tissues and organs and that should explain the autocrine/paracrine actions of Vitamin D on the human body [11]. The metabolic pathway of Vitamin D is summarized later starting from the different Vitamin D sources down to the catabolic pathway that takes place through an hydroxylation reaction on the position 24 of either 25OH Vitamin D or 1,25(OH)2 Vitamin D (Fig. 3). Serum is the most important matrix for the measurement of Vitamin D metabolites. Serum has the advantage of not being contaminated with anticoagulants used for plasma collection, such as EDTA, Heparin, or Citrate. Vitamin D assays being typically sensitive to interferences from sample matrices, these substances have the potential to interfere with each individual assay and appropriate validation must be conducted when plasma is considered for testing. Saliva has been explored by a few research groups using either competitive protein-binding assays [13], immunoassays [14], or physical detection methods [15,16]. Results are not consistent within the different publications and the work of Higashi et al. seems to be the most reliable so far. LC–MS has been used by the Japanese group to quantify 25OH Vitamin D in the saliva of healthy volunteers. They have established a correlation between salivary and serum levels of 25OH Vitamin D, and a postulate is made on the salivary 25OH Vitamin D concentration reflecting the levels of free 25OH Vitamin D in serum. The excretion of Vitamin D metabolites in urine and bile has been observed in adult rats and might be elevated in chronic renal failure [17]. This can be one of the causes of Vitamin D deficiency in this population. Methods also exist to quantify Vitamin D metabolites in food and supplement matrices [18]. HPLC and LC–MS are mainly used by the manufacturers of fortified food and Vitamin D3 and D2 supplements to assess the concentration of the nonhydroxylated Vitamin D in their products.

2.1 Measurement of 25OH Vitamin D As stated in Section 1 of this chapter, 25OH Vitamin D is so far the best biomarker to evaluate the Vitamin D status of individuals. The measurement

Fig. 3 Vitamin D metabolism.

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of 25OH Vitamin D is useful in the diagnosis of Vitamin D insufficiency or deficiency, to help identify individuals who may benefit from Vitamin D supplementation to reach optimal levels, in monitoring response to Vitamin D supplements for the treatment of bone-related diseases, such as rickets (children), osteomalacia, postmenopausal osteoporosis, and renal osteodystrophy or nonbone-related diseases, and in the diagnosis of Vitamin D toxicity, e.g., patients with suspected toxicity (hypercalcemia) [19–24]. Published in 1971, the very first assay used the competitive protein-binding principle with the native Vitamin D-binding protein (VDBP or DBP) as the capture protein [25,26]. The first immunoassay was developed later in 1985 and used a polyclonal antibody and radioactive I125 as the label [27]. Since that time, other methods have emerged and are discussed in this section. Current methods for measuring 25OH Vitamin D are classified into three categories. Physical detection methods, which include high-pressure liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC–MS/MS), are discussed first. The second subsection examines immunoassay methods, covering radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), chemiluminescent immunoassays (CLIAs), lateral flow immunoassays, and assays for clinical chemistry analyzers (CCA) (Fig. 4).

HPLC Physical detection LC–MS

RIA

Measurement of 25OH Vitamin D

ELISA

Immunoassays

CLIA

LFIA

CCA

Fig. 4 Methods for measuring 25OH Vitamin D: Methods for measuring 25OH Vitamin D can be classified in physical detection methods and immunoassays.

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It should be emphasized that the demand for 25OH Vitamin D tests has truly spiraled over the last decade. Different factors can explain this including the tremendous amount of research that is done on Vitamin D and the discovery of nonbone-related actions of Vitamin D. This is illustrated by the figure later plotting the number of publications on Vitamin D each year, in comparison to other vitamins (Fig. 5). The volume of writings on Vitamin D has clearly risen for more than 10 years with an acceleration in the last 5 or 6 years. The next figure further reveals this trend and shows the number of 25OH Vitamin D tests ordered every month at two institutions in the United States over the years (Fig. 6) [28].

Fig. 5 Number of publications Vitamin D: Number of publications for Vitamin A, C, and D, from 2004 to 2015. From Pubmed including only the field title.

A

UIHC

1500 Orders per month

3000 2000 1000 0

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Fig. 6 Increasing Vit D tests: Average number of 25(OH)D orders per month (in 6-month bins) at Weill Cornell Medical College (WCMC) and University of Iowa Hospitals and Clinics (UIHC).

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As stated by Genzen et al. the increase in 25OH Vitamin D testing was most prominent between 2008 and 2010 with annual growth rates of 52–115% between 2008 and 2009. 2.1.1 Physical Detection Methods—25OH Vitamin D High-pressure or high-performance liquid chromatography (HPLC) and LC–MS/MS are part of this category. Both methods are based on LC to physically separate the different Vitamin D metabolites and the other molecules present in human serum or plasma. LC uses the difference of affinity of the molecules toward the solid phase, contained in the so-called column, or toward the liquid phase, a mixture of organic solvents and/or water-based solutions, known as eluent. The polarity but also the size and conformation of the molecules play an important role in this process (Fig. 7). The detection of the separated molecules is different between HPLC and LC–MS. While HPLC uses UV to detect and quantify, i.e., the Vitamin D metabolites [29], LC–MS relies on MS to differentiate between entities and to quantify them [30–32]. UV is a powerful detection method, especially for Vitamin D metabolites, thanks to their strong absorption at 264 nm. The triene moiety present in the molecule is responsible for this high signal and most of the Vitamin D metabolites do incorporate this particular functionality in their structure. However, the various Vitamin D metabolites exhibit similar UV patterns and need to be fully separated by the LC step in order to be detected and quantified separately. In other words, Eluent

