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Neuropathological examination in forensic context
Forensic Science International 146 (2004) 73–81 www.elsevier.com/locate/forsciint
Neuropathological examination in forensic context Hannu Kalimoa,b,c,*, Pekka Saukkod, David Grahame a
Department of Pathology, University of Helsinki and Helsinki University Hospital, FI-00014 Helsingin yliopisto, Finland b Department of Pathology, University of Turku and Turku University Hospital, FI-20520 Turku, Finland c Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE-75185 Uppsala, Sweden d Department of Forensic Medicine, University of Turku, FI-20520 Turku, Finland e Department of Neuropathology, University of Glasgow, Glasgow G51 4TF, Scotland, UK Available online 27 August 2004
Abstract Different diseases of and trauma to the central nervous system (CNS), as well as their consequences are common causes of death and therefore it is important to examine the CNS appropriately in forensic autopsy, bearing in mind that the site of the disease is often as crucial as its nature. The CNS is a complex organ and its examination requires special methods and knowledge and often consultation with a neuropathologist. The prerequisite for the proper examination is correct handling and processing of the CNS. Because of its soft consistency fixation of the CNS in toto before detailed macroscopic analysis is recommended but guidance for an expedited limited examination is also given. The key features to which attention should be paid during the removal and later macroscopic examination of the CNS are described. Processing CNS for microscopy also requires special techniques and in addition to the routine stains both special histological and selected immunohistochemical stainings are often needed to reach the correct diagnosis. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Central nervous system; Neuropathological examination; Laboratory methods
1. Introduction The central nervous system (CNS) is a complex organ composed of neurons, mostly grouped in the grey matter of the cerebral cortex and deep grey matter nuclei precisely and functionally interconnected by myelinated axons of variable length grouped together into tracts. Therefore, in the examination of the CNS it is as important to know where the lesion is located as it is to identify its nature. The same disease in different locations can cause entirely different symptoms and signs and vice versa different diseases in the same location can cause similar symptoms. Thus, the exact neuropathological examination at post mortem requires that the CNS must be examined and studied appropriately, which * Corresponding author. Tel.: +358 2 261 1685; fax: +358 2 333 7459. E-mail address: [email protected] (H. Kalimo).
may take longer and require more complicated processing techniques than the forensic pathologist and/or the medicolegal system might find acceptable due to lack of time, resources or insight. The practice of forensic examination of the CNS varies in different countries. In most centres the autopsy is performed by the forensic pathologist and the volume of the routine workload may restrict or limit the extent to which the brain may be examined. Therefore, in many centres consultations with colleagues in neuropathology have become routine practice. Only in a few larger centres are there neuropathologists who specialize exclusively in forensic neuropathology. Whatever arrangements exist a prerequisite for the proper examination of the specimen is appropriate handling and processing of the CNS. In this article we describe ways in which information can be gained in the most efficient way. Recommendations are given, how to find the answers to the different questions to which the forensic pathologist must be
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.06.022
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H. Kalimo et al. / Forensic Science International 146 (2004) 73–81
able to respond in order to carry out an appropriate examination of the human CNS.
2. Inspections to be made during the removal of the CNS When the brain and spinal cord are removed during the autopsy, attention should be paid to the following aspects of the examination. 2.1. Soft tissue lesions of the scalp Forensic pathologists are generally experienced in such matters but it should be remembered that minor lesions in the soft tissue of the scalp do not exclude significant intracranial injury, a classic example being subdural haematoma, which may occur in the absence of any external signs of an impact to the head. 2.2. Removal of the skull cap Sawing of the calvaria should be done very carefully, keeping in mind the health and safety regulations. For example, use of handsaw is recommended to minimize aerolization of bone dust in cases of possible infectious diseases, such as Creutzfeldt–Jakob’s disease or AIDS. The dura should be left as intact as possible (a test of the skill of the technical assistant), because that guarantees that the brain itself is not damaged. In cases of suspected or known trauma however extra care is needed not to induce or extend any fractures. If the dura is not cut by sawing the calvaria the
skull cap can usually be removed leaving the dura in situ, although it may be difficult in children and aged individuals, because the dura is firmly attached to the skull cap at the extremes of life. Leaving the dura in situ allows an assessment of the tenseness of the intracranial contents by palpation. After removal of the skull cap the intact dura must be cut along the edge of the sawn bone and between the hemispheres to detach falx and then be elevated to allow the upper surface of the brain to be examined in situ (Fig. 1). It is recommended the brain be taken from the skull and examined immediately after the skull cap has been removed. If the intracranial pressure is markedly increased and major cuts have been made in the dura during sawing of the calvaria, underlying brain tissue or its contents may herniate through the incisions (Fig. 2). Furthermore, when the dura is cut the brain easily falls downwards against the edge of the cut occipital bone which may cause post mortem artefactual damage to the occipital lobes or to the brain stem by stretching. The removed brain should not either be left unsupported on a dissecting board and/or in running water. Leaving the dura on the brain while removing the calvaria offers several additional advantages. One example is that when examining for fractures of the skull cap the dura is already detached from the bone. Other advantages are that when the dura remains attached to the brain at the sagittal dural sinus it can be used to suspend the removed brain in the fixative (see below) and the examination of the sagittal sinus is easy. 2.3. Fractures of the skull bones or the spinal column A fracture is not necessarily a significantly dangerous event, but associated complications such as a haemorrhage
Fig. 1. Brain of a diabetic patient who died after cardiac standstill followed by unsuccessful resuscitation. Note the severe swelling and how easily the posterior part of the soft brain becomes damaged against the edge of the skull bone (arrow). The dura has been removed for photographic purposes, normally it should be just lifted up after cutting parallel to the edge of the bone.
