Role of Antitroponin Antibodies and Macrotroponin in the Clinical Interpretation of Cardiac Troponin.

Cardiac troponin is extensively used as a biomarker in modern medicine due to its diagnostic capability for myocardial injury, as well as its predictive and prognostic value for cardiac diseases. However, heterophile antibodies, antitroponin antibodies, and macrotroponin complexes can be observed both in seemingly healthy individuals and patients with cardiac diseases, potentially leading to false positive or disproportionate elevation of cTn (cardiac troponin) assay results and introducing discrepancies in clinical interpretations with impact on medical management. In this review article, we describe the possible mechanisms of cTn release and the sources of variations in the assessment of circulating cTn levels. We also explore the pathophysiological mechanisms underlying antitroponin antibody development and discuss the influence exerted by macrotroponin complexes on the results of immunoassays. Additionally, we explore approaches to detect these complexes by presenting various clinical scenarios encountered in routine clinical practice. Finally, unsolved questions about the development, prevalence, and clinical significance of cardiac autoantibodies are discussed.

Ternary BiOI/Bi2S3/Au Nanosheet Arrays as a Photoelectrochemical Signal Converter for the Detection of Cardiac Troponin I.

Nanosheet arrays with stable signal output have become promising photoactive materials for photoelectrochemical (PEC) immunosensors. However, an essential concern is the facile recombination of carriers in one-component nanoarrays, which cannot be readily prevented, ultimately resulting in weak photocurrent signals. In this study, an immunosensor using gold nanoparticle-anchored BiOI/Bi2S3 nanosheet arrays (BiOI/Bi2S3/Au) as a signal converter was fabricated for sensitive detection of cardiac troponin I (cTnI). The ternary nanosheet arrays were prepared by a simple method in which Bi2S3 was well-coated on the BiOI surface by in situ growth, whereas the addition of Au further improved the photoelectric conversion efficiency and could link more antibodies. The three-dimensional (3D) ordered sheet-like network array structure and BiOI/Bi2S3/Au ternary nanosheet arrays showed stable and high photoelectric signal output and no significant difference in signals across different batches under visible light excitation. The fabricated immunosensor has a sensitive response to the target detection marker cTnI in a wide linear range of 500 fg/mL to 50 ng/mL, and the detection limit was 32 fg/mL, demonstrating good stability and selectivity. This work not only shows the great application potential of ternary heterojunction arrays in the field of PEC immunosensors but also provides a useful exploration for improving the stability of immunosensors.

The role of antibody-based troponin detection in cardiovascular disease: A critical assessment.

Cardiovascular disease has remained the world's biggest killer for 30 years. To aid in the diagnosis and prognosis of patients suffering cardiovascular-related disease accurate detection methods are essential. For over 20 years, the cardiac-specific troponins, I (cTnI) and T (cTnT), have acted as sensitive and specific biomarkers to assist in the diagnosis of various types of heart diseases. Various cardiovascular complications were commonly detected in patients with COVID-19, where cTn elevation is detectable, which suggested potential prognostic value of cTn in COVID-19-infected patients. Detection of these biomarkers circulating in the bloodstream is generally facilitated by immunoassays employing cTnI- and/or cTnT-specific antibodies. While several anti-troponin assays are commercially available, there are still obstacles to overcome to achieve optimal troponin detection. Such obstacles include the proteolytic degradation of N and C terminals on cTnI, epitope occlusion of troponin binding-sites by the cTnI/cTnT complex, cross reactivity of antibodies with skeletal troponins or assay interference caused by human anti-species antibodies. Therefore, further research into multi-antibody based platforms, multi-epitope targeting and rigorous validation of immunoassays is required to ensure accurate measurements. Moreover, in combination with various technical advances (e.g. microfluidics), antibody-based troponin detection systems can be more sensitive and rapid for incorporation into portable biosensor systems to be used at point-of care.

Change in troponin concentrations in patients with macrotroponin: An in vitro mixing study.

