Forensic Sci Med Pathol
DOI 10.1007/s12024-014-9536-9
REVIEW
The role of histopathology in forensic practice: an overview
R. B. Dettmeyer
Accepted: 9 January 2014
 Springer Science+Business Media New York 2014
Abstract The role of forensic histopathology in routine
practice is to establish the cause of death in particular
cases. This is achieved on the basis of microscopic analysis
of representative cell and tissue samples taken from the
major internal organs and from abnormal findings made at
autopsy. A prerequisite of this is adherence to the quality
standards set out for conventional histological/cytological
staining and enzyme histochemical and immunohisto-
chemical methods. The interpretation of histological find-
ings is performed by taking into account macroscopic
autopsy findings and information on previous history.
Histological analysis may prompt postmortem biochemical
and chemical–toxicological investigations. The results of
histological analysis need to be classified by experts in the
context of the available information and the need to
withstand the scrutiny of other experts.
Keywords Forensic histopathology
Histopathological
time estimation
Drug-induced histopathology

Postmortem histopathology
Introduction
Forensic histopathology, performed primarily following
autopsy investigations, has formed an integral part of
diagnosis ever since the inception of forensic medicine [1–
6]. Currently in European forensic medicine, histological
organ and tissue investigations are carried out or ordered
by the authorities [7] in approximately only 50 % of all
R. B. Dettmeyer (&)
Institute of Forensic Medicine, Justus-Liebig University Giessen,
Frankfurter Str. 58, 35392 Giessen, Germany
e-mail: Reinhard.Dettmeyer@forens.med.uni-giessen.de
autopsies; enzyme and immunohistochemical methods are
used even less frequently, while in situ hybridization,
molecular pathological investigations, and electron micro-
scopic diagnosis are less common still. As in general
pathology, microscopy investigations in forensic medicine
serve several purposes:
• To confirm the macroscopic autopsy diagnosis.
• To detect or exclude pathological findings.
• To establish cause of death.
• To detect cells or biological material for further
investigation.
Investigations are often aimed at detecting or confirming
general pathological findings relevant in individual cases to
the cause of death (arteriosclerosis, stenosing coronary
atherosclerosis, obturating coronary thrombus, pulmonary
emphysema, myocardial infarct, cerebral infarct, meningi-
tis, myocarditis, hepatitis, infections such as malaria,
pneumonia, etc.).
In forensic medicine, evidence of vitality at the time of
an event (e.g., infliction of a wound or aspiration of foreign
material) or of the time interval since a finding was caused
(the age of: a wound, myocardial infarction, hemorrhage,
fracture, inflammatory process, cerebral contusion, etc.)
can also be of relevance in an expert forensic medical
appraisal. In such cases, histopathological diagnoses serve
to reconstruct an event and provide additional information
helpful in the overall evaluation; microscopically detected
retinal hemorrhage provides additional evidence of shaken-
baby syndrome, a granulocytic reaction indicates that an
injury was survived, while pathological findings of varying
specificity in the liver or kidneys can be the result of
intoxication.
When, in addition to other investigations, histological
investigations are consistently performed on the major
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Fig. 1 Vaginal swab taken from a 22-year-old woman: abundant
spermatozoa, mostly with sperm tails already detached following an
alleged sex offense (9400)
internal organs following autopsy and on abnormal mac-
roscopic findings, histopathology is able to contribute to
improving cause of death statistics. In some cases, cyto-
logical investigations may be necessary, for example, to
detect spermatozoa, starch granules, or Lycopodium spores,
which can be obtained using laser microdissection fol-
lowing a suspected sex offense [8, 9] (Fig. 1). The detec-
tion of cells on objects, textile fibers in the respiratory tract
in the case of death by asphyxia using a soft cover [10], or
the detection of diatoms in cases where death by drowning
is suspected [11], can also be added to these results.
Thus, against this backdrop, it is hardly surprising that
there are a number of studies on the value of histopathol-
ogy in forensic diagnosis, albeit with varying conclusions
[12–17]. In their 2010 study, de la Grandmaison et al. [12]
demonstrated that macroscopically unidentifiable findings
relevant to the cause of death could be detected histolog-
ically in approximately 40 % of cases and that the cause of
death could be established by means of histology alone in
8.4 % of cases. Histological findings contributed to estab-
lishing cause of death in 13 % of cases, while histology
yielded additional findings in 49 % of cases. Traumatic
lesions could be better documented histologically in 22 %
of cases. In addition to the use of conventional histology in
routine casework, as well as enzyme histochemistry and
immunohistochemistry in specific cases, it should be
pointed out that immunohistochemical methods have been
used increasingly in recent years to answer particular
forensic medical questions, e.g., detection of ischemia
markers in the myocardium and CNS, early detection of
cell and/or tissue necrosis, establishing vitality, and deter-
mining the age of pathological processes.