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Fig. 2. The somewhat careless sawing of the calvaria has resulted in damage to the brain tissue and outflow of the pus from the intracerebral abcesses.
or contusion to the underlying brain or spinal cord may be. Most skull fractures are linear, in the detection of which forensic pathologists are usually quite experienced. The classic method in the search of a fracture is percussion of the skull cap to hear the ‘‘cracked pot’’ sound. Visual detection of fractures is most reliable if the dura is removed (preferably already when removing the calvaria, see above) and immersion in water may facilitate. Particular attention is needed for the two uppermost vertebrae—the atlas and axis, especially for disruption of the transverse ligament of the atlas with atlantoaxial subluxation with of without fracture of the odontoid process of the axis. 2.4. Intracranial haematomas and haemorrhages Extra/epidural haemorrhage rarely occurs without a fracture of the skull, and most commonly in association with fracture of the squamous portion of the temporal bone that causes rupture of the middle meningeal artery. In contrast a subdural haematoma may be a consequence of trivial trauma especially in patients on anticoagulant therapy. For histopathological timing of a subdural haematoma the samples should include the edge of the haematoma where the formation of the capsule begins. Subarachnoid haemorrhage is usually also readily visible in the brain in situ, but detailed examination cannot be made until the brain is removed from the skull (Fig. 3).
2.5. Brain swelling and oedema Palpation through the intact dura provides useful information about the likelihood of raised intracranial pressure. Before removal of the brain the possible asymmetry of the hemispheres, bulging due to a haematoma or tumour, the degree of the flattening of gyri and narrowing of sulci over the hemispheres should be assessed visually (Fig. 1). 2.6. Removal and handling of the CNS In many centres technical assistants are allowed to remove the brain from the skull. They should be trained to do it in the correct way (we have a preference to do it ourselves). Since fresh CNS tissue is soft (and often more so when it is diseased) and therefore easily damaged, it must be handled gently and with care. The blood vessels, cranial nerves and pituitary stalk should be cut as soon as they are exposed to prevent artefactual tearing or damage. The tentorium must be cut carefully along the temporal bone remembering that the cerebellum lies beneath it. While supporting the brain the specimen is rotated out after a transverse cut is made through the upper cervical cord and the tentorium. The cervical spinal cord and spinal nerves should be cut as distally as possible, as the information available in the upper cervical cord may be sufficient for diagnostic purposes unless there are lesions in the other parts of the spinal cord. Removal of the upper segments of the
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Fig. 3. In subarachnoid haemorrhage the blood usually accumulates close to the ruptured aneurysm and within basal cisternae. It is usually easier to search for the aneurysm before fixation, when the clot can be rinsed off. The tiny saccular aneurysm is located in left a. cerebri media (arrow) (courtesy of Dr. Matias Ro¨ ytta¨ ).
cervical cord is greatly facilitated through a Y-shaped removal of the occipital bone. The anterior approach to remove the spinal cord takes 5–10 min and gives ready access to the spinal nerves and dorsal root ganglia. During these procedures it is easy to damage the spinal cord and so care must be exercised at all stages of the procedures (for details see below). After removal additional findings may be made at the base of the brain, most of which can be detailed also after fixation. The dura should be stripped from the base of the skull for identification of fractures, source of infection, etc. However, if the whole brain is not fixed the following findings should be recorded in the fresh brain. Since the volume of the skull can expand only in small children with open sutures, brain oedema, tumours, haematomas and other intracranial space occupying processes can increase intracranial pressure (ICP) and displace the midline structures and cause herniation. A frequent sign of increased ICP is a notch in the oculomotor nerve as it emerges between the posterior cerebral and superior cerebellar arteries. Tentorial herniation causes grooving in the parahippocampal gyri on the under aspect of the medial part of the temporal lobe and the width of these grooves should be measured. In herniations of longer duration haemorrhagic necrosis may be seen (Fig. 4). In cases, where a pressure gradient has developed across the foramen magnum the cerebellar tonsils are displaced downward through the foramen and this causes the grooves on the tonsils and if severe and of longer
duration the tonsils may become necrotic. The external herniations through the skull cap become visible before opening the skull, whereas the internal herniations—subfalcine and tentorial—cannot be assessed fully until the brain is sliced. Subarachnoid haemorrhage (Fig. 3) necessitates a search for the most likely cause, i.e. saccular aneurysm, which may be a difficult task. As a rule of thumb the haemorrhage is most often pronounced near the site of leakage, even though blood does spread readily within the subarachnoid space. Search for an aneurysm is best carried out before fixation of the brain. The blood can be rinsed off with isotonic saline or by hydrogen peroxide treatment (‘‘boiling’’; Dr. I. Alafuzoff, personal communication). Some forensic pathologists use the casting method in which radio-opaque polymerizable rubber solution is perfused into the vasculature, which must, of course, be done before opening the skull. The patency and bleeding of the blood vessels can be detected by X-ray as well as visually by the leakage of the brightly coloured substance [1]. This may be very useful and give good results, but on the other hand, it requires a considerable amount of extra work and the plastic makes processing histological slides much more difficult. The spinal cord may be removed using either the anterior or posterior method. Both approaches have their advantages: In the anterior approach the body does not need to be turned over and the dorsal incision is avoided, unless one has to confirm or exclude blunt trauma to the back (as that cannot
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Fig. 4. The brain of a patient who died of herpes simplex encephalitis. The severe oedema and swelling in the frontal and temporal lobes led to transtentorial herniation. In the right mesial temporal lobe there is a notch with haemorrhagic necrosis of the underlying parenchyma (arrow) caused by compression against the edge of the tentorium.
be done reliably without dissection). The posterior approach is easier in that the ribs are not in the way. Whatever method is practised, it is advisable that the technical assistant is given the opportunity to practise the technique in advance, since sawing the vertebral pedicles (anterior approach) or laminae (posterior approach) at the correct angle requires experience. In the anterior approach after removal of the viscera in the chest and abdomen, the lumbar vertebrae are exposed by detaching the psoas muscles. Then two sawcuts are made on each side of the vertebrae along the line of the intervertebral foramina from the sacrum to the level of III–IV cervical vertebrae, at which levels the longitudinal cuts are united by transverse saw cuts. The column of vertebral bodies is removed from below. The cauda equina and spinal nerve roots are cut distal to the spinal ganglia, after which the spinal cord can be raised to the cervical level. The whole upper cervical cord can be removed, if the dura is cut by a circular incision in the spinal canal as high as possible and thereafter the upper cervical spinal roots within this sleeve of dura are cut, most easily with long narrow scissors from above through the foramen magnum. Thereafter the whole upper cervical cord may be removed from below.
