**5. Cardiac troponin analysis at postmortem**

The first reported use of cTn analysis was in 1998 by Osuna and colleagues who studied 89 cadavers with a mean age of 51.38 ± 2.04y [16]. Subjects were assigned

between four groups, MI (n = 25), asphyxia (n = 30), cranio/multiple trauma (n = 17), and other natural deaths (n = 17). MI was determined by H&E and acridine orange histological staining. The research group determined the concentration of myoglobin, myosin, CK-MB, and cTnI in femoral vein serum samples and PCF in each case. PCF concentrations for all makers were significantly different between each outcome group; however, only myoglobin and myosin demonstrated significance in serum. The PCF cTnI values were higher than serum samples when using the Sanofi Diagnostic Pasteur assay. Values in both matrices were higher in MI patients compared to the other three groups (mean (range) Pericardial cTnI [pg/L]): 2.4 (0.3–6.5); 1.7 (0.03–3.7); 1.1 (0.01–2.3); 0.4 (0.0–1.8) in each group, respectively.

Cina and colleagues [17] demonstrated the utility of cTnT using the then available commercial Cardiac Rapid T (cTnT) lateral flow test from Roche Diagnostics. This device allows testing in the autopsy suite at the time of postmortem with qualitative results (positive or negative test lines) at 15 minutes from sample application. In 40 autopsy cases, 20 were deemed cardiac deaths and 20 were controls (noncardiac related) deaths, diagnosed by gross pathology and histological analysis. 85% (n = 17) of subclavian or femoral blood samples in the cardiac death group were positive for cTnT which was significantly different to control group, where 30% (n = 6) of serum samples were positive for cTnT. The authors deemed these as false-positive results. In subjects aged over 50 years, sensitivity and specificity of cTnT for diagnosis of AMI were 91% and 86%, respectively. The authors noted however the assay was ineffective in frozen blood samples or those with significant haemolysis which was evident at a PMI of >24 h. A similar study of 100 autopsy cases of sudden unexplained death (SUD) was carried out in Chaing Mai, Thailand, and utilised the same rapid cTnT assay [18]. Fifty-two of the deaths were considered cardiac with 20 due to sudden MI and 32 with evidence of old infarction or arrhythmic fibrosis (n = 22), coronary atherosclerosis, >75% luminal without evidence of fibrosis or thrombosis (n = 3), cardiomegaly, and heart weight > 400 g (n = 7) or related to cardiac injury as a result of toxic substances. Thus, subjects were assigned to other cardiac death (SCD), non-cardiac natural death (NCD) or non-natural death (NND). The percentage positivity rate was higher in subclavian blood than femoral blood samples in all three groups. Subclavian blood sensitivity and specificity for SUD were 87.5% and 47%, respectively. Similar to the findings of Cina and colleagues, false-positive rates were associated with increasing PMI.

Davies and colleagues were the first to compare antemortem (<48 h before death) and postmortem concentrations of cTnT (Roche Elecsys 3rd generation electrochemiluminescent assay) and cTnI (Stratus CS fluorometric assay, Dade-Behring [now Siemens healthineers]) in five hospital-based autopsies [19]. One patient suffered cardiac death (myocarditis) with the remaining four were non-cardiac, but moribund before death. Results obtained between antemortem and postmortem samples were erratic. Four of the five subjects (n = 80%) had elevated antemortem cTnT and cTnI samples. The authors concluded postmortem cTn analysis in blood was not suitable due to lack of correlation of cause of death; however, they suggested that elevated antemortem cTn was related to all-cause mortality in those at end of life. A similar conclusion was made by Rahimi and colleagues in 2018 after studying 140 natural and unnatural deaths in Malaysia [20]. Subjects were classified into five groups: cardiovascular death, sudden unexplained death, thoracic trauma, non-thoracic trauma, and other diseases. Median jugular/subclavian/femoral blood cTnT (Roche Elecsys 3rd generation electrochemiluminescent assay) concentrations were 0.51, 0.17, 0.62, 0.90, *Use of Cardiac Troponin for the Diagnosis of Cardiac Pathology in Postmortem Samples Taken… DOI: http://dx.doi.org/10.5772/intechopen.111799*

and 0.51 μg/L, respectively, with no significant difference (p= > 0.05) in relation to cause of death. The authors concluded cTnT lacked specificity in postmortem sampling and is therefore not a useful tool.