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Forensic Sci Med Pathol
Methods
Particularly in view of the standards of evidence in crim-
inal proceedings, it is important to note that, like other
diagnoses, histological diagnoses also need to be verifiable
by a second appraiser at any time. To this end, all sections
need to be suitable for evaluation and meet high quality
standards, including:
• The number of specimens per organ needs to be
sufficiently representative for the diagnosis made.
• Slides should include the most important microana-
tomical structures (e.g., heart: epicardium, subepicar-
dial fatty tissue and coronary artery branches,
myocardium, and endocardium; kidneys: fiber capsule,
renal cortex, renal medulla and papillary tip, renal
pelvis and transitional cell epithelium, insofar as this
latter has not undergone postmortem desquamation).
• The stain selected must possess the appropriate staining
properties to correctly visualize the structures to be
analyzed microscopically.
• For special lines of inquiry, a reliable additional stain
should be selected where possible.
• The slide must not exhibit any artifacts that may impair
its evaluability (e.g., slide artifacts, insufficient stain,
excessive formalin pigment, contamination by tissue
displaced during dissection).
• Particularly when forming conclusions on quantity
(e.g., cell count per high power field), it is essential to
ensure the correct section thickness of between 5 and
8 lm.
• Depending on the stain used, performing positive
controls may be required (e.g., in Ziehl–Neelsen
staining to detect Mycobacterium tuberculosis, in
Prussian blue staining to detect iron, or in Congo red
staining to detect amyloid).
• With immunohistochemical markers, particularly when
positive controls are not possible, withdrawal studies
should be carried out in parallel, i.e., once each without
addition of the primary and of the secondary antibodies.
• The commonly used fixative formaldehyde is accept-
able and adequate in many cases, however, the duration
of fixation, particularly at higher concentrations of
formaldehyde, can make immunohistochemical visual-
ization challenging or impossible due to cross-linkage
of the antigens to be visualized, making appropriate
pretreatment or antigen unmasking necessary (e.g.,
boiling in a citrate buffer, pronase pretreatment, etc.).
• In addition, the importance of the level of experience
of the person performing microscopic analysis should
not be underestimated, although interobserver vari-
ability may be seen even between experienced histopa-
thologists.
Forensic Sci Med Pathol
Table 1 Occurrence of enzyme Vital ATPase Esterase
histochemical reactions [22, 23] wound
age (H)
[16 ? ?
[8 ? ?
[4 ? ?
[2 ? ?
[1 ? ?
• A final point: the investigator can only correctly
interpret those microscopic findings which he/she
knows and recognizes.
Adhering to the above points relating to the quality of
specimens for histopathological evaluation also serves to
protect the appraiser from having their expert opinion
contested.
Conventional histological staining
The vast majority of relevant findings and diagnoses can be
easily visualized using conventional histological staining,
generally hematoxylin-eosin staining. In specific cases, the
spectrum of other conventional staining methods should be
additionally applied. The following applications are often
helpful in forensic practice:
• Prussian-blue staining (or iron staining) to detect:
siderophages as an indication of older injuries,
‘‘heart-failure cells’’ in the lung, or evidence that the
subject survived blood aspiration (in which case more
siderophages are present in the alveolar space) [18].
• Grocott staining to detect fungi (fungal conidia, fungal
hyphae) in, e.g., fungal pneumonia, fungal sepsis, and
fungal colonization of decubitus ulcers.
• Alcian blue staining to detect acid mucopolysaccha-
rides in the aortic wall in dissecting aortic aneurysms
due to idiopathic medial necrosis (combined with
elastica staining to visualize ruptured or irregular
elastic fibers in the aortic wall).
• Sudan III staining (lipid staining) to detect fat embo-
lism in the lungs, the glomerular capillary loops of the
kidneys or in the brain, as well as to grade hepatic
steatosis and visualize fatty degeneration of epithelial
cells in the renal tubules in the case of death by
hypothermia [19].
• Lugol’s staining to detect vaginal epithelial cells
[20].
• PAS staining/Best’s carmine stain to detect glycogen-
positive cells in renal tubular epithelial cells (Arman-
ni–Ebstein cells) in the case of death in diabetic coma
[21].
Amino- Acid- Alkaline Many Many mono-
peptidase phosphatase phosphatase polymorpho- nuclear cells
nuclear cells
? ? ? ? ?