INTRODUCTION: Macrotroponin is a complex formed between endogenous cardiac troponin autoantibodies (cTnAABs) and circulating cardiac troponin (cTn). The potential effect of macrotroponin on current high sensitivity cTn assays has not been fully explored but has recently been identified as a major cause of discrepancy in cTn results between assays. In this study we investigated the effects of mixing troponin (cTn) standards to specimens with and without macrotroponin. METHOD: Macrotroponin was identified in specimens by a recovery of cTnI < 40% following protein A immunoglobulin depletion. Troponin standards containing cTn-IC and cTn-TIC complexes were mixed with serum samples, with (n = 20) and without (n = 10) the presence of macrotroponin. Specimens were tested for cTn before and after mixing by three commercially available high sensitivity cTn assays. Gel filtration chromatography was carried out on five specimens with macrotroponin and each fraction was analzyed by multiple cTn assays. FINDINGS: Following mixing with cTn-TIC standard, all specimens with macrotroponin had a markedly reduced absolute increase in cTnI, indicating negative analytical interference due to macrotroponin. Following mixing with the cTn-IC standard, specimens with macrotroponin demonstrated highly variable changes in cTnI, suggesting significant heterogeneity in macrotroponin complex reactivity between individuals. When the ratio of change, calculated by dividing the absolute change between two cTn assays, was compared between specimens with and without macrotroponin, significant differences were observed (p < 0.001). These findings were supported by variable migration of peak cTn activity on gel filtration chromatography. CONCLUSION: Macrotroponin leads to assay dependent analytical interference affecting current high sensitivity troponin I assays. Furthermore, endogenously occurring cTnAABs are conformationally specific and the analytical effects vary between assays and individuals.

A liquid-crystal-based immunosensor for the detection of cardiac troponin I.

Cardiac troponin I (cTnI) is one of the most sensitive and specific markers of myocardial cell injury, which can detect even minor myocardial damages. It is recognized as the main biochemical marker of the rapid diagnosis of acute myocardial infarction (AMI) and acute coronary syndrome (ACS). In this study, a label-free biosensor that utilizes the birefringence property of a nematic liquid crystal (LC) for the detection of cTnI is demonstrated. A chemically sensitive film with specific molecular recognition ability was decorated on the surface of a substrate, and the LC molecules were arranged in a vertically oriented order under the influence of the sensitive film, and a dark background signal was obtained using a polarizing optical microscope. When the antigen-antibody specifically binds to form a stronger acting force, the orientation of the LC molecules changes, resulting in a bright optical appearance. This LC-based immunosensor not only has the advantages of a facile structure, low cost and excellent specificity but also high sensitivity (a low detection limit of 1 pg ml-1), and has a promising future in biomedical related fields.

A cardiac troponin I photoelectrochemical immunosensor: nitrogen-doped carbon quantum dots-bismuth oxyiodide-flower-like SnO2.

A novel photoelectrochemical (PEC) immunosensor for the determination of cardiac troponin I (cTnI) was constructed. The flower-like stannic oxide (SnO2) with large specific surface area was prepared by hydrothermal synthesis. Nitrogen-doped carbon quantum dots (NCQDs) with excellent surface property were used as a sensitizer for SnO2. Bismuth oxyiodide (BiOI) is a narrow band gap (1.83 eV) nanomaterial, which was firstly modified on NCQDs-sensitized SnO2 through in situ growth method. After NCQDs with small size and BiOI nanoparticles are successively combined with SnO2, the SnO2/NCQDs/BiOI microflower was obtained, which possessed good photochemical properties. Using visible light as excitation source and ascorbic acid (AA) as electron donor, the ultrasensitive and quantitative determination of cTnI was realized by detecting the changes of photocurrent under different concentrations of cTnI. The PEC immunosensor showed a large-scaled response (0.001-100 ng mL-1) and a low detection limit (0.3 pg mL-1) under optimised experimental conditions. The sensor has potential clinical value in the prediction and diagnosis of cardiovascular diseases in elderly patients with diabetes. Graphical abstract.

Interleukin-10 delivered by mesenchymal stem cells attenuates experimental autoimmune myocarditis.

