JC - Cardiac biomarkers journal club....

Journal club

1. JOURNAL CLUB Dr. Reny Agnes .C First year Postgraduate Department of Biochemistry

2. Research Article Prognostic significance of chronic myocardial injury diagnosed by three different cardiac troponin assays in patients admitted with suspected acute coronary syndrome Authors- Ole-Thomas Steiro , Jørund Langørgen , Hilde L. Tjora , Rune O. Bjørneklett , Øyvind Skadberg , Vernon V.S. Bonarjee , Øistein R. Mjelva , Trude Steinsvik , Bertil Lindahl, Torbjørn Omland , Kristin M. Aakre and Kjell Vikenes Place of study- Department of Heart Disease, Haukeland University Hospital, Bergen, Norway

3. Journal- Clinical Chemistry and Laboratory Medicine Published on- 9 Nov 2023 Volume- 62 Issue- 4 Pages- 729- 739 Indexation – pub med, scopus , EMBASE, Medline, Web of Science- Science citation Impact factor- 3.8 Steiro OT, Langørgen J, Tjora HL, Bjørneklett RO, Skadberg Ø, Bonarjee VVS, Mjelva ØR, Steinsvik T, Lindahl B, Omland T, Aakre KM, Vikenes K. Prognostic significance of chronic myocardial injury diagnosed by three different cardiac troponin assays in patients admitted with suspected acute coronary syndrome. Clin Chem Lab Med. 2023 Nov 9;62(4):729-739.

4. Acute Coronary Syndrome Angina to Acute Myocardial Infarction Angina- reduced blood supply ( not severe enough) Acute Myocardial Infarction- Thrombosis in the coronary arteries leading to infarction of cardiac tissue

5. ECG changes

6. Chronic Myocardial Injury Causes- Heart Failure AMI Left ventricular hypertrophy Cardiac fibrosis Myocarditis Severe valvular heart disease Cardiac exposure to metabolic risk factors. Can be candidates for cardioprotective therapy

7. Diagnosis- American Heart Association

8. Cardiac Biomarkers Biomarker is a substance used as an indicator of a biologic state Ideal Cardiac Biomarker High specificity High sensitivity Released rapidly from the heart and persist in circulation for several days Useful in monitoring therapy and prognosis Availability of rapid, easy to perform, cost effective quantitative test

9. Cardiac Troponins: Complex of 3 regulatory proteins M yocardial contraction by regulating the calcium-dependent interaction of actin and myosin. Complex of 3 proteins- TnT TnI TnC

10. Troponin is attached to the protein  tropomyosin  and lies within the groove between  actin  filaments in muscle tissue . Under resting intracellular levels of calcium, tropomyosin covers the active actin sites to which myosin.

11. Action Potential Calcium channel opens in sarcoplasmic reticulum and release calcium Calcium binds to TnC Series of structural changes – Tropomyosin moves away from myosin binding sites Binding of actin and myosin Contraction

12. Troponin C   -binds to calcium ions to produce a conformational change in TnI Troponin T-   binds to tropomyosin, interlocking them to form a troponin-tropomyosin complex Troponin I-   binds to actin in thin myofilaments to hold the actin-tropomyosin complex in place

13. cTnI - additional post translational 31 amino acid residue on its amino terminal end>> Cardiac specific cTnT - 11 amino acid terminal residue It is expressed in Muscular dystrophy, Polymyositis, Dermatomyositis, End stage renal disease TnC - not Cardiac specific Troponins – intracellular- released on cell death Unlike CK MB Troponins are not found in serum of healthy individuals

14. Troponin I Rise- 4 hours Peak- 14 to 24 hours Return to Baseline- 7 to 10 days Initial rise – Cytoplasmic fraction Sustained peak – Release from myofibrils

15. Troponin T Rise- 6 hours Peak- 24 to 48 hours Return to Baseline- 6 to 10 days Blood sample –immediately, 6 to 9 hours , again 12 to 24 hours

16. Maximum Turn Around time- National Academy of Clinical Biochemistry – Standards of Laboratory Practices- Within 1 hour cTn interfering substances– Elevated – ACS, shock, tachycardia, stroke, pulmonary embolism, sepsis, acute heart failure , heterophile antibodies

17. High sensitivity cTn - Overcome Limits of Detection Reported in ng/ml Reference range hsTnI - 0 to 0.4 ng/ml hsTnT - <14 ng/L Method of Estimation - Immunoassay

18. In our Lab- Chemiluminescence Immunoassay hsTnI Reference value- 0 to 34.1pg /ml snd 53.41pg/ml ( female and male) Chemiluminescence Immunoassay- I mmunoassay technique - indicator of the analytic reaction, is a luminescent molecule. S andwich immunoassay based on acridinium ester chemiluminescent technology.

