Advancing excellence in laboratory medicine for better healthcare worldwide

PSEP Report Wahid Ali (IN)

Wahid Ali was awarded an IFCC PSEP and spent two months and half (February-April 2018) at Prof. Sergio Bernardini Clinical Biochemistry and Clinical Molecular Biology Laboratory, Tor Vergata University Hospital, in Rome, Italy.


By Dr. Wahid Ali, Ph.D., MISAR
Chemical Pathology Lab
24-hr Emergency Lab Trauma Centre
OPD Pathology Lab
Central Sample Collection Centre
P.G.Department of Pathology
King George's Medical University
Lucknow India

Analytical measurement of High-sensitivity cardiac troponin I in Acute Coronary Syndrome is a friend or foe: Indian scenario

AIM OF THE VISIT

 Scientific and technological background description of applicant’s and host institutions

 Cardiovascular disease (CVD) is a group of diseases that include both the heart and blood vessels(Mendis et al., 2011). It includes both coronary heart disease (CHD), coronary artery disease (CAD) and acute coronary syndrome (ACS) among several other conditions. According to World Health Organization (WHO), CVDs are the number 1 cause of death globally, more people die annually from CVDs than from any other cause. An estimated 17.7 million people died from CVDs in 2015, representing 31% of all global deaths. Of these deaths, an estimated 7.4 million were due to CHD and 6.7 million were due to stroke. By 2030, researchers project that non-communicable diseases will account for more than three-quarters of deaths worldwide; CVD alone will be responsible for more deaths in low income countries than infectious diseases (including HIV/AIDS, tuberculosis, and malaria), maternal and perinatal conditions, and nutritional disorders combined (Beaglehole and Bonita, 2008). Thus, CVD is today the largest single contributor to global mortality and will continue to dominate mortality trends in the future (Organization, 2009). CHD is the leading cause of death in adults in the U.S., accounting for ~one-third of all deaths in subjects over age 35(Sanchis-Gomar et al., 2016). The 2016 Heart Disease and Stroke Statistics update of the AHA reported that overall death rate from CHD was 102.6 per 100,000 (Mozaffarian et al., 2016). Moreover, from 2003 to 2013, the annual death rate attributable to CHD declined 38.0% and the actual number of deaths declined 22.9% (Mozaffarian et al., 2016).

According to American Heart Association, Angina is chest pain or discomfort caused when your heart muscle doesn't get enough oxygen-rich blood. It may feel like pressure or squeezing in your chest. The discomfort also can occur in your shoulders, arms, neck, jaw, or back. Angina pain may even feel like indigestion.But, angina is not a disease. It is a symptom of an underlying heart problem, usually coronary heart disease (CHD).There are many  types of angina, including microvascular angina, Prinzmetal's angina, stable angina, unstable angina and variant angina.

Angina Pectoris (Stable Angina): Angina pectoris is the medical term for chest pain or discomfort due to coronary heart disease.  It occurs when the heart muscle doesn't get as much blood as it needs. This usually happens because one or more of the heart's arteries is narrowed or blocked, also called ischemia.Angina usually causes uncomfortable pressure, fullness, squeezing or pain in the center of the chest.  You may also feel the discomfort in your neck, jaw, shoulder, back or arm. 

Unstable Angina: Unstable angina or sometimes referred to as acute coronary syndrome (ACS) causes unexpected chest pain, and usually occurs while resting. The most common cause is reduced blood flow to the heart muscle because the coronary arteries are narrowed by fatty buildups (atherosclerosis) which can rupture causing injury to the coronary blood vessel resulting in blood clotting which blocks the flow of blood to the heart muscle.Unstable angina should be treated as an emergency. If you have new, worsening or persistent chest discomfort, you need to go to the ER. You could be having a heart attack which puts you at increased risk for severe cardiac arrhythmias or cardiac arrest, which could lead to sudden death. Cardiovascular diseases (CVDs), especially coronary heart disease (CHD), have assumed epidemic proportions worldwide. Globally, CVD led to 17.5 million deaths in 2012 (Mendis, 2014). More than 75% of these deaths occurred in developing countries. In contrast to developed countries, where mortality from CHD is rapidly declining, it is increasing in developing countries (Kelly and Fuster, 2010).