? ? ? ? -
? ? - - -
? - - - -
- - - - -
Enzyme histochemical staining
Despite being the subject of many studies in the past,
enzyme histochemical methods have gained only scant
acceptance in routine practice. Raekallio [22], for example,
investigated a variety of enzyme markers for the purposes
of determining wound age (see Table 1).
As with immunohistochemical wound age determina-
tion, there are also open questions regarding the validity of
findings resulting from enzyme histochemical methods:
• How representative is the result for the wound overall?
• How many specimens per wound need to undergo
microscopic analysis in order to ensure a reliable
diagnosis?
• Are results reproducible depending on wound age or
postmortem interval?
Naphthol AS-D chloroacetate esterase stain (ASD) is rec-
ommended as an aid to determining the vitality of an injury
and estimating wound age. This staining method enables
neutrophil granulocytes to be readily detected in both fresh
and older wounds, according to my experience, even after a
postmortem interval of several days.
Immunohistochemical methods
In the case of very particular lines of forensic medial
inquiry, the immunohistochemical detection of specific
antigens can be extremely helpful to establishing a diag-
nosis. Examples taken from routine practice include:
• Anti-cytokeratin to assess the extent of amniotic fluid
aspiration, as well as to detect amniotic fluid embolism
[24].
• Anti-IgE to underpin the diagnosis of allergic anaphy-
lactic shock (combined with the detection of eosinophil
granulocytes and degranulated mast cells) [25].
• Detection of C5b-9(m) as an early, relatively autolysis-
resistant necrosis marker in myocardial infarction [26].
• Combinations of immunohistochemically detectable
antigens are recommended as ischemia markers, such
as myoglobin, desmin, cardiac troponin I, and C5b-
9(m) as myocardial ischemia markers [27].
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Fig. 2 Compartment syndrome and rhabdomyolysis following intra-
venous drug use and immunohistochemical detection of myoglobin in
the renal tubules of a 32-year-old deceased male (long-term i.v. drug
use) (anti-myosin 9200)
• Anti-myoglobin to detect rhabdomyolysis including
visualization of myoglobin cylinders in the renal
tubules [28] (Fig. 2).
• b-APP to identify diffuse axonal injury (DAI), e.g.,
following shaken-baby syndrome or in drug-related
deaths [29].
Antibodies against defined pathogens (viruses, bacteria,
fungi) for use in immunohistochemical investigations are
only available to a limited extent. According to my experi-
ence (with molecular-pathological controls on pathogenic
RNA), an immunohistochemical antibody against enterovi-
ruses failed to produce reproducible results. In contrast, an
immunohistochemical antibody against cytomegaloviruses
proved to be highly reliable, as confirmed by a control using
in situ hybridization [30]. However, epithelial cell inclusion
bodies typical of cytomegaly can also be diagnosed using
conventional histology (Fig. 3), e.g., in the parotid gland in
cases of suspected sudden infant death syndrome (SIDS). A
multitude of detection methods ranging from conventional
staining to immunohistochemical methods are discussed in
the literature for complex inflammatory processes and reac-
tions of the organism, extending to shock. The decision
regarding the number of investigations needs to be made on a
case-by-case basis. Where immunohistochemical methods
are used, pretreatment for the purposes of antigen unmasking
in paraffin-embedded tissue specimens may be useful. The
spectrum of methods for antigen unmasking ranges from
proteolytic autodigestion (trypsin, pronase, pepsin, etc.),
heating in a citrate buffer/aluminum chloride, and wet-auto-
claving, to heating in a urea solution [19–34]. Table 2 shows
a selection of other immunohistochemical primary antibodies
used for diagnostic purposes in daily forensic practice.
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Forensic Sci Med Pathol
Fig. 3 Inclusions typical for cytomegaly in the epithelium of the
parotid gland detectable using conventional histology; a 4-month-old
deceased male infant with suspected sudden infant death syndrome
(H&E 9400)
Histopathological findings in specific forensic medical
case groups and investigations
Dealing with often sudden, violent, and non-violent deaths
is at the forefront of routine forensic practice. To this can
be added fatalities where the background is unknown or in
which a malpractice claim is brought against the treating
physician. In this context, it is possible to define case
groups that are dealt with either exclusively or primarily by
forensic medicine:
• Homicide by various means (shooting, stabbing,
drowning, beating to death, poisoning, etc.).
• Deaths associated with the consumption of alcohol,
including the consumption of supposed alcohol substi-
tutes, such as ethylene glycol [35–38] or the consump-
tion of impure drinks with, for example, an excessively
high methanol content.
