SEM examination of human erythrocytes in ... - Wiley Online Library

Journal of Microscopy, Vol. 218, Pt 2 May 2005, pp. 94–103 Received 21 January 2005; accepted 25 February 2005

SEM examination of human erythrocytes in uncoated bloodstains on stone: use of conventional as environmental-like SEM in a soft biological tissue (and hard inorganic material) Blackwell Publishing, Ltd.

P. H O RT O L À Area of Prehistory (CSIC Associate Research Unit), Rovira i Virgili University, Plaça Imperial Tàrraco 1, E-43005 Tarragona, Catalonia, Spain

Key words. Blood smears, haemotaphonomy, red blood cells, scanning electron microscopy, specimen preparation.

Summary Although nowadays the so-called environmental scanning electron microscopes (ESEMs) allow the observation of the samples without metal or carbon coating, many conventional scanning electron microscopes (SEMs) are still in use. On the other hand, the presence of erythrocytes (red blood cells, RBCs) in a smear is considered a blood confirmation. Such a presence has been previously reported even in Lower Stone Age implements. In previous works, I have reported several studies dealing with cytomorphology of RBCs in bloodstains using scanning electron microscopy with standard specimen preparation procedures, i.e. via coating the samples before SEM analysis. In order to explore the potential of conventional SEM as environmental-like SEM in haemotaphonomical studies, two alkaline (limestone) and two acid (flint) rock fragments were smeared with human blood from a male and a female. The bloodstains obtained in this way were then air dried indoors and stored into a non-hermetic plastic box. Afterwards, the smears and their rock substrates were examined directly without coating, via secondary electrons, using a JEOL JSM-6400 scanning electron microscope. Satisfactory results reveal the capability of a conventional SEM to work in secondary-electron mode as an environmental-like SEM on these kinds of biological and inorganic materials, and probably in many other biological and non-biological samples. Received 21 January 2005; accepted 25 February 2005

Introduction Vertebrate blood is a suspension, into a fluid medium (blood plasma), of three types of cells (figured elements): erythrocytes

Correspondence:

Dr

Policarp

Hortolà.

Tel.:

+34 977558 648;

+34 977559 597; e-mail: [email protected]

fax:

(red blood cells, RBCs), leucocytes (white blood cells), and thrombocytes. The most abundant of these corpuscles (i.e. the erythrocytes) were observed during the last half of the 17th century by early optical microscopists, such as Giovanni Alfonso Borelli, Jan Swammerdam, Marcello Malpighi and Antoni van Leeuwenhoek. Unlike the rest of members of the subphylum Vertebrata, blood in the mammals has platelets instead of thrombocytes sensu stricto, and anucleate RBCs instead of nucleate ones (Jain, 1986; Fawcett & Raviola, 1994). Because of their lack of nucleus, under physiological conditions the mammalian erythrocytes are typically shaped as biconcave discs (discocytes); this does not apply to the family Camelidae, where RBCs tend to be both more flattened, and oval (ovalocytes). Other physiological shapes, which are minor or pathological, are echinocytes (burr cells), codocytes (target cells), keratocytes (horn cells), torocytes (doughnut cells), drepanocytes (sickle cells) and many other morphologies (Bessis, 1974; Castoldi, 1981; Barnhart et al., 1983; Jain, 1986; Rozman et al., 1993; Bull & Breton-Gorius, 1995). A smear may be defined as the result of a causal relationship, in which a physical contact (the cause) produces a trace (the effect). The occurrence of (at least morphological) preservation of anucleate, mammalian RBCs from blood smears has been reported even in implements assigned to be c. 2 million years old (Loy, 1998). From the theoretical point of view, a short-time preservation of specimens is a sine qua non precondition for a longer one preservation. An additional interest of bloodstains is that they can be dated by electron spin resonance (Miki et al., 1987). This non-destructive technique requires less than 0.25 g of sample and its dating range could cover all the Quaternary times (Geyh & Schleicher, 1990). The presence of all kind of residues on implements agrees with the forensic well-known Locard’s Principle of Exchange (‘every contact leaves traces’). Aside from this deterministic principle, in a broad sense all experimental framework is based upon the © 2005 The Royal Microscopical Society

