HIGHLY SENSITIVE TROPONIN T Which reference interval ? Gus Koerbin “… practical medicine is basically founded on comparison” Schneider AJ. Pediatrics 1960. 1 “the reference interval is the most widely used medical decisiondecision-making tool” Horn PS, Pesce AJ. Clin Chim Acta 2003 “Reference intervals should be determined in a systematic and scientific manner that provides an adequate degree of confidence for the clinical decision making process.” Horn P. Clinical Chemistry 5th Ed. 2010 2 “If medicine d is to b be scientific, ………..we must also understand the basis of our comparisons” “ ………….a sound basis for these comparisons is often lacking in the clinical laboratory laboratory” Ceriotti F. 2007 Background      Analyte Description Method Pre analytical factors Reference Interval Principle Clinical Significance Jones GRD. Clin Biochem Rev; 2008 3  Analyte description  Useful background information   Within and between person biological variability Method  K factors Key f are:  Accuracy base  Analytical specificity.    Traceability to a method or reference material expressed quantitatively Different methods are appropriately compared. Pre--analytical factors Pre     Serum vs Plasma Haemolysis Icterus Lipaemia AT RELEVENT ANALYTE CONCENTRATIONS Reference Interval Principle  The reference interval principle is usually to take the central 95% of a reference p population. p How to define and determine reference intervals in the clinical laboratory: approved guideline – 2nd ed. CLSI; 20001,2  There are accepted variations from this principle such as the 99th percentile of a healthy population for cardiac troponins Thygesen K, Alpert JS, White HD: Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. Circulation. 2007;116:26342007;116:2634-53. 53. 4 Clinical Significance  Use of reference interval   Confirmation of validity (regardless of process to define that reference interval)   Discussion with experienced p clinicians With clinical colleagues using the test to manage p patients Lack of consultation and acceptance Ignore reference interval  Use outdated or obsolete guidelines  What about Troponin  The guidelines Thygesen K, Alpert JS, White HD et al. Universal definition of myocardial infarction. J Am Coll Cardiol 2007 5 Expert Comments Apple F. HighHigh-Sensitivity Cardiac Troponin Assays: What Analytical and Clinical Issues Need to Be Addressed Before Introduction into Clinical Practice? Clin Chem 2010  From the guidelines   Total assay imprecision is recommended but not required to be ≤10% at the 99th percentile. The literature  Assay imprecision 20% CV at the 99th percentile value does not lead to substantial increases in falsefalsepositive or -negative misclassifications. 6  Guidelines recommend   We should study a normal reference population for calculation of the 99th percentile before assay implementation in clinical practice. However, the specifications of that population      age sex ethnicity race number of individuals included in the “normal” group have not been clearly stipulated. “It appears, however, that no real consensus exists on how to define a “high“highsensitivity assay,” in analytical or clinical terms.” 7 David Morrow  Characterization of the reference population is one of the most important issues we face.  C d t reference Conduct f testing t ti in i a thoroughly characterized set of younger individuals the “normal, healthy population”.  The p population p needs to be of mixed sex and ethnicity and free of any identifiable structural heart disease.   magnetic resonance imaging (MRI) computed tomography (CT) angiography  Significant in defining reference values for the new “high “high-sensitivity” assays that provide  All JJaffe Allan ff sex, age, and ethnicity  Possibly decile age ranges  The real challenge of performing such a study is how to define and select healthy individuals and the number per decile. 8 There is confusion by practitioners over appropriate clinical decision limits. THE ACUTE CORONARY SYNDROME Health minor myocardial i j injury myocardial i f ti infarction The Acute Coronary Syndrome is a Continuum The Current General Opinion  It has been stated unequivocally that troponin release only occurs in the presence of necrosis  This concept that myocytes cannot divide originated g in the 1920s  If this is true then the heart should respond to increases in workload by hypertrophy of existing myocytes.  