Eluent

Eluent

Eluent

Eluent

Sample

Solid phase

Fig. 7 HPLC: Principle of liquid chromatography. A sample containing different analytes is loaded onto the solid phase and an eluent is added. The analytes will progress according to their affinity for the solid phase and for the eluent. In this example, the light gray circles analytes have a higher affinity for the solid phase than the triangle light gray analytes.

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two Vitamin D molecules which would not be fully resolved by the LC part of the method cannot be independently quantified and only the sum of both metabolites can be reported. This is where MS presents a big advantage. In this technique, molecules are detected by their mass-to-charge (m/z) ratio and most of the Vitamin D metabolites have different masses (Fig. 8). They can therefore be separately detected and quantified even if the LC part of the method does not fully resolve them. For molecules having exactly the same mass and having similar affinities for the chromatography solid phase, fragmentation patterns that occur in advanced LC–MS systems are usually different for each individual compound and allow for separated detection and quantification (Fig. 9). This is the case for 1,25(OH)2 Vitamin D and 24,25 (OH)2 Vitamin D, for example. HPLC, and LC–MS even more so, are powerful methods for the quantification of 25OH Vitamin D in human fluids. Benefits of using these techniques for the measurement of 25OH Vitamin D are their high sensitivity (typically below 1 ng/mL for LC–MS), high accuracy, and excellent reproducibility profile (CVs typically range from 2% to 7–8%). Thanks to the detection by MC, LC–MS allows for a good resolution of the multiple Vitamin D metabolites, which makes the method less prone to specific sample interferences such as the one caused by the presence of 24,25(OH)2 Metabolites

Monoisotopic mass

Vitamin D3

384.33

Vitamin D2

396.33

25OH Vitamin D3

400.33

25OH Vitamin D2

412.33

3-epi-25OH Vitamin D3

400.33

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412.33

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400.33

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412.33

1,25(OH)2 Vitamin D3

416.33

1,25(OH)2 Vitamin D2

428.33

24,25(OH)2 Vitamin D3

416.33

24,25(OH)2 Vitamin D2

428.33

25,26(OH)2 Vitamin D3

416.33

25,26(OH)2 Vitamin D2

428.33

Fig. 8 Vitamin D MW: Monoisotopic masses of several Vitamin D metabolites.

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0.6 Signal

• Cannot be resolved by UV • Can be resolved by MS if m/z and/or fragmentation pattern is different

0.5

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Fig. 9 HPLC–LC–MS: Example of a chromatogram of two analytes that are not resolved by liquid chromatography. UV cannot differentiate the two molecules, while MS can discriminate them based on their mass-to-charge ratio.

Vitamin D. HPLC and LC–MS are also able to separately quantify the D2 and D3 forms of 25OH Vitamin D, which represent an interest for specific clinical studies and monitoring of patients supplemented with Vitamin D2. Thanks to its excellent performances LC–MS is currently recognized as the Gold Standard methodology for the measurement of 25OH Vitamin D in human fluids such as serum and plasma. The Vitamin D Standardization Program (VDSP) recognizes three Reference Measurement Procedures (RMPs) (Ghent, NIST, and CDC), which are all LC–MS methods [33,34]. This program aims at promoting the standardization of all 25OH Vitamin D assays, one part being assays produced by manufacturers, and the other part being methods developed by laboratories. The Vitamin D Standardization Certification Program (VDSCP), coordinated by the Centers for Disease Control and Prevention (CDC), is part of these efforts and offers to assay manufacturers and laboratories the possibility to certify the mean accuracy and reproducibility of their methods based on the measurement of multiple sets of blinded samples assessed by one of the Reference Measurement Procedures [35]. However, HPLC and LC–MS also present some drawbacks, which are mainly of a technical nature. They both require the access to high quality water, solvents, and chemicals, and most of the laboratories are developing and maintaining their in-house methods, which poses regulatory challenges