3. Complete or incomplete neuropathological examination? 3.1. Incomplete neuropathological examination The authors understand that a rapid and simple examination of the unfixed brain fresh after its removal from the cranial
cavity is usually all that is needed in cases without indications of CNS involvement. The brain should, of course, be always removed with care. Even in cases with neurological symptoms the forensic pathologist may consider the complete neuropathological examination to be too time consuming and cumbersome. Such cases may all too often lead to difficult consultations with neuropathologists, if the specimens consist of small pieces of tissue removed from an unknown site somewhere in the brain in a haphazard direction, and have been fixed and processed as other body tissues. Even an incomplete examination can be undertaken satisfactorily and provides material of acceptable quality in cases which are considered to be of lesser importance. Since the consistency of even a healthy brain is very soft, the fresh brain should be preferably sectioned while it is still cool. The anatomy is easiest to correlate with imaging and figures in neuroanatomy atlases if the cutting is done in the coronal plane into 1–2 cm thick slices. The knife should be sharp and of sufficient width and length (recommended, e.g. Feather Brain Autopsy Blades No. 325) to make even cuts. The regions of interest should then be sectioned with wide margins and placed flat in a container with fixative (see Fig. 5) to be later trimmed to the size of the cassettes. Placing the sample directly into a cassette is suitable only for small samples (e.g. part of a brain tumour). It is important to write down the exact anatomical location from which the sample has been taken. The use of a simple form with anatomical drawings, for example, one with the vascular territories indicated (see Fig. 6 in Stewart et al., in this issue) is recommended. Such mapping is of great importance when charting the various brain pathologies of trauma (see below).
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Fig. 5. A person murdered by overdose of insulin, who survived 2 months after the injection. Both putamina are necrotic (arrows) and there is a general cortical necrosis. In contrast to ischaemic injury the cortex is necrotic not only in the arterial border zones but also in the central parts of the arterial supply territories (see Figs. 5 and 6 in Stewart et al., in this issue).
3.2. Complete neuropathological examination Neuropathologists favour fixation of the brain in toto, which allows better handling and more exact sampling and localization of lesions. If the findings in the brain are pivotal
for the legal assessment, for example, in trauma cases, this is the optimal way to process the brain for obtaining reliable results. Most commonly the brain and spinal cord are fixed by immersion. The best way is to leave the dura attached in the midline of the brain, through which a string can be
Fig. 6. The brain stem of a child who died of severe brain swelling due to hypo-osmolarity syndrome (water intoxication). The markedly increased intracranial pressure caused compression of the brain stem, respiratory arrest and death. The brain stem is narrower than in normal brain (stippled contour).
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inserted for suspending the brain in ample fixative in a bucket (volume of about 10 l). If dura has become detached from the brain, all the brain can be suspended by a string loosely tied around the midbrain or if the brain stem is important for the examination or it has been damaged, in a cap/bag made of thin gauze which allows easy penetration of the fixative. Routine 4% buffered formaldehyde is by far the most commonly used fixative, which we also recommend as the primary fixative. For optimal results the fixative in the bucket should be renewed after 48 h and 10 days. The spinal cord can be placed at the bottom of the bucket. Usually 10– 14 days of fixation is needed, but even after that the inner parts of a severely swollen brain may be poorly fixed and need additional fixation after sampling. For more rapid fixation in a few centres the brain can be perfusion-fixed through the carotid arteries [2]. The problem is to limit the flow of fixative to structures supplied by a particular vascular territory. In particular fixation of the face should be avoided, especially in those countries where embalming and post mortem display of the deceased person is very important (either for the relatives or the undertakers). During perfusion it is important to pay attention to the osmolality of the fixative as higher buffer concentrations than 0.1 mol/l often cause artefactual shrinkage. Microwaving in the fixative has been introduced as another rapid method for fixation, but the authors do not use this method routinely.
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3.3. Brain cutting Although there are variations in practice the methods of sectioning the brain are similar. The brain stem should be detached by a transverse cut rostral to the pons aiming at the groove between the upper and lower colliculi on the dorsal side of the brain stem. Most neuropathologists section the fixed brain in the coronal plane, some relying on a steady hand, others using a frame, e.g. two L-shaped metal or plastic bars of the desired thicknesses (usually 1–1.5 cm thick) flat on a table and a suitable knife. A special rack designed for sectioning the brain has also been used. If done free-hand or with L-frames the first cut is usually placed through the mamillary bodies and the halves are then sectioned towards their poles. In these sections most anatomical features become easily detectable and sampling for microscopy can be done. Many neuropathology laboratories have the facilities and know-how to make even large whole brain sections, which may be useful, for example, in some trauma cases and in generalized diseases. Immunohistochemical and other special stainings of these large sections is expensive, and therefore the normal small blocks are to be preferred. The sampling sites and the number of blocks for histology depend on the problem to be solved (Table 1). In trauma cases minimum of at least 12 different locations need to be sampled for (normal size slides [3]), whereas for verification of a tumour even a single block may suffice. In cases of
Table 1 Sampling in cases of selected neurodegenerative diseases (for screening purposes, for detailed analyses consult, e.g. [6]) and some other disease entities Region to be sampled
Comments
Findings to search for
Frontal cortex, gyrus medius
Includes frontal lobe arterial border zone
Hippocampus
At the level of lateral geniculate nucleus
Basal ganglia
Including putamen, globus pallidum and cauda of ncl caudatus
AD: senile plaques (silver or specific immunostains), NFTs and NTs; DLB: cortical a-synuclein positive Lewy bodies; GCI: ischaemic nerve cell injury in the border zone AD: senile plaques (silver or specific immunostainings), NFTs; DLB: a-synuclein positive Lewy neurites in CA2–3 region; GCI: ischaemic nerve cell injury especially in CA1 sector MSA: necrosis and accumulation of neuromelanin in putamen (a-synuclein positive oligodendroglial inclusions) WE: necrosis of the neuropil with petechial haemorrhages/siderophages; proliferation of capillaries; relative preservation of neurons MSA: oligodendroglial inclusions, degeneration of Purkinje cells; WE: degeneration of vermis PD and DLB: degeneration of pigmented neurons and Lewy bodies in s. nigra; MSA: degeneration of neurons in s. nigra (MSA-P: SND type) PSP: degeneration of pigmented neurons in locus coerulaeus; NFTs in pontine neurons; MSA: degeneration of pontine neurons (MSA-C: OPCA type); central pontine myelinolysis ALS: degeneration of corticospinal (pyramidal) tracts; MSA: degeneration of inferior olivary nuclei (MSA-C: OPCA type) ALS: degeneration of lateral corticospinal tracts, dilated veins of Foix–Alajouanine syndrome (arterio-venous fistula)
Mamillary bodies Cerebellum Midbrain Pons
Medulla oblongata Spinal cord
Two samples, midline vermis and hemisphere with dentate nucleus Includes substantia nigra and cerebral peduncles Includes pontine nuclei and long descending and ascending tracts, as well as locus coerulaeus Includes pyramidal tracts and inferior olivary nuclei
AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; GCI: global cerebral ischaemia; MSA: multiple system atrophy [parkinsonian (MSA-P): striato-nigral degeneration (SND) type and cerebellar (MSA-C): olivopontocerebeller atrophy (OPCA) types]; neuropil: tissue between cell bodies consisting mainly of neuronal and glial processes; NFT: neurofibrillary tangle; NT: neuropil threads; PSP: progressive supranuclear palsy; WE: Wernicke’s encephalopathy.