When this myocardial hypertrophy reaches a maximum, cell death and heart failure occur. Morrow DA 2007  Why?  The belief is that the heart is a terminally differentiated organ and unable to regenerate working myocytes. 9 Does Troponin p Always y Equal q Necrosis ? The “Healthy Healthy Individual Individual”  There is evidence that there is a continuous turnover of cardiomyocytes in the normal heart  The concept of cardiac remodelling coincided with the development of the concept of apoptosis and the recognition that this phenomenon represents an important mode of cell death in physiological cell turnover and pathological processes.  Apoptotic cells have been described in the normal adult heart suggesting myocyte replication is a significant component of physiological cellular processes. Bergmann 2009 10 What do we know? The cellular location of cardiac troponin  The cardiac troponins are predominantly myofibril bound with only approximately 8% of both TnI and TnT being unbound in the cytosol  In any cardiac myocyte injury it will be this unbound pool of troponin which is released first. Cardiac troponin release during the ACS  There is an interruption of blood flow in the coronary circulation   most commonly involving thrombus formation on a ruptured plaque associated with transitory or prolonged occlusion of a coronary vessel, or distal embolization - leading to myocyte necrosis. The number of myocytes affected may be small to very large and the amount of damage caused is reflected by the rise in troponin concentration in the peripheral blood  The release mechanisms of cTnI and cTnT from cardiocardio-myocytes following reversible or irreversible damage.  Release as a result of leakage from reversibly damaged myocardial membranes.  Left side shows intact nonnondegraded protein chain.  Right side by release of proteolytic troponin degradation products through the intact myocyte membrane. b IIn this hi case, troponins are presumed to be degraded by matrix metalloproteinases (MMP) activated by integrin mediated myocardial stretch. Giannoni et al.: 2009 11 Microbleb formation of adult cultured cardiac myocytes. A: baseline. B: 30 min of anoxia. Hickman 2010, reprinted from Schwartz et al Am J Pathol 1984 Mechanism of troponin release. A. Release of cytosolic troponin followed by structural troponin into bl d ffollowing blood ll i irreversible injury with permanent myocyte membrane damage. B. Release of cytosolic troponin only into blood following reversible injury and bleb formation. Hickman 2010 12 Does Troponin Always Equal Necrosis ? Small amounts of troponin can be released from cardiac myocytes, in apparently healthy subjects, due to a process related to ‘‘physiological renewal or remodelling’’ the myocardium  According to this renewal and remodelling hypothesis, cTnI and cTnT circulating concentrations in healthy subjects should be proportional to the individual’s cardiac mass  Consider   Since the heart mass is proportional to body mass, gender gender-dependent troponin concentrations should be found in healthy subjects Generally, adult males have a greater body mass than females and consequently a larger heart mass 13 Are There Gender Differences in Troponin Distribution ? Distribution of cTnT values in the pooled reference populations. The 99th percentile value was determined to be 13.5 ng/L. The inserts show the distribution of hs hs--cTnT among women (99th population percentile at 10.0 ng/L) and men (99th percentile at 14.5 ng/L). Giannitsis. Clinical Chemistry 56:2254 56:2254– –261 (2010) 14 hs-TnT hs(ng/L) Total (n= 479) Mean 4 1 Median 4 <1 97.5th percentile 11 8 99th p percentile 16 13 Female (n= 215) Mean Median Male (n= 264) TnI - Architect (ng/L) 3 1 3 <1 97.5th percentile 7 7 99th percentile 8 12 Mean 5 1 Median 5 <1 97.5th percentile 14 8 99th percentile 18 13 Notes: Neither assay was normally distributed Mingels A. 2009 97% of samples were less than LOD (9 ng/L) for TnI *LOOK (11 and 12 YO children) Australian cardio-healthy cardiopopulation Aussie Normal (snapshot) Total (n) 487 104 86 4.9 Mean 1.5 6.1 Median <1 6.0 3.1 97.5th p percentile 5.5 11.5 11.7 99th percentile 7.1 12.5 12.6 Female (n) 257 47 37 Mean 1.3 5.3 4.9 Median <1 4.9 3.5 97.5th percentile 4.9 9.1 10.9 99th percentile 56 5.6 11.0 11 0 11.7 11 7 Male (n) 230 57 49 Mean 1.6 6.4 3.9 Median 1.1 5.7 3.6 97.5th percentile 5.8 12.2 11.7 99th percentile 7.7 12.9 13.4 *Concentration <3 ng/L extrapolated using calibration curve with pooled "negative“ sample as TnT zero sample. 