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to the concerned authorities and require time and highly skilled engineers. The cost of an instrument and its maintenance are also often a hurdle in several parts of the world. Last but not least, HPLC and LC–MS are sensitive instruments and serum or plasma needs to be extracted, or at least cleaned up, before analysis. This represents a cumbersome and labor intensive process and should not be neglected when considering the use of these methods for the analysis of hundreds or thousands of samples a day. In terms of performance, C3-epi-25OH Vitamin D remains an issue for many HPLC and LC–MS methods [36]. Because of its similar UV pattern and identical monoisotopic mass and fragmentation patterns it cannot be resolved from 25OH Vitamin D by the UV or MS detection techniques. Therefore, the development of a LC protocol that fully separates the C3-epi metabolite from the desired 25OH Vitamin D is required. Although technical solutions exist and are used by several laboratories, C3-epi-25OH Vitamin D still interferes in many HPLC and LC–MS methods [37]. This was demonstrated in the NIST/NIH Vitamin D Metabolites Quality Assurance Program shown later (Fig. 10). The blinded study sample SRM 972a level 4 was fortified with 3-epi-25-hydroxyvitamin D3 and participants were asked to provide the total concentration of 25OH Vitamin D. LC methods that do not chromatographically separate the 3-epi-25(OH)D3 yield biased results. The majority of the IA methods, on the other hand, do not have cross-reactivity with the 3-epi-25(OH)D3 metabolite and yield an unbiased median result. The technique is evolving quickly and recent developments have been directed toward even more specific methods, multiplexing, and automation of the laborious extraction steps. Jones et al. have developed a LC–MS method that uses derivatization to increase sensitivity and allows the complete profiling of six Vitamin D metabolites using less than 100 μL of sample [38]. The method can be used in human clinical research as well as in animal basic research. Geib et al. reported the use of commercially available 96-well extraction plates for the “one-pot” preparation of serum samples prior to LC–MS analysis [39]. Although the method does not allow for a fully automated sample treatment coupled to LC–MS, it represents one of the multiple efforts to simplify and robotize the cumbersome sample pretreatment steps associated with physical detection methods. 2.1.2 Immunoassays—25OH Vitamin D Immunoassays, i.e., assays based on antibodies, for the measurement of 25OH Vitamin D exist since 1985 and are still the most prevalent methods

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Fig. 10 Figure from NIST: From NIST/NIH Vitamin D Metabolites Quality Assurance Program Report of Participant Results: Summer 2013 Comparability Study (Exercise 7): The blinded study samples consisted of two vials, Vial A and Vial B. Vial B was SRM 972a Vitamin D metabolites in human serum level 4 (SRM 972a L4), which contains endogenous levels of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3), but was fortified with 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). Participants were asked to provide individual concentration values for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 along with a total concentration of 25(OH)D (25(OH)D total ¼ 25(OH)D2 + 25(OH)D3) for the control and each study sample. The results from immunoassay methods are displayed with open dark gray circles (left of the graph), and the results from the LC-based methods are displayed with open light gray circles (right of the graph). For both of the major techniques (IA or LC), the solid lines represent the consensus median, and the dashed lines represent the approximate 95% confidence interval. The red lines represent the NIST value and its associated uncertainty (i.e., value  U95). The LC results are bimodal, where nine reported results agree well with both the NIST value and the reported IA results, but the majority of the LC results (73%) are biased high. SRM 972a L4 was fortified with 3-epi-25(OH)D3, and the NIST-certified value for this vitamin D metabolite is 26.4  2.1 ng/mL. The biological significance of 3-epi-25(OH)D3 is uncertain, and this metabolite is not included in the 25(OH)D total concentration. Therefore, LC methods that do not chromatographically separate the 3-epi-25(OH)D3 yield biased results for 25(OH)D3, and hence 25(OH)D total because the 3-epi-25(OH)D3 and the 25(OH)D3 are diastereomers that are detected by the same multiple reaction monitoring (MRM) ions in MS/MS and absorbance wavelength in UV. The majority of the IA methods, on the other hand, do not have cross-reactivity with the 3-epi-25(OH)D3 metabolite and yield an unbiased median result of 29.6 ng/mL for 25(OH)D total in SRM 972a L4.

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nowadays, although physical detection methods and more particularly LC–MS are more and more present in clinical laboratories. Immunoassays can be classified into three big categories: RIA, ELISA, and CLIA. All three methodologies are competitive immunoassays, i.e., the 25OH Vitamin D present in the sample competes with a labeled 25OH Vitamin D analogue for a limited number of binding sites on an antibody. They differ in the labeling of the competitive antigen and in the detection method. RIA is based on radioactive iodine 125 labeled 25OH Vitamin D analogues and relies on gamma counters for the detection part. ELISA is based on 25OH Vitamin D analogues labeled with an enzyme. Horseradish peroxidase (HRP) is mainly used followed by alkaline phosphatase (ALP). The detection is based on a colorimetric reaction and is quantified by measuring the absorbance in an ELISA reader. CLIA methods are also based on 25OH Vitamin D analogues labeled with an enzyme but the detection is based on the emission of light by a specific substrate and is quantified by a photometer. 25OH Vitamin D immunoassays are made up of two main technological components (Fig. 11). The first one is the release of 25OH Vitamin D from its binding proteins, namely the VDBP or DBP and albumin. The second part is the complexation of the 25OH Vitamin D by the antibody and its competition with the labeled 25OH Vitamin D analogue. Both parts will be discussed later. 25OH Vitamin D is hydrophobic and poorly soluble in human blood. It is therefore linked to two binding proteins, the VDBP or DBP and the albumin. It is necessary to release 25OH Vitamin D from these two proteins prior to analysis and multiple methods have been developed for this purpose (Fig. 12). Historically organic solvents were used to denature and precipitate

25OH Vit D

Release

Vit D labelled

25OH Vit D

Competition

Vit D labelled

25OH Vit D

25OH Vit D

Part I—Release of 25OH Vitamin D from the binding proteins

Part II—Competition between the sample 25OH Vitamin D and the labeled Vitamin D analogue

Fig. 11 25OH Vitamin D immunoassays: Principle of 25OH Vitamin D immunoassays. Left: Release of 25OH Vitamin D from its binding proteins. Right: Complexation of the 25OH Vitamin D by the antibody and its competition with the labeled 25OH Vitamin D analogue.