BACKGROUNDS: Autoimmune myocarditis is characterized by over-activated immune system attacking the cardiomyocytes, resulting in heart function decline. In the current study, we investigated the therapeutic advantages of delivering Interleukin-10 (IL-10) by mesenchymal stem cells (MSCs), both of which had immune suppression functions, in treating experimental autoimmune myocarditis. METHODS: The mouse model of autoimmune myocarditis was established by subcutaneous injection of troponin I in A/J mice. Mouse bone marrow derived mesenchymal stem cells (BM-MSCs) with or without IL-10 overexpression, or the recombinant IL-10 protein were delivered into the mice via tail-vein injection. The inflammation and fibrosis levels of the heart were evaluated with qPCR, ELISA and histological staining. Serum level of anti-troponin-I was assessed by ELISA. Heart function analysis was conducted with echocardiography. RESULTS: BM-MSCs overexpressing IL-10 had enhanced immune suppression functions. They also showed improved therapeutic effects from the perspective of heart function and cardiac fibrosis. The anti-troponin-I level was significantly reduced by MSCs overexpressing IL-10 when comparing with the MSCs or IL-10 protein injection. CONCLUSION: IL-10 delivered by MSCs showed therapeutic advantages in treating experimental autoimmune myocarditis.

Electrochemiluminescent Detection of Proteins Based on Fullerenols Modified Gold Nanoparticles and Triple Amplification Approaches.

In this work, fullerenols were found to be able to enhance the ECL signals of the luminol and H2O2 system and were employed for the first time as a reducing, catalyzing, and stabilizing agent in the one-step fast synthesis of fullerenols@AuNPs in only 5 min. First, the prepared fullerenols@AuNPs were applied to fabricate a label-free immunosensor for the detection of human cardiopathy biomarker (cardiac troponin I, cTnI). Second, using the fullerenols@AuNPs as biolabels to establish a sandwich-type immunosensor and catalyzing in situ copper-stained reaction to generate Cu particles capped on the fullerenols@AuNPs, and then a novel electrochemical stripping chemiluminescent (ESCL) method was developed for detection of cTnI and IgG with about 20 times more sensitive than the former one. At the process of ESCL detection, Cu2+was stripped from Cu@fullerenols@AuNPs with significant increase of the ECL signals. This can be attributed to the fact that the fullerenols@AuNPs nanoparticles and the Cu2+ have excellent conductivity and could facilitate the decomposition of H2O2 to generate various reactive oxygen species (ROSs), thereby accelerating the ECL process. Both immunosensors show high sensitivity and selectivity to cTnI and IgG detection with a wide linear range from fg/mL to ng/mL and the low limits of detection down to fg/mL for cTnI and IgG, respectively.

Sensitive detection of cardiac troponin-I protein using fabricated piezoresistive microcantilevers by a novel method of asymmetric biofunctionalization.

Microcantilever-based sensor platform has attracted a lot of attention over the time in detection of a variety of molecules due to their miniaturized dimensions. Sensitivity enhancement is an important aspect of such sensors, especially when used for point-of-care diagnostic purpose. However, the major concern while operating these sensors in deflection mode is their sensitivity which mainly relies on selective chemical modification protocols employed on these sensor surfaces. One of the ways of getting better sensitivity is through asymmetric (one side) biofunctionalization of the sensor surface. In the presented work here, we have demonstrated a novel approach of asymmetric biofunctionalization of proteins in overall sensitivity enhancement of piezoresistive silicon nitride-oxide microcantilever sensor platform inside a flow chamber. Herein, using our developed surface chemistry, asymmetrically biofunctionalized microcantilevers first exhibited a greater electrical response in terms of piezoresistance change than their symmetric counterpart in the detection of human immunoglobulins (HIgGs) protein. Finally, these microcantilevers were employed to exhibit the enhanced sensitivity towards the detection of a crucial cardiac marker protein, i.e. Troponin-I (cTnI) down to 250 ng ml-1 using asymmetric biofunctionalization process. This study shows that the developed asymmetric biofunctionalization methodology may be used as a general protocol to detect other important biomarkers of clinical applications with improved sensitivity.

Transposing Lateral Flow Immunoassays to Capillary-Driven Microfluidics Using Self-Coalescence Modules and Capillary-Assembled Receptor Carriers.