Lai and colleagues also compared antemortem and postmortem blood sampling. Demonstrating in four cases, a marked proportionate rise in cTnT in postmortem samples compared to antemortem samples. Interestingly, the authors found cTnT values were higher (mean cTnT 5.32 μg/L) in non-cardiac deaths compared to 4.91 μg/L in cardiac-related deaths [21].

In 2006, Zhu and co-workers published two seminal papers in *Legal Medicine* examining the value of postmortem cTnT in cardiac, peripheral blood and PCF in relation to traumatic causes of death [22] and sudden cardiac death pathology [23] in medicolegal autopsies. In traumatic death (n = 405) due to blunt/sharp instrument injury, asphyxiation, drowning, fatal fire, hyper and hypothermia, and toxic poisoning are due to metamphetamine or carbon monoxide. Cardiac blood and PCF cTnT values were lower where PMI was <12 h compared cases where PMI ranged from 12 to 48 h. Elevated cTnT was associated, however, with histological evidence of advanced myocardial damage involving swelling and liquefactive necrosis [22]. In their cohort of sudden cardiac death autopsies (n = 96), 35% were due to AMI, 24% due to recurrent MI, 25% ischemic heart disease without infarction and 16% due to other cardiac causes. The comparative control group (n = 75) consisted of 47% asphyxiation, 36% drowning and 17% cerebrovascular diseases. In agreement with their first study, pericardial cTnT concentrations were higher than blood samples. Differences in cTnT concentrations related to pathological evidence of cardiac damage differed between early (<12 h) and late (12–48 h) postmortem period, concluding that elevations in blood and PCF are dependent not only on the severity of myocardial damage (determined by infarct size, intensity of lesions, interstitial haemorrhage and necrosis) but also by the PMI [22].

Remmer and colleagues [24] focused on postmortem serum and PCF cTnT in relation to PMI from 101 forensic autopsy cases in Estonia. PMI ranged from 8 h to 141 h. Although differences in cTnT were observed between five groups of cause of death (cardiovascular disease; other disease; poisoning; asphyxia; drowning; hypothermia; thoracic trauma, other trauma and fatal fires), significant attention to PMI (**Figure 3**) is important rather than comorbid cardiovascular disease.

In addition to the effects of PMI as demonstrated above, Kumar et al. used SDS-PAGE and Western blotting of cardiac tissue extracts from 10 medicolegal autopsies of burns cases [25]. The authors demonstrated a pattern of cTnT degradation in a time-dependent manner at room temperature (7.3 h, 18.2 h, 30.3 h, 41.2 h, 41.4 h, 54.3 h, 65.2 h and 88.4 h), demonstrating the disappearance of intact cTnT protein and the increasing presence of low-molecular-weight bands related to time (**Figure 4a**). Furthermore, the groups have examined degradation patterns according to the cause of death (**Figure 4b**–**d**) [26].

A study of 20 autopsies of sudden cardiac death and 8 controls (violent noncardiac deaths) demonstrated significantly higher cTnT and cTnI concentrations in pulmonary venous blood. Mean ± SD cTnT were 1826 ± 363 μg/L versus 65 ± 11 μg/L, respectively, and for cTnI were 28 ± 3 mg/L versus 0.14 ± 0.02 μg/L; however, it should be noted that the PMI in all cases was 8 h [27].

The value of cTn as a biochemical marker in relation to sudden cardiac death has been the subject of a systematic review [28] and formal meta-analysis [29]. Whilst both reviews demonstrate the elevation of cTn to be higher in pericardial fluid compared to blood sampling in the postmortem setting (**Figure 5**), blood is

#### **Figure 3.**

*Cardiac troponin T (cTnT) concentration in relation to postmortem interval in 101 medicolegal autopsies. Cause of death were due to cardiovascular disease, other disease, poisoning, asphyxia, drowning, hypothermia, thoracic and non-thoracic trauma and fatal fires. (source: Adapted from [24]).*

susceptible to the effects of haemolysis, postmortem interval, autolysis, and potential bacterial interferences. Pericardial fluid is therefore the preferred sample of choice.

Both reviews also address the issue of cut-off values demonstrating significant difference to cut-off values in the living. Non-cardiac deaths often demonstrate significantly positive cTn values in PCF and blood, thus questioning the sensitivity and specificity at postmortem. Barberi and van den Hondel suggest that more work is required to determine the appropriate cut-off values at postmortem [28].