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Table 2 A list of some useful antibodies Antibody to
Application
a-Synuclein b-Amyloid precursor protein (bAPP) b-Amyloid peptide (Ab) CD31 Different lymphocyte markers CD68 and CD163 Epithelial membrane antigen (EMA) Fibrinogen Glial fibrillary acidic protein (GFAP) Hyperphosphorylated tau-protein Laminin Neurofilament S-100 Synaptophysin and NeuN Ubiquitin
Lewy bodies, inclusions in MSA Axonal pathology in traumatic brain injury Senile plaques (AD), amyloid angiopathy Endothelial cells Inflammatory and haematopoetic malignancies Histiocytes (macrophages), microglial cells Meningeal cells Extravasation of plasma proteins Glial cells (mainly astrocytes and ependymal cells) NFTs, NTs, dystrophic neurites in neuritic plaques, and ‘‘tauopathies’’ Basal laminae of different structures Neurons and their axons Schwann cells, glial cells (among others) Neurons (also primitive and neoplastic) and their axons ALS associated inclusions, Lewy bodies
For abbreviations, see Table 1.
neurodegenerative diseases selected areas should be sampled for screening (Table 1) and complemented when needed according to the clinical suspicions. Ischaemic and hypoglycaemic lesions have definite predilection sites to be sampled (Fig. 5). Epilepsy is more complicated since one should both look for lesions which may cause the epilepsy (e.g. malformations, degenerative alterations and tumours) and lesions resulting from epileptic seizures (e.g. hippocampus and cerebellum). For further details, the readers should consult neuropathology textbooks. It is our practice in cases of traumatic brain injury (TBI) with consent from the legal authorities to take a standardised set of blocks that hopefully when correlated with other information will allow answers to the following questions. (1) (2) (3) (4) (5)
Has there been a head injury? When did it occur? What were the likely mechanisms of injury? What is the nature and distribution of the pathologies? Are there any premorbid lesions that might need to be considered in the clinico-pathological correlations?
The recommended set of blocks for assessing diffuse brain damage including traumatic axonal injury in cases of nonaccidental infant (NAI) head injury are left and right anterior and posterior corpus callosum and associated parasagittal white matter, the medial portions of each temporal lobe, the thalami including posterior limb of the internal capsule, the cerebellar hemispheres, midbrain, upper and lower pons, medulla oblongata and all theupper cervical cord segments [4].
Even the routine stains used for CNS tissue are often somewhat different from those for other tissues and there are several special stains which require experience. The most common general stain is the classic haematoxylin and eosin (H&E), which is often complemented by a stain for myelin (e.g. luxol fast blue and cresyl violet). The vascular and other connective tissue structures are more easily visualised with van Gieson or Herovici stains. The traditional silver staining methods (e.g. modified Bielschowsky [5]) are still very practical and widely used in neuropathology since the axons are beautifully visualized and many of the diagnostic deposits and inclusions of different neurodegenerative diseases are silver-positive and their localization and shape give hints as to their nature. Such are, for example, senile plaques, neurofibrillary tangles, Lewy bodies, Pick bodies and glial inclusions of multiple system atrophy. However, immunohistochemistry in larger measure has replaced many of the classic techniques and allows a more exact characterisation of the different pathologies (a list of some commonly used antibodies are listed in Table 2). It must be emphasized that in immunohistochemistry, for example, specimen fixation, cutting, antibody retrieval methods, antibody dilutions used and selection of secondary antibodies can be extremely critical steps in sample preparation and of paramount importance for reliable results. These methods can rarely be used straight from the ‘‘cook book’’. Therefore a forensic pathology laboratory wishing to produce high quality histopathology slides should always consult an experienced neuropathology laboratory for advice.
3.4. Embedding and stains References Embedding of CNS tissue differs from that of other organs. For example, the high lipid and water content of CNS tissue necessitates longer dehydration times and the paraffin must be of softer quality.
[1] P.J. Karhunen, Neurosurgical vascular complications associated with aneurysm clips evaluated by postmortem angiography, Forensic Sci. Int. 51 (1991) 13–22.
H. Kalimo et al. / Forensic Science International 146 (2004) 73–81 [2] H. Kalimo, J.H. Garcia, Y. Kamijyo, J. Tanaka, J.E. Viloria, J.M. Valigorsky, R.T. Jones, K.M. Kim, W.J. Mergner, R.E. Pendergrass, B.F. Trump, Cellular and subcellular alterations on human CNS. Studies utilizing in situ perfusion-fixation at immediate autopsy, Arch. Pathol. 97 (1975) 352–359. [3] J.F. Geddes, H.L. Whitwell, D.I. Graham, Traumatic axonal injury: practical issues for diagnosis in medicolegal cases, Neuropathol. Appl. Neurobiol. 26 (2000) 105– 116.
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[4] J.F. Geddes, H.L. Whitwell, Head injury in routine and forensic pathological practice in neuropathology. A guide to practising pathologist, in: S. Love (Ed.), Current Topics in Pathology, vol. 95, Springer-Verlag, Berlin, 2001 (pp. 101–124). [5] T.P. Dawson, J.W. Neal, L. Llewellyn, C. Thomas, Neuropathology Techniques, Arnold, London, 2003. [6] D. Dickson (Ed.), Neurodegeneration. The Molecular Pathology of Dementia and Movement Disorders, ISN Neuropath Press, Basel, 2003.