India is a large and socioeconomically diverse country, and there could be evidence of all the stages of this transition in the country (Gupta and Gupta, 2009).  However, this has not been studied. Other striking features of CVD epidemiology in India are high mortality rates, premature CHD, and increasing burden (Gupta et al., 2012).  

Cardiac Markers

Cardiac markers are used in the diagnosis and risk stratification of patients with chest pain and suspected acute coronary syndrome (ACS). The cardiac troponins, in particular, have become the cardiac markers of choice for patients with ACS. Indeed, cardiac troponin is central to the definition of acute myocardial infarction (MI) in the consensus guidelines from the European Society of Cardiology (ESC) and the American College of Cardiology (ACC): These guidelines recommend that cardiac biomarkers should be measured in patients with suspected MI, and that the only biomarker that is recommended to be used for the diagnosis of acute MI at this time is cardiac troponin due to its superior sensitivity and accuracy.

It is convenient to class the biochemical markers in terms of their time to positivity from the onset of symptoms:

Early markers (within 1-6 hours):

  • Myoglobin
  • CK-MB isoforms
  • Heart-type fatty acid binding protein (FABP)
  • Glycogen phosphorylase isoenzyme BB (GPBB)

Middle markers (6-12 hours):

  • CK-MB mass
  • Cardiac troponins (T and I)

Late markers (more than 12 hours):

  • CK-MB mass
  • Cardiac troponins (T and I)

MYOGLOBIN:

Myoglobin is a heme protein found in skeletal and cardiac muscle that has attracted considerable interest as an early marker of MI. Its low molecular weight accounts for its early release profile. Myoglobin typically rises 2-4 hours after onset of infarction, peaks at 6-12 hours, and returns to normal within 24-36 hours. The major limitation of it is lack of specificity (60-95%).

CK-MB ISOFORMS

A creatine kinase-MB (CK-MB) test may be used as a follow-up test to an elevated creatine kinase (CK) in order to determine whether the increase is due to heart damage or skeletal muscle damage. The test is most likely to be ordered if a person has chest pain or if a person's diagnosis is unclear, such as if a person has nonspecific symptoms like shortness of breath, extreme fatigue, dizziness, or nausea.CK and CK-MB were once the primary tests ordered to detect and monitor heart attacks, but they have now been largely replaced by the troponin test, which is more specific for damage to the heart.

Sometimes, the CK test may be used if a heart attack is suspected and a troponin test is not available. In this case, when CK is elevated, a CK-MB test may be used as a follow-up test to determine whether the increase is due to heart damage or skeletal muscle damage.

B type natriuretic peptide (BNP) 

BNP is secreted by right and left ventricular myocytes and released in response to stretch, volume overload, and elevated filling pressures. Serum levels of BNP are elevated in patients with asymptomatic LV dysfunction as well as symptomatic HF. The presence of acute heart failure (HP) in patients with ACS is a well-known predictor of adverse cardiac events16. Therefore it is not surprising that an elevated BNP level is a marker of CHF and is also a predictor of adverse cardiac events in patients with ACS. In addition, the severity of ischaemia is directly proportional to elevation in BNP. BNP levels correlate with the severity of HF and predict survival.

Troponins:

The troponins are regulatory proteins found in skeletal and cardiac muscle. Three subunits have been identified: troponin I (TnI), troponin T (TnT), and troponin C (TnC). The genes that encode for the skeletal and cardiac isoforms of TnC are identical; thus, no structural difference exists between them. However, the skeletal and cardiac sub-forms for TnI and troponin TnT are distinct, and immunoassays have been designed to differentiate between them. It is the most sensitive and specific test for myocardial damage. Because it has increased specificity compared with CK-MB, troponin is a superior marker for myocardial injury.

They are measured in the blood to differentiate between unstable angina and myocardial infarction (heart attack) in people with chest pain or acute coronary syndrome. A person who recently had a myocardial infarction would have an area of damaged heart muscle and elevated cardiac troponin levels in the blood.