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C

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25OH Vit D

Organic solvent

25OH Vit D

25OH Vit D

Digestion enzyme

25OH Vit D

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25OH Vit D

pH shift

25OH Vit D

25OH Vit D

Displacement reagent

25OH Vit D

Fig. 12 25OH Vitamin D release methods: 25OH Vitamin D release methods. (A) Extraction with organic solvents and precipitation of the binding proteins. (B) Enzymatic digestion of the binding proteins. (C) Denaturation of the binding proteins at acidic or alkaline pH. (D) Use of a displacement reagent to release 25OH Vitamin D from the binding proteins.

the serum or plasma proteins, including DBP and albumin [27]. After a separation step 25OH Vitamin D and other hydrophobic molecules were present in the solvent part and could be analyzed with or without additional manipulation steps (Fig. 12A). This method is very efficient and reliable but not compatible with large automated instruments and high-throughput methods. It has been slowly abandoned and other methodologies have been developed over the last 10 years to meet the needs for automated and faster assays. Digestion enzymes such as Proteinase K have been used to decompose the binding proteins and release 25OH Vitamin D in the digestion buffer (Fig. 12B) [40]. This method has not been extensively used because of relatively slow kinetics, the need for optimum enzyme temperature (around 37°C) and the requirement to neutralize the activity of the enzyme in order to avoid the digestion of the antibody itself. The modification of the protein structure through a pH shift is one of the most widely used methods nowadays [41–44]. Modification of the sample pH to mildly acidic or alkaline pH causes an unfolding of the protein structure, releasing the 25OH Vitamin D molecule (Fig. 12C). The pH is then restored to an optimal pH to match the antibody activity. The neutralization of acidic or alkaline pH usually requires an additional step although ingenious methods have been developed to realize this in situ. There is a risk that proteins refold once the pH has been restored and that 25OH Vitamin D is captured by the

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refolded protein and not by the antibody. For these reasons, displacement reagents have been developed very recently [45,46]. These reagents act by displacing the 25OH Vitamin D from its binding site at neutral pH and without any major modification of the protein structure. In presence of the displacement reagent, the binding protein cannot link the 25OH Vitamin D anymore and the immunoreaction with the antibody can take place smoothly (Fig. 12D). Enzymatic digestion of the sample binding proteins was described by Armbruster et al. in 2008 [40]. The serine protease Proteinase K performs the digestion of the sample at 37°C and sodium dodecyl sulfate (SDS) or 8-ANS (8-anilinonaphthalene-1-sulfonic acid) is added to ensure a complete release of 25OH Vitamin D from VDBP and albumin. The activity of the enzyme is then stopped by dilution in a specific buffer containing a calcium scavenger. To illustrate the use of pH shift without the need for a neutralization step, the application filed by Antoni and Vogl describes an ingenious method [47]. The sample binding proteins are denatured by the use of mildly to highly alkaline conditions, releasing the 25OH Vitamin D. At the same time ethylene carbonate is decomposed by the high-pH solution and acidic HCO3  ions are formed. The pH is consequently restored to values around 8.5–9.5 within 10 min. Acidic pH shift has been developed by Yuan et al. in their 25OH Vitamin D assay for CCA [48]. A solution of sodium acetate decreases the pH down to 3.0, releasing the 25OH Vitamin D from its binding proteins. A neutral pH is then restored thanks to the addition of a sodium phosphate buffer. In its application, Uchida et al. described the use of a buffer solution containing a surfactant that has a steroid skeleton combined with another denaturing agent [49]. The best results were obtained when using a mixture of SDS and either CHAPS (3-(3-cholamidepropyl)dimethylammonio-1propanesulfonate) or DC Na (deoxycholate sodium). An efficient release was also obtained when mixing the sample with a combination of guanidine hydrochloride and DC Na. After 25OH Vitamin D has been released from its binding proteins different biological molecules can be involved in immunoassays, including polyclonal antibodies, monoclonal antibodies, and VDBP. Polyclonal antibodies have been used in 25OH Vitamin D immunoassays for more than 30 years. They are produced by immunization of animals with a 25OH Vitamin D analogue coupled to a carrier protein, then

