On the use of Biochemical Markers in Diagnostics of Sudden Cardiac Death
Introduction: Medical examiners frequently examine victims of sudden death. Most often sudden deaths have a cardiovascular cause. To determine the diagnosis of sudden cardiac death based only on morphological findings may be often very difficult. Measurement of blood concentrations of cardiac troponin I (cTnI) and atrial natriuretic peptide (pro-ANP) is now in clinical use in adult patients with heart failure caused by myocardial damage.
Aim: The aim of the study was the estimation wheather cTnI and/or pro-ANP could be markers of sudden cardiac death.
Patients and methods: The study was carried out on 89 necroptic cases, of which 53 were concluded as cardiac-related sudden death, and 36 cases were used as a control group being other than cardiac death cases. Concentrations of markers were determined in blood taken from the left cardiac ventricle and from the right femoral vein. The dependence between the results of biochemical studies and death causes, results of histopathological examination of myocardium, time interval between the death and taking of samples, and resuscitation data was investigated.
Results: Concentrations of cTnI as determined in blood samples from the left ventricle were in most cases very high, largely exceeding the cut-off level, and so were concentrations of pro-ANP. The values of both parameters were significantly lower in peripheral blood. No statistically significant dependences were found between the levels of the studied markers and the cause of death, myocardial histopathological findings, time interval between the death and taking of samples, and resuscitation data.
Conclusion: Based on the results obtained, the study can be concluded that blood is not a suitable medium for determination of biochemical markers of cardial troponin I and atrial natriuretic peptide for post-mortem diagnostics of myocardial damage and for determining the diagnosis of sudden cardiac death in a manner similar to diagnostics of myocardium damage in living patients.
Key words: sudden cardiac death – morphology - biochemical markers – cardiac troponin I – atrial natriuretic peptide
1; V. Parrák
2; P. Kvasnička
3; M. Majdan
2; H. Šidlová
Institute of Forensic Medicine, School of Medicine, Comenius University and Health Care Surveillance Authority, Bratislava, Slovakia
1; Department of Clinical Biochemistry, Teaching Hospital (workplace Petržalka), Bratislava, Slovakia
2; Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia
3; Institute of Pathology, Slovak Medical University and Cytopathos, s. r. o., Bratislava, Slovakia
Vyšlo v časopise:
Soud Lék., 53, 2008, No. 3, p. 31-34
Úvod: Vyšetrovanie prípadov náhlych úmrtí je často predmetom súdnolekárskej praxe. V mnohých prípadoch ide o úmrtia z kardiovaskulárnych príčin. Stanoviť diagnózu náhlej srdcovej smrti len na základe morfologických nálezov môže byť niekedy veľmi obtiažne až nemožné. V diagnostike poškodenia myokardu u živých pacientov sa bežne využívajú biochemické markery kardiálny troponín I (cTnI) a atriálny natriuretický peptid (pro-ANP).
Cieľ: Cieľom práce bolo overiť využitie biochemických markerov cTnI a pro-ANP v diagnostike náhlej srdcovej smrti.
Pacienti a metódy: Predmetom štúdie bolo 89 nekroptických prípadov. V 53 prípadoch išlo o náhlu srdcovú smrť, 36 prípadov s inou ako kardiálnou príčinou smrti slúžilo ako kontrolný súbor. Koncentrácie markerov boli stanovené v krvi z ľavej komory srdca a pravej femorálnej vény. Bola zisťovaná závislosť medzi výsledkami biochemického vyšetrenia a príčinami smrti, morfologickými nálezmi v myokarde, časovým intervalom medzi smrťou a odberom vzoriek biologických materiálov a údajmi o resuscitácii.
Výsledky: Koncentrácie cTnI zistené vo vzorkách krvi z ľavej komory srdca v prevažnej väčšine prípadov mnohonásobne prevyšovali cut-off hodnoty, podobne ako koncentrácie pro-ANP. Hodnoty obidvoch parametrov boli významne nižšie v periférnej krvi. Neboli zistené signifikantné vzťahy medzi hodnotami sledovaných markerov a príčinou smrti, histopatologickými nálezmi v myokarde, časovým intervalom medzi smrťou a odberom vzoriek a údajmi o resucitácii.
Záver: Na základe získaných výsledkov je možné konštatovať, že krv nie je vhodný materiál na stanovenie biochemických markerov kardiálneho troponínu I a atriálneho natriuretického peptidu pre stanovenie diagnózy náhlej srdcovej smrti podobne ako pri zisťovaní poškodenia myokardu u klinických pacientov.