• Fatalities following the use of drugs other than alcohol
(heroin, cocaine, amphetamines, prescription medica-
tion abuse, in particular psychotropic drugs, as well as,
for example, anabolic steroids, etc.).
• Fatalities due to the effects of heat and fire, as well as
hypothermia.
• Intoxications in the context of suicide or accidental
intoxication, e.g., following the consumption of poi-
sonous mushrooms (Fig. 4).
• Sudden unexplained death in neonates and infants
(SIDS).
• In general, sudden fatalities of unexplained (often
natural) cause (sudden death during sports, etc.).
• Other cases of sudden death, e.g., by electric shock and
lightning strike.
Forensic Sci Med Pathol

Table 2 A selection of immunohistochemical primary antibodies and their uses in forensic investigation
Antibodies Target structure/localization Problem

Adhesion molecule, e.g., ICAM-1, VCAM-1 Surface membranes, especially on endothelial
cells for cell–cell interaction
Chromogranin A Enterochromaffin cells, neuroendocrine
tumors
LCA (CD45) Pan-leukocyte marker (leukocyte common
antigen)
Collagens Basal membrane component
Laminin Basal membrane component
CD3 T-lymphocytes
CD45R0 Activated T-lymphocytes
Anti-IgM Immunoglobulin type IgM
Anti-IgG Immunoglobulin type IgG
Anti-fibronectin Fibronectin
Anti-C5b-9(m) complement Complement factor C5b-9(m)
Anti-fibrinogen Fibrinogen
CD68 Macrophages
Desmin Smoothly and horizontally striped muscle
cells, myocardial structure protein
Tenascins Extracellular matrix glycoproteins
Cytokines: generic term for peptide mediators In some cases, many somatic cells, among
with a biological effect on cells, especially others vascular endothelial cells, different
interleukins, interferons, chemokines, TNF- types of leukocytes, including
a, TGF-b, colony-stimulating factors (CSFs) T-lymphocytes, monocytes, macrophages,
T-helper cells, stroma cells, etc.
Heat shock proteins (HSP) Different proteins which help other proteins
maintain their secondary structure; this
means protecting cellular proteins from
denaturation
Vimentin Intermediate filament of mesenchymal cells
(e.g., fibroblasts, endothelial cells, smooth
muscle cells)
Selectins (E-, P-, and L-selectin) E-selectin in plasma membranes of
endothelial cells, P-selectin in endothelial
cells and thrombocytes, L-selectin is made
by all leukocytes: surface molecules to
organize leukocyte invasion: rolling,
trapping, diapedesis
There are numerous other antibodies that have not been checked for suitability in a forensic context, but which are used in individual forensic
studies for defined problems
Activation of leukocyte invasion with
inflammatory processes
Pheochromocytoma
Inflammatory processes
Intact basal membranes
Intact basal membranes
Viral infections
Viral infections
Immunoglobulin deposit in glomerulus loops
with heroin-associated nephropathy
Immunoglobulin deposit in glomerulus loops
with heroin-associated nephropathy
Early myocardial necrosis
Early necrosis marker, e.g., in myocardial
infarct
Early necrosis marker, e.g., in myocardial
infarct
Cellular removal reaction in wound age
determination
Absent in the case of myocardial necrosis
Repair processes around healing lesions,
including myocardial necrosis
For example: increased expression in
inflammatory processes, activation factors
for natural killer cells etc.; thus, TNF-a is
produced primarily by monocytes/
macrophages
Increased expression following cellular stress
caused by heat, radiation, toxins, etc.
Wound healing in skin lesions
Pro-inflammatory marker, in inflammatory
processes

• Protracted death following serious injury (traffic acci-
dents, other severe trauma, late effects such as embo-
lism, sepsis, hemorrhage, etc.).
• Fatalities involving allegations that medical malprac-
tice led to a patient’s death.
The importance of histological investigations needs to be
seen against the backdrop of these relatively specific
forensic medical groups, while bearing the validity of
evidence in criminal proceedings in mind! In this context,
the spectrum of histological, enzyme histochemical,
immunohistochemical, and other microscopy investiga-
tions can often yield diagnoses crucial to establishing cause
of death, chronology of disease processes, wound vitality,
intoxication, and postmortem intervals of varying length.

