Sem analysis of red blood cells in aged human bloodstains

doi:10.1016/0379-0738(92)90120-L

Forensic Science International

Volume 55, Issue 2, August 1992, Pages 139-159

https://doi.org/10.1016/0379-0738(92)90120-L Get rights and content

Abstract

Mammal red blood cells (RBC) in bloodstains have been previously detected by light microscopy on stone tools from as early as 100 000 ± 25 000 years ago. In order to evaluate the degree of morphological preservation of erythrocytes in bloodstains, an accidental human blood smear on white chert and several experimental bloodstains on hard substrates (the same stone - white chert; another type of stone - graywacke; a non-stone support - stainless steel), were stored in a room, in non-sterile and fluctuating conditions, for lengths of time ranging from 3 to 18 months. Afterwards, the specimens were coated with gold and examined by a Cambridge Stereoscan 120 scanning electron microscope. Results revealed a high preservation of RBC integrity, with the maintenance of several discocytary shapes, a low tendency to echinocytosys and a frequent appearance of a moon-like erythrocytary shape in the thinner areas of the bloodstains.

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Cited by (24)

Microscopic imaging of human bloodstains: testing the potential of a confocal laser scanning microscope as an alternative to SEMs
2020, Micron
The forensic interest on human bloodstains derives from their relation to crime investigation, whereas an archaeological and ethnographic concern arises from their occurrence because of warfare and ritual. The development of digital reflected light microscopes provided an opportunity to use ligh microscopy to study surface topographies in a more accurate way than previously. However, this enhancement has been focused on increasing magnification rather than resolution. An advanced type of light microscope is the confocal laser scanning microscope (CLSM). Its potential as an alternative to scanning electron microscopes (SEMs) for imaging human bloodstains was tested. A fragment of stone (brown chert) was smeared with human peripheral blood, air-dried, and stored indoors. After nearly two years, the sample was examined and imaged using an Olympus LEXT OLS4000 CLSM. The surface detail of CLSM images appeared to be comparatively lower than that of SEM micrographs of coated bloodstains taken at high-vacuum mode and high accelerating voltage, similar to that of SEM micrographs of uncoated bloodstains taken at low-vacuum mode and high accelerating voltage, and similar to or even higher than that of SEM micrographs of uncoated bloodstains taken at high-vacuum mode and low accelerating voltage. These results suggest that a CLSM is a practical alternative to SEMs for imaging human bloodstains when a very-high level of surface detail is not required.
In the pursuit of the holy grail of forensic science – Spectroscopic studies on the estimation of time since deposition of bloodstains
2018, TrAC - Trends in Analytical Chemistry
Citation Excerpt :
The process of bloodstain formation itself has been recently studied by researchers dealing with timeline reconstruction of events, with the hope that better understanding of the blood desiccation mechanisms will enhance the assessments based on BPA results [25–28]. After the completion of water evaporation process, the most abundant components in formed bloodstains are RBC, and hence the vast number of developed dating approaches were based on the changing properties, either morphological, mechanical [29–34], or chemical [15–21,35,36] of erythrocytes. In fact, it is not even RBC that has been given a particular attention, but one of its major constituents, appreciated by forensic scientists for its dynamical nature.
Bloodstains can serve as a source of high-value information, allowing for the reconstruction of bloodshed events or identification of the sample donor. However, from the forensic perspective, the evidential potential of blood traces is not fully exploited. This is because despite significant research efforts, to date, no reliable method for estimating time elapsed since bloodstain deposition has been established. Nonetheless, over the last few years (2011–2017), some noteworthy advances have been made in the field of bloodstain dating, therefore the objective of the following paper is to provide a critical review of recently developed methods, with a particular emphasis on spectroscopy-based approaches. Finally, impediments to applying established procedures in routine forensic practice, along with perspectives to improve the future developments of bloodstain dating techniques, are also discussed.
Human bloodstains on bone artefacts: an SEM intra- and inter-sample comparative study using ratite bird tibiotarsus
2016, Micron
Citation Excerpt :
Although usable SEM micrographs of bloodstains have previously been obtained without coating the sample when using a (high-vacuum) conventional SEM working at very low accelerating voltage (Hortolà, 2005, 2008, 2013a), usually ESEMs or variable-pressure SEMs working in low-vacuum mode and high accelerating voltage are employed when examining an uncoated sample. According to previous observations (Hortolà, 1992, 2002, 2013b), because macrocracks materialise only in thick areas of bloodstains, the thinness of a bloodstained area can be evidenced by the lack of such macrocracks. Moon-like shapes (hecatocytes) are in fact a type of microcracks outlining the (whole or partial) cell perimeter (e.g. Hortolà, 2002: Fig. 1-c).