Point-of-care (POC) immunodiagnostic tests play a crucial role in enabling rapid and correct diagnosis of diseases in prehospital care, emergency, and remote settings. In this work, we present a silicon-based, capillary-driven microfluidic chip integrating two microfluidic modules for the implementation of highly miniaturized immunoassays. Specifically, we apply state-of-the-art microfluidic technology to demonstrate a one-step immunoassay for the detection of the cardiac marker troponin I in human serum using sample volumes of ∼1 µL and with a limit of detection (LOD) of ∼4 ng mL-1 within 25 min. The microfluidic modules discussed here broadly map functionalities found in standard lateral flow assays. We implement a self-coalescence module (SCM) for the controlled reconstitution and delivery of inkjet-spotted and dried detection antibodies (dAbs). This allows for homogeneous dissolution of 1.3 ng of fluorescently labeled dAbs in 416 nL of the sample used for the assay. We also show how to immobilize receptors inside closed microfluidic devices in <30 s using bead lane modules inside which microbeads functionalized with capture antibodies (cAbs) are self-assembled. The resulting bead lane module, with a volume of ∼3 × 10-5 mm3, is positioned across the flow path and holds ∼300 5 µm-diameter microbeads. Altogether, these capillary-driven elements allow for the manipulation of samples and reagents with an unprecedented precision and control, paving the way for the next generation of POC immunodiagnostics.

Reduced graphene oxide-gold nanoparticles-catalase-based dual signal amplification strategy in a spatial-resolved ratiometric electrochemiluminescence immunoassay.

A novel spatial-resolved electrochemiluminescent (ECL) ratiometry for cardiac troponin I (cTnI) analysis was developed using resonance energy transfer (RET) and a coreactant consumption strategy for signal amplification. Specifically, the spatial-resolved dual-disk glassy carbon electrodes were modified with CdS nanowires (CdS NWs) and luminol-gold nanoparticles (L-Au NPs) as potential-resolved ECL emitters, respectively. After stepwise immobilization of anti-cTnI and bovine serum albumin on the dual-disk electrodes, the CdS NWs-based electrode, with varied concentrations of cTnI, was used to provide a working signal, whereas the L-Au NPs-based electrode, with a fixed amount of cTnI, was employed to provide the reference signal. To efficiently amplify the working signal on the CdS NWs-based electrode, an anti-cTnI-reduced graphene oxide-gold nanoparticles-catalase probe (anti-cTnI-rGO-Au NPs-CAT) was loaded onto the electrode to form a sandwich immunocomplex. The RET from CdS NWs to Au NPs and the coreactant (i.e. H2O2) consumption by the CAT generate a significant ECL decrease on the CdS NWs-based electrode in the presence of cTnI. This novel and sensitive ratiometric detection mode for cTnI was achieved using the ratio values of the working signal of the CdS NWs-based electrode and the reference signal of the L-Au NPs-based electrode. The integration of RET and coreactant consumption strategy in the designed spatial-resolved ratiometric platform endows the immunosensor with a wide linear range of 5.0 × 10-13 - 1.0 × 10-7 g mL-1 and a low detection limit of 0.10 pg mL-1 for cTnI. Furthermore, the method exhibits high accuracy and sensitivity for cTnI determination in human serum samples.

A wavelength-resolved electrochemiluminescence resonance energy transfer ratiometric immunosensor for detection of cardiac troponin I.

In this study, a wavelength-resolved electrochemiluminescence resonance energy transfer (ECL-RET) ratiometric immunosensor from Au nanoparticle functionalized graphite-like carbon nitride nanosheets (Au-g-C3N4) to Au nanoclusters (Au NCs) has been constructed for the first time. At a working voltage of 0 to -1.2 V, Au-g-C3N4 showed a strong cathodic ECL emission with a peak at 460 nm, which overlapped well with the absorption spectra of Au NCs thus stimulating the fluorescence emission of Au NCs at 610 nm. Moreover, within this voltage range, the Au NCs showed no ECL signal; therefore, they would not interfere with the detection of the system. We used cardiac troponin I (cTnI) as an analytical model to construct a sandwich immunosensor based on the ECL-RET ratiometric strategy. By measuring the responses of the ECL460 nm/FL610 nm ratio at different cTnI concentrations, the sensitive detection of cTnI with a wide range of 50 fg mL-1 to 50 ng mL-1 and a low detection limit of 9.73 fg mL-1 can be achieved. This work enriches the wavelength-resolved ECL-RET system and provides an innovative reference for the development of more efficient and sensitive ECL-RET ratiometry.