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recurrent bleedings of these animals and isolation of the serum containing the antibodies against 25OH Vitamin D, also called antiserum [27,50]. Rabbits and goats are the most common animals but chickens, guinea pigs, hamsters, horses, mice, rats, and sheep can also be used. Multiple animals are typically involved as the immune system of each animal is different and will lead to polyclonal antibodies having different properties. Before proceeding with the bleedings small serum samples are collected from each animal to evaluate the level of 25OH Vitamin D antibodies present and their performance profile. The specificity of the antibodies, i.e., the cross-reactivity against various Vitamin D metabolites, is one of the key criteria during the animal selection process. Modern 25OH Vitamin D assays must be able to quantify 25OH Vitamin D3 and D2, as both molecules can be present in patient samples, without interference from 1,25(OH)2 Vitamin D; 24,25 (OH)2 Vitamin D; 25,26(OH)2 Vitamin D; and C3-epi-25OH Vitamin D. Polyclonal antibodies typically lack the requested specificity, although some examples of specific polyclonal antibodies have been reported. The major drawback of polyclonal antibodies is the impossibility to reproduce them identically. Large volumes of antiserum can be generated from one animal or even from pooling different animal bleedings with similar performance profiles. However, the increasing demand for 25OH Vitamin D test has required assay manufacturers to scale-up their production processes and larger and larger volumes of antibodies are used nowadays, making the use of polyclonal antibodies a hazardous situation for companies willing to supply 25OH Vitamin D assays with stable quality over time. Monoclonal antibodies relieve this issue [51]. The first step of their development is identical to the polyclonal antibodies although mice are used in most cases. Animals are immunized with a 25OH Vitamin D—protein conjugate, then screened on a regular basis by evaluation of serum samples. Once the required response is obtained, the animals are sacrificed and the spleen cells are collected and fused with myeloma cells to form a hybridoma. The hybridoma is immortal and can be stored in liquid nitrogen for life. A screening process ensures that the selected hybridoma feature the requested performance characteristics. When needed an aliquot of the hybridoma is cultured with classical cell culture techniques and monoclonal antibodies can be harvested and purified within a few weeks. Thanks to this technique, highly specific monoclonal antibodies can be produced on demand and with a perfect lot-to-lot consistency. Most of the contemporary immunoassays use monoclonal antibodies or VDBP as described later.

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The third category of biological molecule that can be used in 25OH Vitamin D immunoassays is the binding proteins [52]. Extracted native VDBP or its recombinant form both have the ability to capture the released 25OH Vitamin D from the samples. The role of VDBP in the human body is to transport and store multiple Vitamin D metabolites. It has therefore the capacity to bind both the D2 and the D3 forms of 25OH Vitamin D. However, interferences from other Vitamin D metabolites can be foreseen although the affinity toward the two forms of 25OH Vitamin D is the highest. Depending on the assay format the antibodies or the binding proteins are immobilized on a plastic surface or on paramagnetic particles. Released 25OH Vitamin D from the patient samples will compete for the biological molecule with a labeled 25OH Vitamin D analogue. RIA use a radioactive isotope of iodine namely iodine 125 as the label. Iodine 125 is incorporated on the 25OH Vitamin D analogue onto a tyramine or a histamine moiety by electrophilic addition. As the iodine 125 is relatively small compared to the 25OH Vitamin D analogue structure the competition with the released 25OH Vitamin D is very well balanced and that is the reason why RIA are still among the most sensitive assays. ELISAs involve HRP or ALP, two enzymes capable of transforming a substrate into a product with the formation of color. As both HRP and ALP are large biological molecules, the balance of the competition between the native and the labeled 25OH Vitamin D is disturbed in favor of the native antigen and assay development tricks must be used to overcome this issue. For this reason, however, ELISAs are usually less sensitive than, e.g., RIA. CLIAs are also based on enzyme conjugates but the substrates are different and produce the emission of light that is read by a photometer. Other assay formats exist in which the antibodies are present in the liquid phase and are precipitated using a neutral solid phase such as long-chain polyethylene glycol [53]. The Vitamin D analogue can also be immobilized on a solid phase and the antibody is therefore labeled [54]. More recently sandwich-based immunoassays have emerged [55]. A classical sandwich immunoassay is not possible for small molecules such as 25OH Vitamin D, as only one paratope is available for complexation by a single antibody. But the change of conformation of an antibody upon complexation of 25OH Vitamin D can be exploited to be bound by a second antibody. The latter only recognizes the first antibody conformationally altered by the presence of 25OH Vitamin D.

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Finally, the position of the 25OH Vitamin D molecule on which the carrier protein for immunization or the label is conjugated is crucial. Immunization of animals with a 25OH Vitamin D analogue conjugated to the hydroxyl group on position 3 will produce antibodies directed toward the opposite side of the molecule, i.e., the side chain. These antibodies will be very selective for different Vitamin D metabolites bearing different side chains but will suffer from a lack of selectivity for Vitamin D metabolites that differ from the position 3 region [27]. On the other hand, antibodies produced from a protein conjugated on the side chain will have an opposite specificity profile [51]. In both cases, the labeled 25OH Vitamin D analogue must be conjugated at the same position as the immunogen used during the antibody development so that the same region of the molecule is exposed to the antibody during the assay. The figure later shows the distribution between the different methods participating in the Vitamin D External Quality Assessment Scheme (DEQAS) in July 2012 and in July 2015 (Fig. 13) [56]. Chemiluminescencebased assays (CLIA) represent the biggest part of the pie with a 9% increase over the last 3 years. The fact that these methods are fully automated and have high throughput is certainly one of the main reasons for this success. ELISA was the second method in July 2012 but the number of participants has strongly diminished by more than 50%, while LC–MS has become more and more popular during the same period. RIA has continued to diminish and HPLC and other methods have remained stable at relatively low numbers. It should be emphasized that this does not represent the real distribution of 25OH Vitamin D methods in clinical laboratories worldwide, but it shows at least the general trends in the field.