SEM AS ESEM IN BLOODSTAINS ON STONE

geological Lyell’s Principle of Actualism (‘the present is the key to the past’). In forensic analysis, the presence of RBCs in a smear is considered a blood confirmation (Fiori, 1962). Despite the diagnostic value of RBCs and the ancient erythrocyte evidence, interest in bloodstain analysis has been focused largely on the molecular level, and the knowledge of the morphological characteristics of stain-origin RBCs had not been taken into account. Scanning electron microscopes (SEMs) have a high resolution for bulk objects, large depth of field, and shadow-relief effect of electron contrast. Furthermore, their capability of examining objects at very low magnification represents a very useful tool in either forensic or archaeological studies (Goldstein et al., 1992). A typical feature of (high-vacuum) conventional SEM is the previous conductive coating of samples. It has been widely assumed that non-conductive specimens, such as non-dehydrated biological samples, are subject to charging, so they should be coated prior to SEM examination in order to make them conductive. Thus image formation becomes possible. Further technological developments led to the (low-vacuum) environmental SEMs (ESEMs), which allow the examination of samples without coating. As well as their capability of examining objects at very low magnification, shared with SEMs, their lack of necessity for sample coating is also very useful in either archaeological and forensic specimens when they may be exhibited in museums or in court. The coating materials used in conventional SEM (gold, gold-palladium, carbon … ) are difficult to remove from samples. Nevertheless, despite this undesirable characteristic of specimen preparation, many conventional SEMs are still in use. In preceding papers, I have reported SEM studies dealing with the cytomorphology of mammalian (whether or not human) RBCs in bloodstains (Hortolà, 1992a,b, 1994, 2001a,b, 2002). The SEM examination of mammalian bloodstains brings to the eye a ‘strange’ world of multiple erythrocyte shapes, which are susceptible to systematization (Hortolà, 2002). All this haemotaphonomical research was carried out using conventional SEMs with previous sample coating. In this paper, I report the use of such a conventional SEM as an environmental-like SEM to examine RBCs in blood smears on rock substrates, that is to say, a soft biological tissue as well as inorganic material, without any specimen coating.

95

Table 1. Working conditions tested in the blood smears. The list is shown in increasing value for each operating parameter, independently of the concrete combinations amongst all the possible combinations, carried out.

Accelerating voltage (kV) 0.5 1.0

Working distance (mm)

Probe current (A)

Image-recording resolution (pixels)

11 12 13 14 15 19 21 22 25

6·10−12 3·10−11 6·10−11 1·10−10

256 × 208 512 × 416 1024 × 832

Magnification (×) 20 850 1000 1500 1700 2200 3000

that of ‘contact’. All the bloodstains were air dried indoors without direct sunlight or wind for about 1.5 hours, and then stored in a non-hermetic plastic box. Afterwards, the set of bloodstained rock fragments was affixed to a SEM sample holder by a removable Leit-C-Plast plastic conductive carbon cement (Bal-Tec, Münster, Germany) and then the specimens were directly examined without coating, via secondary electrons, by a JSM-6400 scanning electron microscope (JEOL Ltd, Tokyo, Japan). Micrographs were recorded as digital images by an INCAEnergy system (Oxford Instruments Analytical Ltd, Bucks, U.K.). In order to obtain good imaging quality with the minimal damage and electrostatic charging effects to the uncoated bloodsmears − the rock material was assumed to be less sensitive to these effects than the biological one – several combinations of working conditions, using always low accelerating voltages and magnifications, were tested (Table 1). Each blood smear was examined several times under different working conditions. Once the working conditions were standardized, a magnification test for the bloodstained areas was carried out using a broader range of magnifications (850, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7000 and 10 000×). The examination of the blood smears took place from 1 day to 4.5 months after bloodstaining. Unbloodstained areas of the stone substrates were examined later.

Materials and methods Two fragments from a single stock of an alkaline rock (a limestone, L) and other two from a single stock of an acid rock (a black chert usually known as flint, F) were smeared with peripheral human blood of an adult male (m) and an adult female (f). Both rock types were used for both individuals, resulting in four single bloodstains. Blood extraction was carried out by puncturing the end of the right-hand forefinger with a sterile clinical lancet. The smearing mechanism was © 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94–103

Results and discussion The best erythrocyte images were obtained under the following conditions: 0.5 kV accelerating voltage, 12 mm working distance, 3·10−11 A probe current, and 1024 × 832 pixels image-recording resolution. Therefore, these conditions were taken as standard for all subsequent SEM examinations (Figs 1–4). Figure 1 shows a partial view of each bloodstain (bs) and its substrate (sb) at low magnification. A macrocracking

96

P. H O RTO L À

Fig. 1. Partial view of the bloodstains and their substrates at low magnification. bs = bloodstained area, L = limestone (smear’s substrate), F = flint (smear’s substrate), f = female’s blood, m = male’s blood, sb = substrate area (i.e. unbloodstained rock surface). All original magnifications were 20×. All scale bars are 2 mm.