19. Point of Care testing- To reduce turn around time For better management of patients Dry chemistry-based fluorescence or colorimetric immunosensors

20. Immunostrip technique for Cardiac Troponin T-

21. Research Article

22. Objectives- To assess prevalence and association of CMI with long term prognosis using three different high sensitivity cTn assays To assess whether CMI identified by different hsTn assays serve as a uniform and relevant marker of elevated cardiovascular risk

23. Materials and Methods Study design- Prospective and Cross Sectional Observational study Population - Total – 1,147 presenting with chest pain Approved by Regional Ethics Committee

24. Inclusion Criteria- Age >= 18 years admitted with symptoms suggesting acute coronary syndrome Final diagnosis by 2 cardiologist based on clinical information and routine laboratory test ( hs - TnT ) Exclusion Criteria- Patients who had acute myocardial injury based on the sex specific cutoff values of any assay were excluded from the current analysis Non-coronary cardiac diseases included diseases such as pericarditis, myocarditis, and heart failure. Noncardiac chest pain included myalgia, esophageal disease, and pleural diseases.

25. Cardiac Troponin analysis cTnT - fresh material Cobas 602 and Cobas 801 by Roche cTnI – bio banked material (-80 ℃ during 2 different time points) . Architect platform (2/3 ) and Alinity platform (1/3 ) by Abbott .Atellica platform by Siemens Healthineers The recommended percentiles- by manufacturers

26. CMI- cTn concentration at presentation above sex specific 99 th percentile Upper reference limit without rise or fall >20% in subsequent blood samples percentile URL 99 th percentile URL cTnT (Roche) - 9 and 16.8 ng/L ( men and women) ( LoD ) of 5 ng/L cTnI (Abbott) -15.6 and 34.2 ng /L ( men and women) LoD of 1.9 ng/L cTnI ( Siemens) – 38.6 and 53.5 ng/L (men and women ) LoD of 1.6 ng/L

27. Outcomes- Medium follow up time-4.1 years Primary prognostic end point- Cardiovascular death , AMI after discharge, revascularization Secondary end point- hospitalization for heart failure , stroke Tertiary end point- Death from any cause Mortality- Norwegian Cause of Death Registry Readmission, Diagnosis and Procedures- Norwegian Patient Registry

28. Statistical Analysis Baseline characteristics were reported as means (±2 SD) for normally distributed data, median with 25- and 75 percentiles for nonnormally distributed data The correlation (r) between cTn measured by different assays was assessed by Pearson´s correlation test The prognostic performances of cTn as continuous variables were assessed by the receiver operating characteristics curve (ROC) for all three endpoints. The Youden Index was used to identify the optimal cTn cutoff points for risk stratification.

29. Sensitivity, specificity, predictive values, and odds ratio (OR ) were calculated for the primary and secondary endpoints using the between-assay equivalent cutoff values, the calculated optimal cutoff values (Youden Index), the LoD , 90th, 95th, 97.5th, and 99th percentiles as provided by the manufacturer p-values <0.05 – statistically significant IBM SPSS Statistics version 26.0.0.1 and Medcalc version 17.6

30. Results 3 / more blood samples Men>Women detectable cTn Correlation between cTn concentration cTnI – Abbott and Siemen's =high ( r = 0.876) cTnT and cTnI = high but weak ( r=0.724) The Pearson correlation coefficient r is a measure of any linear trend between two variables. The value of r ranges between −1 and 1. r = zero, it means that there is no linear association between the variables

31. All (n=1,147) CMI a  (n=218) No CMI (n=929) p-Value Age, median 61 (51–71) 76 (68–84) 58 (49–67) <0.001 Female gender, n (%) 458 (40.0 %) 111 (50.9 %) 347 (37.4 %) <0.001 Cardiovascular risk factors Obesity b , n (%) 159 (26.7 %) 27 (25.7 %) 132 (26.9 %) 0.806 Active smoker, n (%) 215 (18.7 %) 32 (14.7 %) 183 (19.7 %) 0.087 Former smoker, n (%) 504 (43.9 %) 102 (46.8 %) 402 (43.3 %) 0.346 Hyperlipidemia, n (%) 228 (19.9 %) 58 (26.6 %) 170 (18.3 %) 0.006 Diabetes mellitus, n (%) 128 (11.2 %) 44 (20.2 %) 84 (9.0 %) <0.001 Insulin-dependent, n (%) 35 (3.1 %) 19 (8.7 %) 16 (1.7 %) <0.001 Hypertension, n (%) 454 (39.6 %) 122 (56.0 %) 332 (35.7 %) <0.001 Family history of CAD, n (%) 212 (18.5 %) 22 (10.1 %) 190 (20.5 %) <0.001 Baseline Characteristics