Neuropathological examination in forensic context Hannu Kalimoa,b,c,*, Pekka Saukkod, David Grahame a
Department of Pathology, University of Helsinki and Helsinki University Hospital, FI-00014 Helsingin yliopisto, Finland b Department of Pathology, University of Turku and Turku University Hospital, FI-20520 Turku, Finland c Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE-75185 Uppsala, Sweden d Department of Forensic Medicine, University of Turku, FI-20520 Turku, Finland e Department of Neuropathology, University of Glasgow, Glasgow G51 4TF, Scotland, UK Available online 27 August 2004
Abstract Different diseases of and trauma to the central nervous system (CNS), as well as their consequences are common causes of death and therefore it is important to examine the CNS appropriately in forensic autopsy, bearing in mind that the site of the disease is often as crucial as its nature. The CNS is a complex organ and its examination requires special methods and knowledge and often consultation with a neuropathologist. The prerequisite for the proper examination is correct handling and processing of the CNS. Because of its soft consistency fixation of the CNS in toto before detailed macroscopic analysis is recommended but guidance for an expedited limited examination is also given. The key features to which attention should be paid during the removal and later macroscopic examination of the CNS are described. Processing CNS for microscopy also requires special techniques and in addition to the routine stains both special histological and selected immunohistochemical stainings are often needed to reach the correct diagnosis. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Central nervous system; Neuropathological examination; Laboratory methods
1. Introduction The central nervous system (CNS) is a complex organ composed of neurons, mostly grouped in the grey matter of the cerebral cortex and deep grey matter nuclei precisely and functionally interconnected by myelinated axons of variable length grouped together into tracts. Therefore, in the examination of the CNS it is as important to know where the lesion is located as it is to identify its nature. The same disease in different locations can cause entirely different symptoms and signs and vice versa different diseases in the same location can cause similar symptoms. Thus, the exact neuropathological examination at post mortem requires that the CNS must be examined and studied appropriately, which * Corresponding author. Tel.: +358 2 261 1685; fax: +358 2 333 7459. E-mail address: [email protected] (H. Kalimo).
may take longer and require more complicated processing techniques than the forensic pathologist and/or the medicolegal system might find acceptable due to lack of time, resources or insight. The practice of forensic examination of the CNS varies in different countries. In most centres the autopsy is performed by the forensic pathologist and the volume of the routine workload may restrict or limit the extent to which the brain may be examined. Therefore, in many centres consultations with colleagues in neuropathology have become routine practice. Only in a few larger centres are there neuropathologists who specialize exclusively in forensic neuropathology. Whatever arrangements exist a prerequisite for the proper examination of the specimen is appropriate handling and processing of the CNS. In this article we describe ways in which information can be gained in the most efficient way. Recommendations are given, how to find the answers to the different questions to which the forensic pathologist must be
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.06.022
74
H. Kalimo et al. / Forensic Science International 146 (2004) 73–81
able to respond in order to carry out an appropriate examination of the human CNS.
2. Inspections to be made during the removal of the CNS When the brain and spinal cord are removed during the autopsy, attention should be paid to the following aspects of the examination. 2.1. Soft tissue lesions of the scalp Forensic pathologists are generally experienced in such matters but it should be remembered that minor lesions in the soft tissue of the scalp do not exclude significant intracranial injury, a classic example being subdural haematoma, which may occur in the absence of any external signs of an impact to the head. 2.2. Removal of the skull cap Sawing of the calvaria should be done very carefully, keeping in mind the health and safety regulations. For example, use of handsaw is recommended to minimize aerolization of bone dust in cases of possible infectious diseases, such as Creutzfeldt–Jakob’s disease or AIDS. The dura should be left as intact as possible (a test of the skill of the technical assistant), because that guarantees that the brain itself is not damaged. In cases of suspected or known trauma however extra care is needed not to induce or extend any fractures. If the dura is not cut by sawing the calvaria the
skull cap can usually be removed leaving the dura in situ, although it may be difficult in children and aged individuals, because the dura is firmly attached to the skull cap at the extremes of life. Leaving the dura in situ allows an assessment of the tenseness of the intracranial contents by palpation. After removal of the skull cap the intact dura must be cut along the edge of the sawn bone and between the hemispheres to detach falx and then be elevated to allow the upper surface of the brain to be examined in situ (Fig. 1). It is recommended the brain be taken from the skull and examined immediately after the skull cap has been removed. If the intracranial pressure is markedly increased and major cuts have been made in the dura during sawing of the calvaria, underlying brain tissue or its contents may herniate through the incisions (Fig. 2). Furthermore, when the dura is cut the brain easily falls downwards against the edge of the cut occipital bone which may cause post mortem artefactual damage to the occipital lobes or to the brain stem by stretching. The removed brain should not either be left unsupported on a dissecting board and/or in running water. Leaving the dura on the brain while removing the calvaria offers several additional advantages. One example is that when examining for fractures of the skull cap the dura is already detached from the bone. Other advantages are that when the dura remains attached to the brain at the sagittal dural sinus it can be used to suspend the removed brain in the fixative (see below) and the examination of the sagittal sinus is easy. 2.3. Fractures of the skull bones or the spinal column A fracture is not necessarily a significantly dangerous event, but associated complications such as a haemorrhage
Fig. 1. Brain of a diabetic patient who died after cardiac standstill followed by unsuccessful resuscitation. Note the severe swelling and how easily the posterior part of the soft brain becomes damaged against the edge of the skull bone (arrow). The dura has been removed for photographic purposes, normally it should be just lifted up after cutting parallel to the edge of the bone.
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Fig. 2. The somewhat careless sawing of the calvaria has resulted in damage to the brain tissue and outflow of the pus from the intracerebral abcesses.
or contusion to the underlying brain or spinal cord may be. Most skull fractures are linear, in the detection of which forensic pathologists are usually quite experienced. The classic method in the search of a fracture is percussion of the skull cap to hear the ‘‘cracked pot’’ sound. Visual detection of fractures is most reliable if the dura is removed (preferably already when removing the calvaria, see above) and immersion in water may facilitate. Particular attention is needed for the two uppermost vertebrae—the atlas and axis, especially for disruption of the transverse ligament of the atlas with atlantoaxial subluxation with of without fracture of the odontoid process of the axis. 2.4. Intracranial haematomas and haemorrhages Extra/epidural haemorrhage rarely occurs without a fracture of the skull, and most commonly in association with fracture of the squamous portion of the temporal bone that causes rupture of the middle meningeal artery. In contrast a subdural haematoma may be a consequence of trivial trauma especially in patients on anticoagulant therapy. For histopathological timing of a subdural haematoma the samples should include the edge of the haematoma where the formation of the capsule begins. Subarachnoid haemorrhage is usually also readily visible in the brain in situ, but detailed examination cannot be made until the brain is removed from the skull (Fig. 3).