pattern, due to the typical scales in (totally or partially) thick bloodstains, can be seen. In Fig. 2, erythrocytes from central, non-peripheral areas of the smears are displayed at a sufficient magnification level for clear visualization. A macrocracked area is shown in Lm and Lf. The Lm micrograph exhibits a slight banding. From previous experience, deep macrocracks in bloodstains result often in a banding, which can sometimes also be observed in some microcracks depending upon magnification. Banding is probably due to interference in electron conductiveness. It has also been observed by me in goldcoated thick-film bloodstains, so this phenomenon would be independent of whether or not a bloodstain sample is coated. Furthermore, this banding does not inhibit the visualization of surface RBCs in the smears (e.g. Fig. 4: Ff, and Fig. 6:

1000×ori−5000×ori). Figure 3 shows, at the same magnification as Fig. 2, the naked smear substrates, in order to compare with bloodstained areas at a same magnification. In all of them, the fine-grained substrate textures may be seen. In addition, the different substrate roughness is evidenced, with L appearing rougher than F. In Fig. 4, erythrocytes in rock edge areas of the smears are displayed at higher magnification than Fig. 2. In Lf, the part of the blood film closest to the edge – probably only composed of plasma – has risen from the substrate. In Fm and Ff, most of the RBCs exhibit a morphology of hecatocytes (moon-like shapes). Figure 5 displays, at the same magnification as Fig. 4, the naked smear substrates from the same zones as those of Fig. 3, in order to compare with bloodstained areas at a same magnification. © 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94– 103


97

Fig. 2. Erythrocytes in central, non-peripheral areas of the smears, at a sufficient magnification level for clearly visualizing the surface RBCs. Legend as in Fig. 1. All original magnifications were 850×. All scale bars are 60 µm.

Concerning the magnification, usable erythrocyte images were obtained up to 10 000× (Figs 6, 7). Figure 6 exhibits some results for Lm. Banding in Lm: which appeared up to 5000× (Fig. 6) but not at higher magnifications, is similar to the banding shown in Lm at 850× (Fig. 2). In Fig. 7, some results, at the same magnifications as in Fig. 6, are displayed for Fm. Here, with respect to 7000×ori, the 10 000×ori micrograph was taken at a contiguous, more informative area with some discocytes that are also visible from 1000×ori to 3000×ori. In both cases, magnifications over 10 000× were not attempted because they were considered to be of little use in the examination of human erythrocytes, the mean diameter of which is 7.49 µm according to SEM stereo micrographs reported by LeBlond & Shoucri (1978). © 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94–103

Moreover, the smallest mammalian RBCs are those of chevrotain (Tragulus javanicus), at 2.1 µm (Handley & Ponder, 1961), so a magnification of 10 000× should be sufficient to clearly visualize the erythrocytes in any mammalian bloodstain. Despite the examination of the blood smears taking place up to 4.5 months after bloodstaining, this time span could not affect per se the degree of bloodstain preservation, according to previous findings in very much older bloodstains (Hortolà, 2002). The magnification test was carried out in the bloodstains from a sole individual on different substrates, because, as expected, for the same substrate type no differences in blood smears were found between genders. The male blood smears were chosen with the aim of following the

98

P. H O RTO L À

Fig. 3. Naked smear substrates as compared with a bloodstained area at the same magnification that Fig. 2. Legend as in Fig. 1. All original magnifications were 850×. All scale bars are 60 µm.

same examination order used throughout the study. Finally, it can be pointed out that, despite the reiterated secondaryelectron SEM examination of the uncoated bloodstain samples, no appreciable damage was detected in them at any time during this study. Conclusions ESEMs represent an important advance in electron microscopy due to their low-vacuum working mode, which means that there is no necessity for sample coating. As many conventional SEMs are still in use, their potential deserves be explored. The satisfactory results obtained in this study reveal the capability of a conventional SEM to work in secondary-electron mode like an ESEM in either soft biological tissue as well as hard

inorganic material, at least in erythrocytes in bloodstains on stone and probably in many other kinds of biological and nonbiological samples. Acknowledgements J. Vallverdú and M. Mosquera (colleagues at the Area of Prehistory, Rovira i Virgili University) supplied me a little of their (respectively, male and female) blood for this study. The Service of Scientific and Technical Resources of the Rovira i Virgili University provided the use of its electron microscopy facility. This work was supported by project grants CICYT No. BOS2003-08938-C03-03 (Spanish Government) and DGR No. 2001SGR-00313 (Government of the Commonwealth of Catalonia). © 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94– 103


99

Fig. 4. Erythrocytes in rock edge areas of the smears, at higher magnification than Fig. 2. Legend as in Fig. 1. All original magnifications were 1500×. All scale bars are 30 µm.