15 Are There Gender Differences in Troponin Distribution ? There is evidence that there are statistically significant differences in troponin T concentrations and gender. What about TnI ? 16 A significant difference was found between the cTnI values in men and women. • men: median di 0.011 mg/L, / range from undetectable values to 0.196 mg/L; • women: median 0.007 mg/L, range from undetectable values to 0 130 mg/L) 0.130 Distribution of cTnI concentration values measured by the ADVIA TnI--Ultra method in the reference population. The approximation TnI to a log log--normal distribution is indicated by a dotted curve. Clerico A. et al.. al.. Clin Chem Lab Med 2008 No statistical gender differences seen using a prototype highly sensitive TnI assay from Beckman Coulter Venge P. 2009 17 Are There Age Differences in Troponin Distribution ? Male (n = 57) Female (n = 47) Median (range) 99th Percentile Median (range) 99th Percentile p-Value All 5.7 (<3 - 13.3) 12.9 5.0 (<3 - 12.4) 11.0 < 0.01 ≤ 60 years 5.0 (<3 - 10.7) 10.5 4.2 (<3 - 5.7) 5.6 < 0.01 >60 years 6.9 (<3 - 13.3) 13.1 5.2 (<3 - 12.4) 11.7 < 0.01 p-Value (between ≤60 and >60 years)) < 0.01 0 01 < 0.01 0 01 Koerbin 2010 in press 18 > 144 13-144 12-133 11-122 10-111 9-100 Kids 8-99 Male 6-77 5-66 4-55 3-44 2-33 1-22 Female 7-88 60% 50% 40% 30% 20% 10% 0% <1 % of P opulatio n TnT in Healthy Children and Adults TnT (ng/L) Koerbin unpublished LOOK (11 and 12 YO children) Australian cardio-cardio healthy Aussie Normal population (snapshot) > 99th percentile URL (12.5 ng/L) 1% 1% 1% > LOD (5 ng/L) 4% 41% 36% > LOB (3 ng/L) 14% 74% 63% < LOB 86% 26% 37% 19 What about TnI ? Population Males (n = 311) Females (n = 381) Whole ≤50 years >50 years p-Value* Median (range) 99th percentile 0.011 (0– (0–0.196) 0.081 0.010 (0– (0–0.130) 0.081 0.014 (0– (0–0.196) 0.130 <0.0001 (between ≤50 and >50 years) Median (range) 99th percentile 0.007 (0– (0–0.130) 0.065 0.006 (0– (0–0.070) 0.028 0.009 (0– (0–0.130) 0.091 <0.0001 (between ≤50 and >50 years) Clerico 2008 20 ADVIA TnITnI-Ultra Mean and SEM concentrations. Gender and age in healthy males (black bars) and females (white bars) in deciles. Clerico 2008 Venge P. 2009 21 Are There Age Differences in Troponin Distribution ? There is evidence that there are statistically significant differences in troponin T and I concentrations and age. Is there Consensus on Detection Limits and 99% Upper Reference Limitss 22 DL (ng/L) 99th URL (ng/L) Number of subjects Ultra Advia Centaur 6 72 638 Clerico A. Clin Chem Lab Med 2008 Ultra Advia Centaur 6 39 309 Collinson PO. Clin Chem 2009 Beckman Ultra Accu TnI NR 80 442 Venge P.Clin Chem 2009 cTnI - Abbott Architect 9 13 479 Mingles A. Clin Chem 2009 Elecsys hsTnT 2 14 533 Hermsen D. Clin Lab 2007; Jarausch J. Clin Chem 2008; Beyrau R. Clin Chem Lab Med 2009 Elecsys hsTnT 1 16 479 Mingles A. Clin Chem 2009 5 10.0 (female) 14.5 (Males) 616 Giannitsis E. Clin Chem 2010 NR 11.0 (female) 12.9 (Males) 104 Koerbin in press NR 5.6 (Girls) 7.7 (Boys) 487 Koerbin unpublished (LOOK) Method References Giannoni et al.: 2009 Elecsys hsTnT Elecsys hsTnT Elecsys hsTnT How Much Troponin T is in a “Troponin Free” Sample ? TnT (ng/L) Koerbin unpublished Roche Multi Diluent 5.8 Abbott Zero Calibrator <1.0 Human Albumin 8.5 Saline 10.0 Distilled H2O Pooled "TnT Negative" serum 10.1 <1.0 23 Hruska 2009 A Comment from Per Venge “…we may now adopt conventional criteria for the definition of what are normal or abnormal levels of cTnI, that is, use the 97.5th percentiles instead of the 99th percentiles because of the inherent difficulties of accurate definition of the latter…” “… we may define levels below or above these levels to meet specific clinical needs…” 24 Which reference interval ?   Central 95% or the 97.5 percentile of a “normal healthy” reference population A decision limit   Single point (99th percentile URL) Rise and fall     Gender specific Age specific      Half life estimation Partitioning < 50 years, >50 yrs < 60 years, >60 yrs Children Deciles COMBINATION OF SOME OF THE ABOVE ? Conclusion 25