2.5. Brain swelling and oedema Palpation through the intact dura provides useful information about the likelihood of raised intracranial pressure. Before removal of the brain the possible asymmetry of the hemispheres, bulging due to a haematoma or tumour, the degree of the flattening of gyri and narrowing of sulci over the hemispheres should be assessed visually (Fig. 1). 2.6. Removal and handling of the CNS In many centres technical assistants are allowed to remove the brain from the skull. They should be trained to do it in the correct way (we have a preference to do it ourselves). Since fresh CNS tissue is soft (and often more so when it is diseased) and therefore easily damaged, it must be handled gently and with care. The blood vessels, cranial nerves and pituitary stalk should be cut as soon as they are exposed to prevent artefactual tearing or damage. The tentorium must be cut carefully along the temporal bone remembering that the cerebellum lies beneath it. While supporting the brain the specimen is rotated out after a transverse cut is made through the upper cervical cord and the tentorium. The cervical spinal cord and spinal nerves should be cut as distally as possible, as the information available in the upper cervical cord may be sufficient for diagnostic purposes unless there are lesions in the other parts of the spinal cord. Removal of the upper segments of the
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Fig. 3. In subarachnoid haemorrhage the blood usually accumulates close to the ruptured aneurysm and within basal cisternae. It is usually easier to search for the aneurysm before fixation, when the clot can be rinsed off. The tiny saccular aneurysm is located in left a. cerebri media (arrow) (courtesy of Dr. Matias Ro¨ ytta¨ ).
cervical cord is greatly facilitated through a Y-shaped removal of the occipital bone. The anterior approach to remove the spinal cord takes 5–10 min and gives ready access to the spinal nerves and dorsal root ganglia. During these procedures it is easy to damage the spinal cord and so care must be exercised at all stages of the procedures (for details see below). After removal additional findings may be made at the base of the brain, most of which can be detailed also after fixation. The dura should be stripped from the base of the skull for identification of fractures, source of infection, etc. However, if the whole brain is not fixed the following findings should be recorded in the fresh brain. Since the volume of the skull can expand only in small children with open sutures, brain oedema, tumours, haematomas and other intracranial space occupying processes can increase intracranial pressure (ICP) and displace the midline structures and cause herniation. A frequent sign of increased ICP is a notch in the oculomotor nerve as it emerges between the posterior cerebral and superior cerebellar arteries. Tentorial herniation causes grooving in the parahippocampal gyri on the under aspect of the medial part of the temporal lobe and the width of these grooves should be measured. In herniations of longer duration haemorrhagic necrosis may be seen (Fig. 4). In cases, where a pressure gradient has developed across the foramen magnum the cerebellar tonsils are displaced downward through the foramen and this causes the grooves on the tonsils and if severe and of longer
duration the tonsils may become necrotic. The external herniations through the skull cap become visible before opening the skull, whereas the internal herniations—subfalcine and tentorial—cannot be assessed fully until the brain is sliced. Subarachnoid haemorrhage (Fig. 3) necessitates a search for the most likely cause, i.e. saccular aneurysm, which may be a difficult task. As a rule of thumb the haemorrhage is most often pronounced near the site of leakage, even though blood does spread readily within the subarachnoid space. Search for an aneurysm is best carried out before fixation of the brain. The blood can be rinsed off with isotonic saline or by hydrogen peroxide treatment (‘‘boiling’’; Dr. I. Alafuzoff, personal communication). Some forensic pathologists use the casting method in which radio-opaque polymerizable rubber solution is perfused into the vasculature, which must, of course, be done before opening the skull. The patency and bleeding of the blood vessels can be detected by X-ray as well as visually by the leakage of the brightly coloured substance [1]. This may be very useful and give good results, but on the other hand, it requires a considerable amount of extra work and the plastic makes processing histological slides much more difficult. The spinal cord may be removed using either the anterior or posterior method. Both approaches have their advantages: In the anterior approach the body does not need to be turned over and the dorsal incision is avoided, unless one has to confirm or exclude blunt trauma to the back (as that cannot
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Fig. 4. The brain of a patient who died of herpes simplex encephalitis. The severe oedema and swelling in the frontal and temporal lobes led to transtentorial herniation. In the right mesial temporal lobe there is a notch with haemorrhagic necrosis of the underlying parenchyma (arrow) caused by compression against the edge of the tentorium.
be done reliably without dissection). The posterior approach is easier in that the ribs are not in the way. Whatever method is practised, it is advisable that the technical assistant is given the opportunity to practise the technique in advance, since sawing the vertebral pedicles (anterior approach) or laminae (posterior approach) at the correct angle requires experience. In the anterior approach after removal of the viscera in the chest and abdomen, the lumbar vertebrae are exposed by detaching the psoas muscles. Then two sawcuts are made on each side of the vertebrae along the line of the intervertebral foramina from the sacrum to the level of III–IV cervical vertebrae, at which levels the longitudinal cuts are united by transverse saw cuts. The column of vertebral bodies is removed from below. The cauda equina and spinal nerve roots are cut distal to the spinal ganglia, after which the spinal cord can be raised to the cervical level. The whole upper cervical cord can be removed, if the dura is cut by a circular incision in the spinal canal as high as possible and thereafter the upper cervical spinal roots within this sleeve of dura are cut, most easily with long narrow scissors from above through the foramen magnum. Thereafter the whole upper cervical cord may be removed from below.