Kľúčové slová: náhla srdcová smrť – morfológia – biochemické markery – kardiálny troponín I – atriálny natriuretický peptid
Medical examiners frequently
examine victims of sudden death. From a forensic point of view,
sudden death is currently most often defined as rapid, unexpected and
natural death occurring within 1 h of the onset of final symptoms
(5). The time elapsing between the onset of final symptoms and death
is controversial, settled at 24, 6 or 1 h according to different
definitions. However, such determination is often useless because
sudden death is unwitnessed in about 40% of cases (16), for example
when it occurs during sleep. The time period between onset of
symptoms and death is therefore frequently unknown. Most often sudden
deaths have a cardiovascular cause when the time period is short
(5), although extra cardiac causes may be encountered.
From a general perspective,
morphological diagnostics of cardiac-related sudden death can be
divided into three categories. In some cases, a firm diagnosis
of cardiac-related death can be made based on conclusive gross and
histopathological findings. In many other cases, we find evidence
supportive, but not diagnostic, of cardiac death (e.g.,
atherosclerotic coronary artery disease, cardiomegaly, myocardial
scarring). The third cohort comprises cases of sudden death with
minimal to mild cardiac disease, no other significant pathology, and
negative toxicology studies (3).
In cases where sudden death
occurs at a very early stage of infarction, the myocardial
lesions cannot be easily detected by traditional macroscopic
examination or routine histopathological staining. Thus, examination
of haematoxylin-eosin-coloured slides will not, with certainty,
reveal ischaemic lesions until more than 6 h post-infarction (9, 10).
In an attempt to improve the detection of early myocardial
infarctions, several immunohistochemical techniques have been
introduced and have contributed some improvement into the detection
of early ischemic lesions (4, 7, 11). Nevertheless, a marker
with both high sensitivity and high specificity has not been found.
Several biochemical markers, such
as creatine kinase MB, myoglobin, cardiac troponins, natriuretic
peptides and others are used for the diagnosis of myocardial injury
in clinical practice (13).
In forensic medicine, there is
a need for sensitive biochemical markers for the post-mortem
diagnosis of cardiac-related sudden death.
Troponins (T, I, C) are
structural proteins bound to tropomyosin of striated muscles. They
take part in regulation of muscle contraction. The primary structure
of myocardial troponin was found to differ from that of the skeletal
muscle troponin, and the two species can be immunochemically
differentiated. This provides a cardiospecific method of
differentiating myocardial and skeletal muscle lesions. So far, the
largest body of experience has been acquired with determinations of
cardial troponins T (cTnT) and I (cTnI). They are markers
of microscopic myocardial necroses (14). The release of troponins
into circulation as a result of traumatic heart lesion has been
repeatedly reported (1, 2, 6).
Atrial natriuretic peptides ANP,
BNP, and CNP are pro-hormones produced by cardiomyocytes of cardiac
atria. Together with their receptors A, B, and C play an important
role in maintaining the homeostasis. They have vasodilating, diuretic
and natriuretic actions and regulate the systemic resistance. These
peptides have been proved to contribute to pathogenesis of many
diseases, for example heart failure, hypertension, acute coronary
events etc. They are markers of myocardial ischaemia and, in clinical
medicine, their concentrations in adult patients with cardiac failure
are monitored (12).
The aim of the study was to
evaluate and compare the diagnostic efficacy of post-mortem cTnI and
pro-ANP determinations in the serum of heart and peripheral blood, to
compare these results with structural findings in myocardium, and to
estimate whether these biochemical markers could be used for
post-mortem diagnosis of cardiac-related sudden death.
The study was carried out on 89
necroptic cases, of which 53 were concluded as cardiac-related sudden
death, and 36 cases were used as a control group being other
than cardiac death cases. The time interval from the death to taking
of biological samples (blood and myocardium) was 6 to 105.5 hours. We
also recorded data on resuscitation.