2.2 Measurement of 1,25(OH)2 Vitamin D Basics of HPLC, LC–MS, and Immunoassays have been discussed in the previous section on the measurement of 25OH Vitamin D and most of them apply for the measurement of 1,25(OH)2 Vitamin D as well. In some aspects the measurement of 1,25-dihydroxyvitamin D or 1,25 (OH)2 Vitamin D is similar to the measurement of 25OH Vitamin D. Some other aspects are very different. 1,25(OH)2 Vitamin D links in the human body to the Vitamin D receptor (VDR) and is therefore the biologically active form of the Vitamin D family [57]. Because of its rapid production and transformation metabolism, and of its tight regulation by hormonal control, the

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July 2015 3%

1%

1% 6%

16%

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Fig. 13 DEQAS July 2015: Distribution of 25OH Vitamin D methods participating to the DEQAS program in July 2015 and in July 2012.

concentration of 1,25(OH)2 Vitamin D is not a reliable marker of an individual’s Vitamin D status, and the measurement of 25OH Vitamin D plays this role [12]. However, 1,25(OH)2 Vitamin D can be useful as a secondorder test in the assessment of Vitamin D status, especially in patients with renal disease [58], in case of investigation of some patients with clinical evidence of Vitamin D deficiency [59], in the differential diagnosis of hypercalcemia [60], in primary hyperparathyroidism [61], and in physiologic hyperparathyroidism secondary to low-calcium or Vitamin D intake, for

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Physical detection

Measurement of 1,25(OH)2 Vitamin D

LC–MS

RIA

Immunoassays

ELISA

CLIA

Fig. 14 Methods for measuring 1,25(OH)2 Vitamin D: Methods for measuring 1,25(OH)2 Vitamin D can be classified in physical detection methods and immunoassays.

patients with granulomatous diseases [62] and malignancies containing nonregulated 1-alpha hydroxylase in the lesion, and in hypoparathyroidism [63]. The concentration of 1,25(OH)2 Vitamin D in the serum or plasma of healthy individuals is typically 20–50 pg/mL, that being 1000 times less than the concentration of 25OH Vitamin D and to a lower extent than the concentration of Vitamin D, 24,25(OH)2 Vitamin D, and other Vitamin D metabolites. Measuring 1,25(OH)2 Vitamin D is like fishing for the right specimen in a shoal of similar fish. Scientists have, therefore, developed specific extraction and separation methods to allow the quantification of 1,25 (OH)2 Vitamin D without interference from other structurally close molecules. Contrary to 25OH Vitamin D, 1,25(OH)2 Vitamin D has much less affinity with the VDBP and the extraction methods ensure an efficient and reliable release. Immunoassays largely dominate the field, with RIA, ELISA, and CLIA methods. More recently, the physical detection method LC–MS has appeared and is slowly expanding, due to strong technical hurdles (Fig. 14). 2.2.1 Physical Detection Methods—1,25(OH)2 Vitamin D LC–MS represents this category [64–68]. Despite its high performances LC–MS suffers from the low sensitivity of the technique against 1,25(OH)2 Vitamin D. Therefore, derivatization is often used to increase the dose response of the signal. Extraction and separation steps are also mandatory to ensure sufficient specificity and accuracy. Immunoaffinity columns are today the most widely used method to separate 1,25(OH)2 Vitamin D from the other Vitamin D metabolites. Antibodies against 1,25(OH)2 Vitamin D are immobilized on a solid phase and specifically capture and

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release this molecule according to the elution conditions. On-line sample extraction and 96-well plate solid-phase extraction sample preparation have also been described. Overall the extraction, separation, LC, and MS steps make LC–MS a complex and technically challenging method for the measurement of 1,25(OH)2 Vitamin D. 2.2.2 Immunoassays—1,25(OH)2 Vitamin D Immunoassays are the most widespread method for the measurement of 1,25 (OH)2 Vitamin D. They can be classified into three big categories: RIA, ELISA, and CLIA. The RIA and ELISA methods are competitive immunoassays and are all based on extraction and separation protocols to isolate the 1,25(OH)2 Vitamin D from the other Vitamin D metabolites and various matrix components. Extraction is generally accomplished with the use of an organic solvent or of a mixture of solvents, such as methanol, acetonitrile, cyclohexane, diisopropyl ether, or ethyl acetate [69,70]. The separation of 1,25(OH)2 Vitamin D can then be performed using two apparently similar but very different techniques. Classical chromatography on silica or C18OH cartridges is one of these. The extracted sample is applied on the cartridge and the different molecules present in the extract will progress, or elute, through the solid phase at different speeds, mainly depending on their polarity. The use of multiple specific solvents, or eluents, ensures a clean separation of 1,25(OH)2 Vitamin D from the other molecules. The fraction containing 1,25(OH)2 Vitamin D is then used as such, or after a drying—reconstitution step, in the RIA or ELISA (Fig. 15). The second technique that is used to isolate 1,25(OH)2 Vitamin D is the immunoaffinity separation [71–73]. Specific antibodies against 1,25(OH)2 Vitamin D are immobilized on a solid phase and the extracted sample is applied through this solid phase. Based on the affinity of the antibodies toward 1,25(OH)2 Vitamin D, this molecule is retained in the cartridge while the others flow through quickly. Conditions that will release 1,25 (OH)2 Vitamin D from the antibody are then applied to recover the target molecule and this fraction is used as such, or after a drying—reconstitution step, in the RIA or ELISA (Fig. 16). Although they may look very similar, the chromatography and the immunoaffinity techniques are very different in their principle as the first rely on different elution speeds according to physicochemical parameters, mainly polarity, while the second uses the capacity of an immobilized antibody to selectively bind 1,25(OH)2 Vitamin D. The specificity of the