Apart from their forensic significance in crime investigation, human bloodstains have an anthropological interest due to their occurrence on certain traditional weapons and ritual objects. Previously, a guiding study of erythrocytes in experimental samples including domestic sheep (Ovis aries) tibia was carried out using a scanning electron microscope (SEM). Here, a comparative SEM study to reveal the potential differences in bloodstain surface morphology as a function of intra-sample (smear region) and inter-sample (individual smear, smearing mechanism, bone origin) parameters is reported. A fragment of emu (Dromaius novaehollandiae) tibiotarsus was smeared with an adult man’s peripheral blood. After air-drying and storing indoors, the boundary and neighbouring inner areas of the three individual bloodstains obtained were examined via secondary electrons in a variable-pressure SEM working in low-vacuum mode. As a whole, desiccation microcracks were present, the limits between the smear and the substrate appeared poorly defined, and no erythrocyte negative replicas were observed in the examined areas. In addition, a putative fibrin network, more or less embedded in the dried plasma matrix, was observed in the smears’ boundary. Regarding the smear region in sliding smears, the periphery and boundary revealed to be different, while the head and tail were similar. Considering individual sliding smears, they had similar characteristics. Relating to the smear region as a function of the smearing mechanism, the periphery was different whether sliding or touching, while the boundary was similar in sliding and touching smears. Concerning the smear region as a function of the bone origin, the periphery revealed to be similar in both ratite and mammalian bone, while the boundary did different in ratite and mammalian bone. The results of this study show that SEM examination can be used fruitfully to detect bloodstains on ratite bone. Combined with previous SEM results in domestic sheep bone, they suggest, further, that blood remains can be detected on objects made of bone irrespectively of the mammalian or ratite origin of this raw material.
Extracellular methemoglobin primes red blood cell aggregation in malaria: An in vitro mechanistic study
2013, FEBS Letters
Citation Excerpt :
The images were cropped again and scaled for final display. RBC hematocrit (5%) treated with metHb (0.2 mg/ml) for 1 h at 37 °C and samples for scanning electron microscope (SEM) were prepared as mentioned earlier with minor modification [30]. In brief, metHb treated or untreated cells were washed with PBS and fixed in 1% glutaraldehyde solution for 24 h at 4 °C.
Toxic byproducts from infected RBC cause rheological alteration and RBC aggregation. Malaria culture supernatant has the ability to exhibit RBC aggregation. Ammonium sulfate fractionation and immunodepletion of methemoglobin from culture supernatant confirms methemoglobin as a major aggregant. In vitro treatment of RBC with methemoglobin induces irreversible high order RBC aggregates, resistant to shear stress and physical forces. Methemoglobin-mediated ROS generation in the external micro-environment to develop oxidative stress close to RBC membrane seems to be responsible for initiating and forming high order RBC aggregates through phosphatidyl-serine externalization. Removal of oxidative stress through antioxidant treatment abolishes high order RBC aggregate formation. In conclusion, we discovered a novel pathway of methemoglobin-mediated RBC aggregation and its potential role in patho-physiological effects during malaria.
Methemoglobin exposure produces toxicological effects in macrophages due to multiple ROS spike induced apoptosis
2013, Toxicology in Vitro
Citation Excerpt :
DNA fragments were visualized under UV-light and images were captured with a Kodak Gel Logic 1500 imaging system. Macrophage sample for SEM was prepared as described previously (Hortola, 1992). In brief, MetHb treated or untreated cells were washed with PBS and fixed in 2.5% glutaraldehyde solution for 24 h at 4 °C.
Macrophages are an integral part of the immune system, required to produce a robust immune response against an infectious organism. Presence of methemoglobin in body fluids such as blood, cerebrospinal fluid and urine is associated with tissue damage. We tested cytotoxic effects of MetHb and underlying molecular events in mouse macrophage cell line J774A.1. MetHb exposure dose dependently reduced macrophage viability in an MTT assay. Light microscopy and scanning electron microscopic (SEM) observation of MetHb treated macrophage indicated death (less number of cells per field), severe damage to membrane structure and accumulation of particulate matter in the cytosol. The macrophage death during MetHb exposure was due to induction of apoptosis as indicated by annexin-V/FITC staining and DNA fragmentation analysis. MetHb treatment generated a periodic ROS spikes with time in the macrophage cytosol to develop oxidative stress. Scavenging ROS spikes with NAC, mannitol or PBN dose dependently protected macrophages against MetHb induced toxicity, apoptosis and cellular membrane damage. Our work highlighted the contributions of MetHb mediated toxicity toward macrophage and its potential role in tissue damage and immune depression during malaria and other hemolytic disorders.
Scanning electron microscopy as an auxiliary method in the study of exhumed bones
2011, Forensic Science International
Citation Excerpt :
Furthermore, the analysis of other regions of the skeleton did not show the same elements visualized in that location. The red blood cells remain intact for a long period of time and have been found through scanning electron microscopy, even in stone instruments of the pre-historic period [3]. Therefore, it is possible to analyze exhumed bone fragments after long periods of inhumation.
Scanning electron microscopy (SEM) has been used in forensic science in many ways. The reports of cases in which SEM has been used as an auxiliary method in the investigation of exhumed bones are rare. In this article, we report an exhumation that was made to determine if a seized weapon could have been used in a homicide. We used SEM to analyze a fracture in the interior of the skull of the victim. The findings described in this article showed us that it is possible to develop new researches in this field.

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Sem analysis of red blood cells in aged human bloodstains

Abstract

Pan Am. Geol.

Recent advances in blood residue analysis

Prehistoric blood residues: detection on tool surfaces and identification of species of origin

Science

Blood residue analysis at Çayönü Tepesi, Turkey

J. Field Archaeol.

Compendio de Histologia humana

Schumacher, Grundriss der Histologie des Menschen

Hematology For Medical Technologists

Erythrocytes

Red blood cells

Los indicios en Medicina legal

Étude comparative des différentes techniques de taille du silex et des roches dures

Anthropologie