Electrochemiluminescence Immunosensor Based on Au Nanocluster and Hybridization Chain Reaction Signal Amplification for Ultrasensitive Detection of Cardiac Troponin I.

Measurement of cardiac troponin I in the blood is crucial for the early diagnosis of acute myocardial infarction. Herein, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor has been developed for determination of cardiac troponin I (cTnI) by using Au nanoclusters and hybridization chain reaction (HCR) signal amplification. In this ECL immunosensor, Au nanoclusters were dual-labeled at each end of hairpin DNA (H1 and H2) and acted as the luminophore. DNA initiator strands (T1) and secondary antibody (Ab2) were conjugated on Au nanoparticles (AuNPs) to obtain a smart probe (Ab2-AuNP-T1). In the presence of target cTnI, the sandwiched immunocomplex composed of cTnI, Ab1, and Ab2-AuNP-T1 was formed. Then the initiator strands T1 of Ab2-AuNP-T1 opened the hairpin DNA structures and triggered a cascade of hybridization events. Consequently, a large number of Au NCs were indirectly modified on the surface of the electrode, which could react with the coreactant (K2S2O8) and emit a strong ECL signal. Under the optimal conditions, the immunosensor exhibited a wide detection range for cTnI from 5 fg/mL to 50 ng/mL and a low detection limit of 1.01 fg/mL (S/N = 3). Because of the excellent specificity, stability, and reproducibility of the proposed ECL-HCR sensor, it has a great application prospect for cTnI detection in clinical diagnosis.

SERS-based immunoassay using gold-patterned array chips for rapid and sensitive detection of dual cardiac biomarkers.

Cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) are important diagnostic biomarkers for acute myocardial infarction (AMI). Many efforts have been undertaken to develop highly sensitive detection methods for the quantitative analysis of these dual targets. However, current immunoassay methods are inadequate for accurate measurement of cTnI and CK-MB, due to their limited detection sensitivity. Thus, there is still an urgent demand for a new technique that will enable ultrahigh sensitive detection of these biomarkers. In this study, we developed a surface-enhanced Raman scattering (SERS)-based sandwich immunoassay platform for the ultrasensitive detection of cTnI and CK-MB. In this study, a monoclonal-antibody-immobilized gold-patterned chip was used as a SERS active template. Target samples and polyclonal-antibody-conjugated Au@Ag core-shell nanoparticles were then added. Using this SERS platform, the concentration of biomarkers could be quantified by monitoring the characteristic Raman peak intensity of Raman reporter molecules. Under optimized conditions, the limits of detection (LODs) were estimated to be 8.9 pg mL-1 and 9.7 pg mL-1 for cTnI and CK-MB, respectively. Thus, the proposed SERS-based immunoassay has great potential to be an effective diagnostic tool for the rapid and accurate detection of cTnI and CK-MB.

Cardiac troponin I autoantibody induces myocardial dysfunction by PTEN signaling activation.

BACKGROUND: The objective of the current study was to study the molecular mechanism(s) underlying cardiac troponin I autoantibody (cTnIAAb) binding to cardiomyocyte and resultant myocardial damage/dysfunction. METHODS: cTnIAAb was purified from serum of 10 acute myocardial infarction (AMI) patients with left ventricular remodeling. Recombinant human cTnI was used to generate three mouse-derived monoclonal anti-cTnI antibodies (cTnImAb1, cTnImAb2, and cTnImAb3). The target proteins in cardiac myocyte membrane bound to cTnImAb and effect of cTnIAAb and cTnImAb on apoptosis and myocardial function were determined. FINDINGS: We found that cTnIAAb/cTnImAb1 directly bound to the cardiomyocyte membraneα-Enolase (ENO1) and triggered cell apoptosis via increased expression of ENO1 and Bax, decreased expression of Bcl2, subsequently activating Caspase8, Caspase 3, phosphatase and tensin homolog (PTEN) while inhibiting Akt activity. This cTnIAAb-ENO1-PTEN-Akt signaling axis contributed to increased myocardial apoptosis, myocardial collagen deposition, and impaired systolic dysfunction. INTERPRETATION: Results obtained in this study indicate that cTnIAAb is involved in the process of ventricular remodeling after myocardial injury. FUND: The National Natural Science Foundation of China (Grant#: 81260026).