3. Complete or incomplete neuropathological examination? 3.1. Incomplete neuropathological examination The authors understand that a rapid and simple examination of the unfixed brain fresh after its removal from the cranial
cavity is usually all that is needed in cases without indications of CNS involvement. The brain should, of course, be always removed with care. Even in cases with neurological symptoms the forensic pathologist may consider the complete neuropathological examination to be too time consuming and cumbersome. Such cases may all too often lead to difficult consultations with neuropathologists, if the specimens consist of small pieces of tissue removed from an unknown site somewhere in the brain in a haphazard direction, and have been fixed and processed as other body tissues. Even an incomplete examination can be undertaken satisfactorily and provides material of acceptable quality in cases which are considered to be of lesser importance. Since the consistency of even a healthy brain is very soft, the fresh brain should be preferably sectioned while it is still cool. The anatomy is easiest to correlate with imaging and figures in neuroanatomy atlases if the cutting is done in the coronal plane into 1–2 cm thick slices. The knife should be sharp and of sufficient width and length (recommended, e.g. Feather Brain Autopsy Blades No. 325) to make even cuts. The regions of interest should then be sectioned with wide margins and placed flat in a container with fixative (see Fig. 5) to be later trimmed to the size of the cassettes. Placing the sample directly into a cassette is suitable only for small samples (e.g. part of a brain tumour). It is important to write down the exact anatomical location from which the sample has been taken. The use of a simple form with anatomical drawings, for example, one with the vascular territories indicated (see Fig. 6 in Stewart et al., in this issue) is recommended. Such mapping is of great importance when charting the various brain pathologies of trauma (see below).
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Fig. 5. A person murdered by overdose of insulin, who survived 2 months after the injection. Both putamina are necrotic (arrows) and there is a general cortical necrosis. In contrast to ischaemic injury the cortex is necrotic not only in the arterial border zones but also in the central parts of the arterial supply territories (see Figs. 5 and 6 in Stewart et al., in this issue).
3.2. Complete neuropathological examination Neuropathologists favour fixation of the brain in toto, which allows better handling and more exact sampling and localization of lesions. If the findings in the brain are pivotal
for the legal assessment, for example, in trauma cases, this is the optimal way to process the brain for obtaining reliable results. Most commonly the brain and spinal cord are fixed by immersion. The best way is to leave the dura attached in the midline of the brain, through which a string can be
Fig. 6. The brain stem of a child who died of severe brain swelling due to hypo-osmolarity syndrome (water intoxication). The markedly increased intracranial pressure caused compression of the brain stem, respiratory arrest and death. The brain stem is narrower than in normal brain (stippled contour).
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inserted for suspending the brain in ample fixative in a bucket (volume of about 10 l). If dura has become detached from the brain, all the brain can be suspended by a string loosely tied around the midbrain or if the brain stem is important for the examination or it has been damaged, in a cap/bag made of thin gauze which allows easy penetration of the fixative. Routine 4% buffered formaldehyde is by far the most commonly used fixative, which we also recommend as the primary fixative. For optimal results the fixative in the bucket should be renewed after 48 h and 10 days. The spinal cord can be placed at the bottom of the bucket. Usually 10– 14 days of fixation is needed, but even after that the inner parts of a severely swollen brain may be poorly fixed and need additional fixation after sampling. For more rapid fixation in a few centres the brain can be perfusion-fixed through the carotid arteries [2]. The problem is to limit the flow of fixative to structures supplied by a particular vascular territory. In particular fixation of the face should be avoided, especially in those countries where embalming and post mortem display of the deceased person is very important (either for the relatives or the undertakers). During perfusion it is important to pay attention to the osmolality of the fixative as higher buffer concentrations than 0.1 mol/l often cause artefactual shrinkage. Microwaving in the fixative has been introduced as another rapid method for fixation, but the authors do not use this method routinely.
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3.3. Brain cutting Although there are variations in practice the methods of sectioning the brain are similar. The brain stem should be detached by a transverse cut rostral to the pons aiming at the groove between the upper and lower colliculi on the dorsal side of the brain stem. Most neuropathologists section the fixed brain in the coronal plane, some relying on a steady hand, others using a frame, e.g. two L-shaped metal or plastic bars of the desired thicknesses (usually 1–1.5 cm thick) flat on a table and a suitable knife. A special rack designed for sectioning the brain has also been used. If done free-hand or with L-frames the first cut is usually placed through the mamillary bodies and the halves are then sectioned towards their poles. In these sections most anatomical features become easily detectable and sampling for microscopy can be done. Many neuropathology laboratories have the facilities and know-how to make even large whole brain sections, which may be useful, for example, in some trauma cases and in generalized diseases. Immunohistochemical and other special stainings of these large sections is expensive, and therefore the normal small blocks are to be preferred. The sampling sites and the number of blocks for histology depend on the problem to be solved (Table 1). In trauma cases minimum of at least 12 different locations need to be sampled for (normal size slides [3]), whereas for verification of a tumour even a single block may suffice. In cases of
Table 1 Sampling in cases of selected neurodegenerative diseases (for screening purposes, for detailed analyses consult, e.g. [6]) and some other disease entities Region to be sampled
Comments
Findings to search for
Frontal cortex, gyrus medius
Includes frontal lobe arterial border zone
Hippocampus
At the level of lateral geniculate nucleus
Basal ganglia
Including putamen, globus pallidum and cauda of ncl caudatus
AD: senile plaques (silver or specific immunostains), NFTs and NTs; DLB: cortical a-synuclein positive Lewy bodies; GCI: ischaemic nerve cell injury in the border zone AD: senile plaques (silver or specific immunostainings), NFTs; DLB: a-synuclein positive Lewy neurites in CA2–3 region; GCI: ischaemic nerve cell injury especially in CA1 sector MSA: necrosis and accumulation of neuromelanin in putamen (a-synuclein positive oligodendroglial inclusions) WE: necrosis of the neuropil with petechial haemorrhages/siderophages; proliferation of capillaries; relative preservation of neurons MSA: oligodendroglial inclusions, degeneration of Purkinje cells; WE: degeneration of vermis PD and DLB: degeneration of pigmented neurons and Lewy bodies in s. nigra; MSA: degeneration of neurons in s. nigra (MSA-P: SND type) PSP: degeneration of pigmented neurons in locus coerulaeus; NFTs in pontine neurons; MSA: degeneration of pontine neurons (MSA-C: OPCA type); central pontine myelinolysis ALS: degeneration of corticospinal (pyramidal) tracts; MSA: degeneration of inferior olivary nuclei (MSA-C: OPCA type) ALS: degeneration of lateral corticospinal tracts, dilated veins of Foix–Alajouanine syndrome (arterio-venous fistula)
Mamillary bodies Cerebellum Midbrain Pons
Medulla oblongata Spinal cord
Two samples, midline vermis and hemisphere with dentate nucleus Includes substantia nigra and cerebral peduncles Includes pontine nuclei and long descending and ascending tracts, as well as locus coerulaeus Includes pyramidal tracts and inferior olivary nuclei
AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; GCI: global cerebral ischaemia; MSA: multiple system atrophy [parkinsonian (MSA-P): striato-nigral degeneration (SND) type and cerebellar (MSA-C): olivopontocerebeller atrophy (OPCA) types]; neuropil: tissue between cell bodies consisting mainly of neuronal and glial processes; NFT: neurofibrillary tangle; NT: neuropil threads; PSP: progressive supranuclear palsy; WE: Wernicke’s encephalopathy.