32. Medical history All (n=1,147) CMI a  (n=218) No CMI (n=929) p-Value Previous AMI, n (%) 204 (17.8 %) 64 (29.4 %) 140 (15.1 %) <0.001 Previous PCI, n (%) 214 (18.7 %) 55 (25.2 %) 159 (17.1 %) 0.006 Previous CABG, n (%) 67 (5.8 %) 31 (14.2 %) 36 (3.9 %) <0.001 Atrial fibrillation, n (%) 120 (10.5 %) 53 (24.3 %) 67 (7.2 %) <0.001 Previous stroke, n (%) 30 (2.6 %) 12 (5.5 %) 18 (1.9 %) 0.003 Renal failure c , n (%) 119 (10.4 %) 79 (36.2 %) 40 (4.3 %) <0.001 Peripheral arterial disease, n (%) 24 (2.1 %) 10 (4.6 %) 14 (1.5 %) 0.004 Known heart failure, n (%) 33 (2.9 %) 21 (9.6 %) 12 (1.3 %) <0.001

33. Clinical and laboratory parameters All (n=1,147) CMI (n=218) No CMI (n=929) p-Value Systolic blood pressure, mmHg 145 (144–146) 147 (144–150) 145 (144–146) 0.140 Diastolic blood pressure, mmHg 84 (83–85) 82 (79–85) 84 (84–85) 0.012 Pulse, bpm 74 (73–75) 78 (75–80) 73 (72–74) <0.001 Total chol , mmol/L (<5.2) 4.9 (4.8–4.9) 4.5 (4.3–4.7) 5.0 (4.9–5.0) <0.001 LDL, mmol/L(<3.4) 3.1 (3.0–3.2) 2.7 (2.5–2.9) 3.2 (3.1–3.3) <0.001 HDL chol , mmol/L (>1.5) 1.4 (1.4–1.5) 1.5 (1.4–1.6) 1.4 (1.4–1.5) 0.034 Hemoglobin, g/dL 14.2 (14.1–14.4) 13.5 (13.3–13.7) 14.4 (14.3–14.5) <0.001 Glucose, mmol/L(3.9-5.6) 6.2 (6.1–6.3) 6.9 (6.6–7.2) 6.1 (6.0–6.2) <0.001 CRP, mg/L, median(1) 2.0 (0.7–4.0) 2.0 (0.9–4.5) 1.0 (0.7–4.0) 0.020 proBNP, ng/L, median 81 (34–230) 88 (32–310) 80 (35–226) 0.263 eGFR,(>90) mL/min/1.37 m 2 (>90) 84.7 (83.5–85.8) 65.9 (62.7–69.0) 88.7 (87.7–89.7) <0.001

34. Laboratory parameters hs-cTnT (Roche), median (N<9ng/L) 6 (3–11) 20 (15–29) 5 (3–7) <0.001 hs-cTnI (Abbott), median (N<15 ng/L) 2.7 (1.5–5.5) 9.7 (5.3–19.8) 2.2 (1.5–3.8) <0.001 hs-cTnI (Siemens), median (N< 38ng/L) 4.4 (2.7–8.8) 15.3 (8.0–36.9) 3.8 (2.4–6.3) <0.001 All (n=1,147) CMI (n=218) No CMI (n=929) p-Value

35. Outcome within follow-up All (n=1,147) CMI (n=218) No CMI (n=929) p-Value All-cause mortality, n (%) 91 (7.9 %) 53 (24.3 %) 38 (4.1 %) <0.001 Cardiovascular death, n (%) 26 (2.3 %) 15 (6.9 %) 11 (1.2 %) <0.001 AMI, n (%) 35 (3.0 %) 16 (7.7 %) 19 (2.0 %) <0.001 Revascularization, n (%) 58 (5.1 %) 10 (4.6 %) 48 (5.2 %) 0.725 Heart failure, n (%) 26 (2.3 %) 20 (9.2 %) 6 (0.6 %) <0.001 Stroke, n (%) 1 (0.1 %) 1 (0.5 %) 0 (0.0 %) 0.039

36. Diagnostic Inconsistencies using URL by manufacturers Total of 218 patients had CMI Prevalence – 4.5 times > according to cTnT assay than cTnI by Abbott

37. Linear regression and Scatter Plot

38. Distribution of patients diagnosed with CMI according to three assays Only 29/218 patients (13.3 %) had CMI according to all three assays

39. During a median of 4.1 years follow-up, 93 patients (8.1 %) reached the primary endpoint 162 patients (14.1 %) reached the secondary endpoint 91 patients (7.9 %) reached the tertiary endpoint Increased cTnT, but cTnI below the 99th percentile, was significantly associated with reaching the secondary and tertiary endpoints, but not the primary endpoint.