Concentrations of markers were
determined in blood taken from the left cardiac ventricle and from
the right femoral vein. The samples were centrifuged, the supernatant
was divided in 3 parts and stored at -20 °C. Using an
immunochemical chemiluminiscence assay, we determined the
concentration of cTnI using mouse monoclonal antibodies ACS-180
(Bayer, Germany) with threshold concentration (cut-off for healthy
people – 0.15 ng/ml), and the concentration of pro-ANP using sheep
polyclonal antibodies – LIAmat assay SERISTRA diagnostic kits
(B.R.A.H.M.S. AG, Germany) with cut-off of 200 pmol/l. At the same
time, samples of myocardium were taken; these were fixed in formalin,
processed using a routine procedure by embedding in paraffin
blocks and staining with haematoxylin-eosin and phosphotungstic acid
according to Mallory. The samples were examined using light
We investigated the dependence
between the results of biochemical studies and death causes, results
of histopathological examination of myocardium, time interval between
the death and taking of samples, and resuscitation data.
For observed marker levels we
report basic statistics (mean, standard deviation, range, and
median). We note that source data in this study were seriously
censored. The reason is that the observed levels were frequently
perceived as unreasonably large. Pairwise comparisons between groups
were carried out using the Wilcoxon-Mann-Whitney U-test and the
Kolmogorov-Smirnov two-sample test (17). As the results were
identical, only the results of U-test are reported. To evaluate the
diagnostic potential of the two markers, we report maximum absolute
deviation (MAD) between the empirical distribution functions of the
sudden cardiac death and control groups. This quantity is known as
a test statistic in the Kolmogorov-Smirnov two-sample test. For
the purposes of this paper, its important property is that it gives
the probability of correct classification of a case (sudden
cardiac death vs. other cause of death) based on observed level of
a marker. A diagnostically usable marker would provide
values of MAD close to 1 (meaning correct classification with
probability close to 1). Due to censoring in the data, we constructed
the empirical distribution functions used in the calculation of MAD
as Kaplan-Meier estimates (8). The calculations were carried out in
the R framework (15).
Concentrations of the two markers
as determined in blood samples from all cases examined are shown in
Table 1. Data are compared by cause of death (sudden cardiac death
vs. other), and we report, along with basic descriptive statistics,
results of the Wilcoxon-Mann-Whitney U-test and maximum absolute
deviations between distribution functions. The concentrations of cTnI
in the blood from the left ventricle were in most cases very high,
and so were concentrations of pro-ANP. The values of both parameters
were significantly lower in peripheral blood. However, even these
were by at least an order of magnitude higher than the cut-off values
mentioned above. An interesting finding was a low level of
pro-ANP in the blood from v. femoralis in the subgroup (n = 12)
with asphyxia as cause of death, as shown in Table 2.
The MAD values indicate that the
observed levels of the two markers have poor predictive value as
regards the cause of death. Due to the broad distributions of
observed levels, a specific level of a marker bears
practically little information about the cause of death. This holds
irrespective of whether the differences between the sudden cardiac
death group and control group are found significant.
No statistically significant
dependences were found between the levels of the studied markers and
the cause of death, time interval between the death and taking of
samples, or resuscitation data. Similarly, no significant correlation
was identified between the marker levels and the findings of
histopathological examination of myocardium, which provided a broad
range of findings, from negative through minor ischaemic alterations
to developed infarctions – that is, necrosis.
We observed that, in all cases
and irrespective of the cause of death, the levels of the two markers
studied covered a wide range largely exceeding the cut-off level
for the respective marker. The effect was less pronounced for pro-ANP
than for cTnI.
High concentrations of the
markers in blood samples from the left cardiac ventricle in both the
sudden cardiac death and control group can be attributed to
post-mortem alterations, such as haemolysis, which can take place
shortly after death. The reason for positive findings and increased
levels also in peripheral blood samples is largely unclear.
Though the levels of both markers
were in most cases significantly higher in the sudden cardiac death
group compared to the control group, the ranges of values were always
largely overlapping, making diagnosis based on marker levels
impossible, as confirmed by the MAD values.
Based on our observations, we
conclude that blood is not a suitable medium for determination
of biochemical markers of cardial troponin I and atrial
natriuretic peptide for post-mortem diagnostics of myocardial damage
and for determining the diagnosis of sudden cardiac death in a manner
similar to diagnostics of myocardium damage in living patients. This
overall conclusion pertains to both cTnI and pro-ANP.
The interesting finding of
close-to-normal levels of pro-ANP in peripheral blood in cases who
died of asphyxia is worth further study.
Šidlo, MD., PhD.
of Forensic Medicine, School of Medicine, Comenius
University and Health Care Surveillance Authority, Antolská
11, 857 01 Bratislava, Slovakia
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