The troponin complex is situated on the thin filament of the striated muscle contractile apparatus and consists of troponin T (39 kD), troponin I (26 kD), and troponin C (18 kD), each coded by a separate gene(Frey et al., 1998). Specific cardiac and skeletal muscle isoforms are expressed in cardiac and skeletal striated muscle in adults. Troponins are mainly bound to the myofibrils, although 6–8% of cTnT and 2.8–4.1% of cTnI is cytosolic(Wu and Feng, 1998). This affects release kinetics. There is rapid early release of cytosolic cTnT after ischaemic injury, followed by more prolonged release of myofibrillar troponin, resulting in a biphasic release pattern. As cTnI has a smaller cytosolic pool, release is likely to be monophasic. Concentrations of both begin to rise in the 4–8 hours following injury and peak at 12–24 hours(Wu and Feng, 1998, Collinson, 1998). cTnT may remain raised for more than two weeks and cTnI for more than 5–7 days.

Troponin is a complex of 3 protein subunits:

  • Troponin C – the calcium binding component
  • Troponin I - the inhibitory component
  • Troponin T – the tropomyosin-binding component

The subunits exist in a number of isoforms, and cardiac-specific troponin T (cTnT) and cardiac-specific troponin I (cTnI) isoforms have been identified.

TROPONIN T: In contrast to cTnI, assays for determination of cTnT are only patented and marketed by 1 company (Roche diagnostics) and therefore no standardization bias exists for cTnT. However, antibodies used in the first generation ELISA cTnT assay showed some cross-reactivity with skeletal muscle TnT, and caused falsely increased cTnT in 30-50% of severely uremic patients in the absence of increased cTnI and evidence of MI. 2nd generation cTnT assay has been developed with monoclonal antibodies against cTnT that do not show any cross-reactivity against skeletal muscle TnT, but uremic patients still show a 12-17% false positive rate.

TROPONIN I: Troponin I is absolutely cardiac-specific and can be used to eliminate false clinical impression of AMI in patients with increased CK-2 concentrations. cTnI is the preferred cardiac marker to use in patients with renal disease.

With reference to above literature, we aimed to measure the High-sensitivity cardiac troponin I (hsTrop I) in Acute Coronary Syndrome in North Indian patients.

Professional Exchange Programme Objectives

  1. To learn the basic analytical differences between cTnT and cTnI in the population of host institution.
  2. To standardize the laboratory methodology for hsTropI assay in our setup.
  3. To characterize the silent features and superiority of hsTropI over routine TropT test.
  4. To explore the molecular mechanism for the raised hsTropT in acute MI subjects.
  5. To correlate the molecular and biochemical indices further to recommend the hsTropI in ACS patients ofnorthernIndia.

Project Plan (technique, time schedule)

Time line

Work plan

0-30 day

To observe and learn the basic technique of hsTropI assay by following SOP of the host institute.

 

30-60 day

30-60 day: To explore the molecular profiling for hsTropI and further to correlate the molecular assay with biochemical indices.

 

60-90 day

To compile summary of observations and project report that can be submitted to host institute as well as recommendation to be submitted to IFCC and base institution 


Impact of the project on the participating institutions

The markers that are well suited for the early diagnosis of AMI within the time interval 0–6 hours after symptom onset are myoglobin, H-FABP and CK-MB isoforms. Although all have been shown to be excellent sensitive early markers, there are still significant issues concerning its specificity. H-FABP is more cardio-specific than myoglobin and the use of H-FABP as a marker for the early diagnosis of AMI seems preferable. CK-MB mass measurement is suitable in the 6–24 hours interval; CK-MB based on activity measurement is more sensitive in the 12–24 hours interval, and the other cardiac markers like total CK, cTnT, and cTnI are most reliable after 12 hours from symptom onset. The prolonged diagnostic window of cardiac troponins of several days that is highly sensitive and specific obviates the needs for less specific markers with long diagnostic window like aspertate aminotransferase (ALT) and CK. Based on the recent recommendations by the ESC/ACC, cTnI and cTnT are the best markers for the confirmation of AMI. CK-MB (preferably mass) is the second best marker in the absence of troponins assays.