Extracted sample

Eluent A

Eluent A

Eluent B

Eluent B

1,25(OH)2 Vitamin D Other extracted molecules

Solid phase

Fig. 15 Chromatography 1,25: Separation of 1,25(OH)2 Vitamin D by chromatography. The extracted sample is applied on the cartridge and the different molecules progress according to their affinity or polarity for the solid phase or for the different eluents.

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Extracted sample 1,25(OH)2 Vitamin D Other extracted molecules

Solid phase

Fig. 16 Immunoaffinity 1,25: Separation of 1,25(OH)2 Vitamin D by immunoaffinity. The extracted sample is applied on a solid phase coated with specific antibodies against 1,25 (OH)2 Vitamin D. Based on the affinity of the antibodies toward 1,25(OH)2 Vitamin D, this molecule is retained in the cartridge, while the others flow through quickly.

chromatographic separation depends on the quality of the solid phase and on its packing, and on the strict respect of the elution protocol. On the contrary, immunoaffinity separation is a more robust technique with respect to the experimental conditions, but the specificity of the method is highly dependent on the choice of the antibody during assay development. The comprehension of these differences is necessary when using, comparing, or troubleshooting these methods. Immunoaffinity can also be applied to automated assays [74,75]. In this case, the separation antibody is immobilized on a paramagnetic particle and different buffers are used to perform the capture or the release of 1,25(OH)2 Vitamin D from the particles (Fig. 17). Washing steps involving a magnet to retain the particles in the bottom of the separation cell lead to the separation of 1,25(OH)2 Vitamin D from the other molecules. The solution containing 1,25(OH)2 Vitamin D is then transferred into a second test cell where another paramagnetic particle coated with an antibody against 1,25(OH)2 Vitamin D is used for the immunoassay part. Recently, a novel CLIA method has been developed and released on the market. This innovative assay relies on the VDR to selectively bind the 1,25 (OH)2 Vitamin D. Due to its high instability in assay conditions a more stable fragment of the VDR is used to capture the 1,25(OH)2 Vitamin D. A conformation change of the VDR fragment occurs upon binding of the 1,25(OH)2 Vitamin D and a supported monoclonal antibody selectively recognizes the conformationally changed molecule. After a washing step under magnetic field, a second antibody directed toward another region of the recombinant VDR is added and contains the label that will lead to the chemiluminescent signal. This novel methodology involves no true physical separation step, and therefore allows the assay to be fully automatable.

A

B

C

D

E

F

Extracted sample Separation cell

1,25(OH)2 Vitamin D Other extracted molecules

* Immunoassay cell

*

Fig. 17 Particles immunoaffinity 1,25: Separation of 1,25(OH)2 Vitamin D by immunoaffinity with particles. The sample in incubated with paramagnetic particles coated with a specific antibody against 1,25(OH)2 Vitamin D (A). 1,25(OH)2 Vitamin D is captured by the antibody (B). A magnetic field is applied to attract the particles and the remaining solution is washed away (C). The magnetic field is stopped and 1,25(OH)2 Vitamin D is released from the particles thanks to a specific buffer (D). The supernatant containing the 1,25(OH)2 Vitamin D is transferred to a second cuvette and incubated with paramagnetic particles coated with a specific antibody against 1,25(OH)2 Vitamin D and a labeled 1,25(OH)2 Vitamin D conjugate (E). The competition takes place and the signal is read after another washing cycle (F).