© 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94–103

100

P. H O RTO L À

Fig. 5. Naked smear substrates from the same zone as Fig. 3, as compared with a bloodstained area at the same magnification as Fig. 4. Legend as in Fig. 1. All original magnifications were 1500×. All scale bars are 30 µm.

© 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94– 103


101

Fig. 6. Magnification test for the male’s blood smear on limestone. Some intermediate (2500×, 4000×) or previously shown magnifications (850×, 1500×) are omitted. ori = original magnification.


102

P. H O RTO L À

Fig. 7. Magnification test for the male’s blood smear on flint. The same magnifications as in Fig. 6 are omitted. ori = original magnification.

© 2005 The Royal Microscopical Society, Journal of Microscopy, 218, 94– 103


References Barnhart, M.I., Wallace, M.A. & Lusher, J.M. (1983) Red blood cells. Biomedical Research Applications of Scanning Electron Microscopy, Vol. 3 (ed. by G. M. Hodges and K. E. Carr), pp. 171–243. Academic Press, London. Bessis, M. (1974) Corpuscles. Atlas of Red Blood Cell Shapes. Springer, Berlin. Bull., B.S. & Breton-Gorius, J. (1995) Morphology of the erythron. Williams Hematology, 5th edn (ed. by E. Beutler, M. A. Lichtman, B. S. Coller and T. J. Kipps), pp. 349–363. McGraw-Hill, New York. Castoldi, G.L. (1981) Erythrocytes. Atlas of Blood Cells. Function and Pathology (ed. by D. Zucker-Franklin, M. F. Greaves, C. E. Grossi and A. M. Marmont), pp. 35–145. Ermes/Lea & Febiger, Milan/Philadelphia. Fawcett, D.W. & Raviola, E. (1994) Bloom and Fawcett, a Textbook of Histology, 12th edn. Chapman & Hall, New York. Fiori, A. (1962) Detection and identification of bloodstains. Methods of Forensic Science, Vol. 1 (ed. by F. Lundquist), pp. 243–290. John Wiley & Sons, New York. Geyh, M.A. & Schleicher, H. (1990) Absolute Age Determination. Physical and Chemical Dating Methods and Their Application. Springer-Verlag, Berlin. Goldstein, J.I., Newbury, D.E., Echlin, P., Joy, D.C., Romig, A.D. Jr, Lyman, C.E., Fiori, C. & Lifshin, E. (1992) Scanning Electron Microscopy and X-Ray Microanalysis. A Text for Biologists, Materials Scientists, and Geologists, 2nd edn. Plenum Press, New York. Handley, C.O. Jr & Ponder, E. (1961) Erythrocyte diameters: mammals. Blood and Other Body Fluids (ed. by D. S. Dittmer), pp. 119–120. Federation of American Societies for Experimental Biology, Washington D.C. Hortolà, P. (1992a) SEM analysis of red blood cells in aged human bloodstains. Forensic Sci. Int. 55, 139–159.


103

Hortolà, P. (1992b) SEM characterization of blood stains on stone tools. The Microscope 40, 111–113 (Errata, 40(3), vi). Hortolà, P. (1994) Application of SEM to the study of red blood cells in forensic bloodstains. Microsc. Anal. (UK) 40, 19–21 and Microsc. Anal (EU), 28, 21–23. Hortolà, P. (2001a) Experimental SEM determination of game mammalian bloodstains on stone tools. Environ. Archaeol, 6, 99–104. Hortolà, P. (2001b) Morphological characterisation of red blood cells in human bloodstains on stone: a systematical SEM study. Anthropologie 39, 235–240. Hortolà, P. (2002) Red blood cell haemotaphonomy of experimental human bloodstains on techno-prehistoric lithic raw materials. J. Archaeol. Sci. 29, 733–739. Jain, N.C. (1986) Schalm’s Veterinary Hematology, 4th edn. Lea & Febiger, Philadelphia. LeBlond, P.F. & Shoucri, R. (1978) Calculation of surface area and volume of human erythrocytes from scanning electron micrographs. J. Microsc. 113, 161–170. Loy, T.H. (1998) Organic residues on Oldowan tools from Sterkfontein Cave, South Africa. Dual Congress of the International Association for the Study of Human Paleontology, and International Association of Human Biologists (ed. by M. A. Raath, H. Soodyall, K. L. K. D. Barkhan and P. V. Tobias), pp. 74–75. University of the Witwatersrand Medical School, Johannesburg. Miki, T., Kai, A. & Ikeya, M. (1987) Electron spin resonance of bloodstains and its application to the estimation of time after bleeding. Forensic Sci. Int. 35, 149–158. Rozman, C., Woessner, S., Feliu, E., Lafuente, R. & Berga, L. (1993) Cell Ultrastructure for Hematologists. Doyma, Barcelona.