The evolutionarily conserved C-terminal peptide of troponin I is an independently configured regulatory structure to function as a myofilament Ca2+-desensitizer.

The C-terminal end segment of troponin subunit I (TnI) is a structure highly conserved among the three muscle type-specific isoforms and across vertebrate species. Partial deletion or point mutation in this segment impairs cardiac muscle relaxation. In the present study, we characterized the C-terminal 27 amino acid peptide of human cardiac TnI (HcTnI-C27) for its role in modulating muscle contractility. Biologically or chemically synthesized HcTnI-C27 peptide retains an epitope structure in physiological solutions similarly to that in intact TnI as recognized by an anti-TnI C-terminus monoclonal antibody (mAb TnI-1). Protein binding studies found that HcTnI-C27 retains the binding affinity for tropomyosin as previously shown with intact cardiac TnI. A restrictive cardiomyopathy mutation R192H in this segment abolishes the bindings to mAb TnI-1 and tropomyosin, demonstrating a pathogenic loss of function. Contractility studies using skinned muscle preparations demonstrated that addition of HcTnI-C27 peptide reduces the Ca2+-sensitivity of myofibrils without decreasing maximum force production. The results indicate that the C-terminal end segment of TnI is a regulatory element of troponin, which retains the native configuration in the form of free peptide to confer an effect on myofilament Ca2+-desensitization. Without negative inotropic impact, this short peptide may be developed into a novel reagent to selectively facilitate cardiac muscle relaxation at the activated state as a potential treatment for heart failure.

Paper-based cascade cationic isotachophoresis: Multiplex detection of cardiac markers.

Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care.

The Troponin-I fast skeletal muscle is reliable marker for the determination of vitality in the suicide hanging.

Troponin I (TnI) is the inhibitory subunit of the troponin complex in the sarcomeric thin filament of striated muscle and plays a central role in the calcium regulation of contraction and relaxation. Vertebrate TnI has evolved into three isoforms encoded by three homologous genes: TNNI1 for slow skeletal muscle TnI, TNNI2 for fast skeletal muscle TnI and TNNI3 for cardiac TnI, which are expressed under muscle type-specific and developmental regulations in both the atrium and ventricle of the heart. Skeletal muscle TnI (both sTnI iso-forms) have been proposed as a sensitive and fast fiber-specific serum marker of skeletal muscle damage; fsTnI concentration in increased peripheral blood when fast twitch fibers were damaged. In our study we investigate if the 'Troponin I, fast skeletal muscle' can also be used as a reliable diagnostic tool in forensic practice, to perform differential diagnosis about vitality in suicide by hanging and simulated hanging (suspension of the victim after murder). We selected 8 women and 13 men, mean age 52.2 years, who died from suicidal hanging. The ligature material used for hanging was soft material in 11 cases and hard material in 10 cases. We chose cases as a control group of adults (n = 10; six women, four men, mean age 47.3 years) that died from opioid overdose (n = 2), car accident (n = 3) and sudden cardiac death (n = 5). Those deaths were characterized by their rapidity. To test the Anti-Troponin I fast skeletal muscle Antibody (Abcam clone-134,838), we used a case of a subject who died of myocardial infarction (timing infarct dated to 24-36 h prior to death). The reactions to Troponin I (namely the amount and extent of marker depletion) was scored for each section from 0 to -3: 0 = no loss of staining; -1 = minimal decrease in staining, compared to normally stained tissue; -2 = clear decrease in staining with some positivity (brown color) remaining; and -3 = no positive (brown) staining. The set of results obtained leads us to believe that the use of this antibody (Anti-Troponin I fast skeletal muscle antibody) is very promising to be able to make a certain differential diagnosis between antemortem and postmortem hangings. It should be emphasized that the present study seems to open new and promising horizons in the possibility to discriminate between suicidal hanging and simulated hanging (suspension of the victim after murder).