Troponin I is as effective as cTnT in diagnosing myocardial necrosis in the setting of trauma and coronary bypass grafting (Swaanenburg et al., 1998, Bonnefoy et al., 1998). In percutaneous transluminal coronary angioplasty/stent, and in association with congestive heart failure, there are reports of raised cTnT and cTnI (Genser et al., 1997, Missov et al., 1997, Rao et al., 1998). Troponin I have been shown to be cost effective. From the published literature it is clear that in the management of ACS and acute MI in clinical practice, cTnI is comparable in diagnostic and prognostic efficacy to cTnT. Any variation in results is likely to be caused by differences in patient populations, blood sampling timing, and analytical methods. In renal impairment, even against second generation cTnT assays, cTnI is superior. In muscle damage, cTnI is as least as useful as cTnT. Therefore we will emphasis on clinical application of cTnI on patients suffering from ACS in north Indian population due to its superiority over cTnT. After standardization of hsTropI in our ethnic group i.e. North Indian subject with the history of acute MI, we will recommend the cardiologists as well as physicians in state of Uttar Pradesh for maximum utilization of hsTropI as a sensitive and cost effective for ACS patients.After successful completion of this project may change the clinical diagnosis to rate out the false negative.

Outcome Report:

  1. The basic analytical differences between cTnT and cTnI in the population of host institution were analysed.
  2. The laboratory methodology for hsTropI assay in our setup was standardized.
  3. The salient features and superiority of hsTropI over routine TropT test were characterized.
  4. The molecular mechanism for the raised hsTropT in acute MI subjects was explored.
  5. The molecular and biochemical indices were correlated and hsTropI assay is recommended the in ACS patients of northern India.
  6. The laboratory sketch and the things learned are being implemented in our set up to produce good results.

References

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BONNEFOY, E., FILLEY, S., KIRKORIAN, G., GUIDOLLET, J., RORIZ, R., ROBIN, J. & TOUBOUL, P. 1998. Troponin I, troponin T, or creatine kinase-MB to detect perioperative myocardial damage after coronary artery bypass surgery. Chest, 114, 482-486.

COLLINSON, P. 1998. Troponin T or troponin I or CK-MB (or none?). European heart journal, 19, N16-24.

FREY, N., MÜLLER-BARDORFF, M. & KATUS, H. A. 1998. Myocardial damage: the role of troponin T. Developments in Cardiovascular Medicine, 205, 27-40.

GENSER, N., MAIR, J., TALASZ, H., PUSCHENDORF, B., CALZOLARI, C., LARUE, C., FRIEDRICH, G., MOES, N. & MUEHLBERGER, V. 1997. Cardiac troponin I to diagnose percutaneous transluminal coronary angioplasty-related myocardial injury. Clinica chimica acta, 265, 207-217.

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MENDIS, S. 2014. Global status report on noncommunicable diseases 2014, World health organization.

MENDIS, S., PUSKA, P. & NORRVING, B. 2011. Global atlas on cardiovascular disease prevention and control, World Health Organization.

MISSOV, E., CALZOLARI, C. & PAU, B. 1997. Circulating cardiac troponin I in severe congestive heart failure. Circulation, 96, 2953-2958.

MOZAFFARIAN, D., BENJAMIN, E. J., GO, A. S., ARNETT, D. K., BLAHA, M. J., CUSHMAN, M., DAS, S. R., DE FERRANTI, S., DESPRÉS, J.-P. & FULLERTON, H. J. 2016. Executive summary: heart disease and stroke Statistics—2016 update. Circulation, 133, 447-454.

ORGANIZATION, W. H. 2009. World health statistics 2009, World Health Organization.

RAO, A., NAEEM, N., JOHN, C., COLLINSON, P., CANEPA-ANSON, R. & JOSEPH, S. 1998. Direct current cardioversion does not cause cardiac damage: evidence from cardiac troponin T estimation. Heart, 80, 229-230.

SANCHIS-GOMAR, F., PEREZ-QUILIS, C., LEISCHIK, R. & LUCIA, A. 2016. Epidemiology of coronary heart disease and acute coronary syndrome. Annals of translational medicine, 4.

SWAANENBURG, J. C., KLAASE, J. M., DEJONGSTE, M. J., ZIMMERMAN, K. W. & TEN DUIS, H. J. 1998. Troponin I, troponin T, CKMB-activity and CKMB-mass as markers for the detection of myocardial contusion in patients who experienced blunt trauma. Clinica chimica acta, 272, 171-181.

WU, A. & FENG, Y. 1998. Biochemical differences between cTnT and cTnI and their significance for diagnosis of acute coronary syndromes. European heart journal, 19, N25-9.

 

 
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