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2.3 Free and Bioavailable 25OH Vitamin D Due to its hydrophobic nature 25OH Vitamin D, and other Vitamin D metabolites, circulate on binding proteins. About 90% of the total circulating 25OH Vitamin D is bound to the so-called VDBP or DBP. VDBP, also known as group-specific component globulin, is a 58 kDa glycosylated alpha-globulin. It binds Vitamin D metabolites with the following relative affinities: 25OHD-23,26-lactone > 25OHD ¼ 24,25(OH)2D ¼ 25,26(OH)2D > > 1,25(OH)2D > Vitamin D[76–78]. The remaining 10% are bound to albumin, the main protein of human blood plasma. Although the affinity of albumin toward 25OH Vitamin D is much lower than the affinity of VDBP, the high concentration of albumin compensates for this difference. A tiny fraction representing 0.04% of the total 25OH Vitamin D concentration circulates as the free form [79]. The sum of the free form and the fraction bound to albumin is called bioavailable 25OH Vitamin D [80]. The sum of all three fractions is called total 25OH Vitamin D although the term “total” often refers to the sum of the D2 and D3 forms of the Vitamin D metabolites (Fig. 18). The conversion of 25OH Vitamin D into the biologically active 1,25 (OH)2 Vitamin D takes place into the cells and so requires the internalization of 25OH Vitamin D from the extracellular fluid. Different transport mechanisms are likely to be involved and some of them involve the concentration of the free ligand as one of the important parameters. In these cases, the fraction of free 25OH Vitamin D relates to the biological activity of Vitamin D, and therefore may better reflect the physiological action of Vitamin D than the total concentration of 25OH Vitamin D (Fig. 19) [81]. The fraction of free 25OH Vitamin D represents about 0.04% of the total concentration of 25OH Vitamin D. However, this percentage is not constant and varies according to different conditions. Although the level of

25OH

90% DBP

Total 25OH Vitamin D Albumin

25OH

10% Bioavailable 25OH Vitamin D

25OH

0.04%

Free 25OH Vitamin D

Fig. 18 Free and bioavailable Vitamin D: Free, bioavailable, and total 25OH Vitamin D.

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Fig. 19 Transport mechanisms: Membrane receptor-mediated and receptorindependent mechanisms for the cellular uptake of vitamin D. Vitamin D metabolites are bound to DBP in serum and extracellular fluid. Intracellular uptake of vitamin D metabolites may occur via one of four different mechanisms outlined in the scheme.

albumin tends to be stable among individuals, the concentration of VDBP can fluctuate in several conditions, therefore, influencing the fraction of free 25OH Vitamin D[82–88]. Pregnancy leads to an increase of the VDBP levels by about 50% while, e.g., liver failure and chronic kidney disease both result in a decrease of the VDBP concentration by also about 50%. In case of elevated binding proteins concentration, the fraction of free 25OH Vitamin D is lower, and vice-versa in the case of low-binding proteins concentration. In these conditions, and in the conditions listed later, the measurement of free 25OH Vitamin D may be a better marker of the Vitamin D activity than the classical measurement of total 25OH Vitamin D (Table 1). In addition to variable concentrations VDBP also exists as different polymorphic forms (Table 2). The affinity of the different VDBP forms toward 25OH Vitamin D may vary although this is still under debate [76–78]. A polymorphic form with a high affinity for 25OH Vitamin D will decrease the fraction of free 25OH Vitamin D available. Conversely, a low-affinity VDBP form will result in higher levels of free 25OH Vitamin D. The major form of VDBP present in individuals depends on the ethnicity. For example,

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Vitamin D Testing

Table 1 VDBP Concentration: Vitamin D-Binding Protein Concentration According to Different Clinical Conditions Reported DBP Concentration Clinical Condition Range (mg/L)

Normal women (Bouillon)

333  58

Normal men and women (Bikle)

405  128

Normal

436  33

Pregnancy

18  4 weeks

613  142

32  3 weeks

683  82

35  1 weeks

688  104

40  1 weeks

616  84

Use of oral contraceptive

488  90

Normal weight women

266  104

Obese women

320  121

Liver disease

178  92

Peritoneal dialysis

203  14

Nephrotic patients

371  46

Dialysis population

158 (69–217)

Pediatric renal disease

CKD stage 2/3 206 (136–287) CKD stage 4/5 208 (101–282) Dialysis

168 (99–242)

Table 2 VDBP Forms: Affinity Constants of DBP for 25OH Vitamin D, for the Six Different Phenotypes, According to Arnaud and Constans Diplotype Phenotype Binding Coefficient

GC/GC

Gc-1S/Gc-1S

6  108

GC/TC

Gc-1S/Gc-1F

4.8  108

GC/TA

Gc-1S/Gc-2

8.6  108

TC/TC

Gc-1F/Gc-1F

3.6  108

TC/TA

Gc-1F/Gc-2

7.4  108

TA/TA

Gc-2/Gc-2

11.2  108

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N. Heureux

among blacks the predominant form is Gc1F, while among whites, the predominant form is Gc1S. If the difference in affinity really exists, the measurement of free 25OH Vitamin D may again be a better marker of the Vitamin D action than the measurement of total 25OH Vitamin D [89–91]. Free 25OH Vitamin D can be either measured or estimated through calculations. Indirect measurement was first demonstrated by the group of Bikle in 1986 [86]. Centrifugal ultrafiltration of serum samples spiked with radioactive compounds led to the evaluation of the percentage of free 25OH Vitamin D. Its concentration was then calculated from the concentration of total 25OH Vitamin D, as obtained by another assay. Although the methodology proved to be quite accurate and made it possible to establish large differences in the percentage of free 25OH Vitamin D between a normal population and a population of patients presenting liver failures, the technique presented several drawbacks. It required large volumes of samples and the manipulation of tritium and C14-based reagents, and consumed a lot of time and precious manpower. Direct measurement of free 25OH Vitamin D first became available in 2013 through the development of a super sensitive ELISA [79]. The method presents a limit of detection of