40. Hazard ratio for reaching end points A measure of how often a particular event happens in one group compared to how often it happens in another group, over time.

41. Continuous variables and optimal cTn cutoff values When evaluating as continuous variables, the prognostic accuracy- Abbott cTnI assay (AUC 0.718, 95 % CI: 0.691–0.744) The Roche cTnT assay (AUC 0.697, 95 % CI: 0.670–0.724) The Siemens cTnI assay (AUC 0.662, 95 % CI: 0.634–0.689) For the secondary endpoint, there were no differences in AUC between the Abbott cTnI assay and the cTnT assay For the tertiary endpoint (all-cause mortality), the cTnT assay had higher prognostic value than the cTnI assays ,

42. The Area Under the ROC ( receiver operating characteristics curve) Curve (AUC) that summarizes the overall performance of a binary classification model. Range of AUC: The AUC value ranges from 0 to 1. An AUC of 0.5 indicates a model that performs no better than random chance. An AUC closer to 1 indicates a model with excellent performance.

43. Youden index The Youden's index is the value that maximizes the sensitivity and specificity of any continuous variable, helping to choose an appropriate cut point for dichotomization. AUC increased for all assays if cut off values were lowered from 99 th to 90 th percentile ( cut offs moved closer to Youden index) Improved sensitivity but reduced specificity

44. Assay 99 th percentile 97.5 th percentile 95 th percentile 90 th percentile   Roche cobas 9.0/16.8 8.4/13.3 6.9/11.3 6.1/9.2 Abbott Architect 15.6/34.2 14.1/27.0 8.0/13.0 4.8/7.6 Siemens Atellica 38.6/53.5 17.8/26.3 10.1/15.9 6.3/9.2

45. Prognostic precision at optimal cut off by Youden index

46. Receiver operating characteristics curve for primary end point A receiver operating characteristic curve, or ROC curve, is a graphical plot that illustrates the performance of model at varying threshold values

47. Receiver operating characteristics curve for secondary end point and tertiary end points

48. Discussion CMI is diagnosed several times more often by the cTnT assay. The optimal cTn cutoff value for predicting future CV events was lower than the 99th percentile URL, and much lower for cTnI compared to cTnT Troponin URLs must be harmonized, or the diagnostic definition of CMI should be reconsidered The calculated 99th percentile URL for different cTn assays is highly dependent on selection of reference group

49. When echocardiography or biomarkers such as NT- proBNP , eGFR or HbA1C are used for screening before selecting the healthy cohort, the 99th percentile can be reduced by 50 % The mean age and ethnic composition of the reference group can also affect 99th percentile The optimal prognostic threshold for cTnI was as low as 2.9 and 3.5 ng/L for the Abbott and Siemens cTnI assays. Using such a low threshold for risk assessment would reduce the specificity

50. Conclusion CMI was diagnosed four times more often by the cTnT assay than the two cTnI assays. The cTnT assay had a higher performance for risk prediction at the 99th percentile. This indicates that the diagnostic definition of CMI should be reconsidered to reduce assay-dependent differences. Using a lower percentile derived from a healthy cohort, bioequivalent cTn cutoff values, or cutoff values based on prognosis may be considered to harmonize the classification and prognostication within a high-risk patient group

51. Merits Detailed study with additional supplementary material for cross reference 90 th ,95 th and 97 th percentile values for each analyzer is mentioned Detailed statistical analysis on use of various URL for better sensitivity and specificity Baseline characteristics and serial measurements are taken into consideration for diagnosis The final reference limit that can be used for prognostic accuracy is obtained

52. Limitations Details of sampling methods is not mentioned Time intervals between each sample is not specified The cohort does not represent cardiomyopathies, arrhythmias, cardiac remodeling, and fibrosis. Data on patient outcomes were collected through patient registries, which is less robust than information verified by clinical adjudication. The number of patients with CMI is limited to 218 patients. The possible discrepancy between the assays and the possible effect on risk assessment and treatment should be assessed in a larger study evaluating more hs-cTn assays

53. References Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics Michael L Bishop Clinical Chemistry DM Vasudevan Textbook of Biochemistry Steiro OT, Langørgen J, Tjora HL, et al. Prognostic significance of chronic myocardial injury diagnosed by three different cardiac troponin assays in patients admitted with suspected acute coronary syndrome. Clin Chem Lab Med. 2023 Nov 9;62(4):729-739. Kenji Nakata, Yuri Tanaka, Minako Harada, et al,Association between Myocardial Oxygen Supply and Demand and Myocardial Injury in Patients with End-Stage Kidney Disease, Journal of Atherosclerosis and Thrombosis.2024 31:540-549 Stark M, Kerndt CC, Sharma S. Troponin, StarPearls Treasure Island StatPearls Publishing; 2024 Jan Internet

54. Thankyou