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Table 2 A list of some useful antibodies Antibody to
Application
a-Synuclein b-Amyloid precursor protein (bAPP) b-Amyloid peptide (Ab) CD31 Different lymphocyte markers CD68 and CD163 Epithelial membrane antigen (EMA) Fibrinogen Glial fibrillary acidic protein (GFAP) Hyperphosphorylated tau-protein Laminin Neurofilament S-100 Synaptophysin and NeuN Ubiquitin
Lewy bodies, inclusions in MSA Axonal pathology in traumatic brain injury Senile plaques (AD), amyloid angiopathy Endothelial cells Inflammatory and haematopoetic malignancies Histiocytes (macrophages), microglial cells Meningeal cells Extravasation of plasma proteins Glial cells (mainly astrocytes and ependymal cells) NFTs, NTs, dystrophic neurites in neuritic plaques, and ‘‘tauopathies’’ Basal laminae of different structures Neurons and their axons Schwann cells, glial cells (among others) Neurons (also primitive and neoplastic) and their axons ALS associated inclusions, Lewy bodies
For abbreviations, see Table 1.
neurodegenerative diseases selected areas should be sampled for screening (Table 1) and complemented when needed according to the clinical suspicions. Ischaemic and hypoglycaemic lesions have definite predilection sites to be sampled (Fig. 5). Epilepsy is more complicated since one should both look for lesions which may cause the epilepsy (e.g. malformations, degenerative alterations and tumours) and lesions resulting from epileptic seizures (e.g. hippocampus and cerebellum). For further details, the readers should consult neuropathology textbooks. It is our practice in cases of traumatic brain injury (TBI) with consent from the legal authorities to take a standardised set of blocks that hopefully when correlated with other information will allow answers to the following questions. (1) (2) (3) (4) (5)
Has there been a head injury? When did it occur? What were the likely mechanisms of injury? What is the nature and distribution of the pathologies? Are there any premorbid lesions that might need to be considered in the clinico-pathological correlations?
The recommended set of blocks for assessing diffuse brain damage including traumatic axonal injury in cases of nonaccidental infant (NAI) head injury are left and right anterior and posterior corpus callosum and associated parasagittal white matter, the medial portions of each temporal lobe, the thalami including posterior limb of the internal capsule, the cerebellar hemispheres, midbrain, upper and lower pons, medulla oblongata and all theupper cervical cord segments [4].
Even the routine stains used for CNS tissue are often somewhat different from those for other tissues and there are several special stains which require experience. The most common general stain is the classic haematoxylin and eosin (H&E), which is often complemented by a stain for myelin (e.g. luxol fast blue and cresyl violet). The vascular and other connective tissue structures are more easily visualised with van Gieson or Herovici stains. The traditional silver staining methods (e.g. modified Bielschowsky [5]) are still very practical and widely used in neuropathology since the axons are beautifully visualized and many of the diagnostic deposits and inclusions of different neurodegenerative diseases are silver-positive and their localization and shape give hints as to their nature. Such are, for example, senile plaques, neurofibrillary tangles, Lewy bodies, Pick bodies and glial inclusions of multiple system atrophy. However, immunohistochemistry in larger measure has replaced many of the classic techniques and allows a more exact characterisation of the different pathologies (a list of some commonly used antibodies are listed in Table 2). It must be emphasized that in immunohistochemistry, for example, specimen fixation, cutting, antibody retrieval methods, antibody dilutions used and selection of secondary antibodies can be extremely critical steps in sample preparation and of paramount importance for reliable results. These methods can rarely be used straight from the ‘‘cook book’’. Therefore a forensic pathology laboratory wishing to produce high quality histopathology slides should always consult an experienced neuropathology laboratory for advice.
3.4. Embedding and stains References Embedding of CNS tissue differs from that of other organs. For example, the high lipid and water content of CNS tissue necessitates longer dehydration times and the paraffin must be of softer quality.
[1] P.J. Karhunen, Neurosurgical vascular complications associated with aneurysm clips evaluated by postmortem angiography, Forensic Sci. Int. 51 (1991) 13–22.
H. Kalimo et al. / Forensic Science International 146 (2004) 73–81 [2] H. Kalimo, J.H. Garcia, Y. Kamijyo, J. Tanaka, J.E. Viloria, J.M. Valigorsky, R.T. Jones, K.M. Kim, W.J. Mergner, R.E. Pendergrass, B.F. Trump, Cellular and subcellular alterations on human CNS. Studies utilizing in situ perfusion-fixation at immediate autopsy, Arch. Pathol. 97 (1975) 352–359. [3] J.F. Geddes, H.L. Whitwell, D.I. Graham, Traumatic axonal injury: practical issues for diagnosis in medicolegal cases, Neuropathol. Appl. Neurobiol. 26 (2000) 105– 116.
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[4] J.F. Geddes, H.L. Whitwell, Head injury in routine and forensic pathological practice in neuropathology. A guide to practising pathologist, in: S. Love (Ed.), Current Topics in Pathology, vol. 95, Springer-Verlag, Berlin, 2001 (pp. 101–124). [5] T.P. Dawson, J.W. Neal, L. Llewellyn, C. Thomas, Neuropathology Techniques, Arnold, London, 2003. [6] D. Dickson (Ed.), Neurodegeneration. The Molecular Pathology of Dementia and Movement Disorders, ISN Neuropath Press, Basel, 2003.