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Fig. 4 A 46-year-old deceased male: significant cell and nuclear
polymorphism in gastric mucosa epithelial cells following death cap
intoxication (H&E 9400)
However, autolysis and putrefaction are limiting factors
that need to be considered in forensic practice and also in
histopathological diagnosis; also of relevance is the ques-
tion of whether histological findings on exhumation fol-
lowing a postmortem interval of several weeks or in some
cases years can still be evaluated. Table 3 lists a selection
of histological findings that can still be made following
lengthy postmortem intervals.
However, the quality of histopathological diagnosis also
depends on the quality of information on previous history
(type of injury, circumstances of death, drug and medica-
tion use, etc.). Certain grades and classifications can be
helpful when formulating a forensic medical expert opin-
ion, if additional information is available at the same time.
Quantifying pulmonary fat embolism
Pulmonary fat and bone marrow embolisms are seen fol-
lowing, for example, traffic accident-related trauma
(increasingly so after longer survival times), as well as in
the setting of surgical procedures such as total endopros-
thesis following femoral neck fracture, usually in elderly
individuals. In some cases, fat and bone marrow embolisms
may be the immediate cause of death if extensive embo-
lism can be established histologically, combined with signs
of acute right heart failure or existing cardiac damage
found at autopsy. The classification according to Falzi
et al., modified from Janssen, can be helpful here [1, 19]
(Table 4). In all cases of proven pulmonary fat embolism,
particularly when a victim is known to have survived for a
short period of time, it is necessary to check whether fat
embolism to the brain and capillary loops of the renal
glomerulus can be detected, particularly in the case of
antemortem kidney failure.
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Forensic Sci Med Pathol
Determining the age of a thrombus
or thromboembolism
It may be of interest to determine the age of a thrombus or
thromboembolus in the case of a two-staged onset of symp-
toms reported in the medical history or for comparison with a
given time of a traumatic effect. Primary orientation is taken
here from signs of thrombus organization, starting from the
vascular wall. Orienting statements on the age of a thrombus
are often possible using conventional histological staining,
i.e., fresh, recent but no longer fresh, somewhat older (days–
weeks), and old (largely organized). Where possible, several
specimens are needed from the region of the vascular wall to
which the thrombus adheres. Alongside other histological
criteria, the first step is to check for continuity of the basal
membrane, fibroblast and fibrocyte invasion, and the
appearance of siderophages, as well as looking for branched
capillary blood vessels [40]. Other histological criteria can
include the detection of a fine fibrin fiber network at the
center of the thrombus or thromboembolus and the suscep-
tibility of the cell nuclei of enclosed leukocytes to staining.
Determining the age of myocardial infarction
Literature data on the histopathological determination of
myocardial infarct age vary [41]. Thus, taking specimens from
the peripheral region of the infarct, as well as from its center, is
always advised. Electron microscopic changes have been
described in the early phase of myocardial infarction (up to
60 min), while immunohistochemical findings of myoglobin
loss and fibrinogen detection have been described in animal
models [42]. Contraction band necrosis in chromotrope anilin
blue staining (CAB) as an expression of collapse of the myo-
fibril apparatus has also been described [43]; where this is the
case, hemorrhagic demarcation of the infarct area may have
already been visible macroscopically. Homogeneous eosino-
philic areas in cardiomyocytes appear from around 2–3 h [5].
Unequivocal immunohistochemical findings with early
necrosis markers such as C5b-9(m) and fibrinogen in the case of
simultaneous desmin and myoglobin loss can be expected from
a myocardial infarct age of 4–5 h. Using light microscopy, the
necrotic zone in the infarct region then becomes increasingly
distinct with darkened cell nuclei and patent granular degra-
dation of myocardial cells; this is also seen at the infarct center
for up to 9 h. Parallel to this, cellular infiltration by leukocytes
begins at the margins and may also be detected in the central
sections of the myocardial infarct after around 24 h, where a
dense leukocyte infiltrate may form over subsequent days
(increasing the risk of cardiac wall rupture). Well-differentiated
granulation tissue as an expression of infarct organization is
seen after approximately 2–3 weeks, with branched capillary
blood vessels, fibrocytes, fibroblasts, lymphocytes, macro-
phages, possibly siderophages and scant granulocytes. Later,
Forensic Sci Med Pathol

Table 3 Detectability of histological findings according to post- Table 3 continued

mortem intervals taken from a selection of data in the literature (for
further examples and related literature references see [39]) Finding Approximate
postmortem period
Finding Approximate
postmortem period Fatty degeneration of the liver 3 months
10 years
Electrical burn 3 weeks
Fatty liver hepatitis 2 weeks
Peritonitis, sepsis, septicopyemia 17 days/42 days
Septic spleen 8 days
Polynuclear alveolar cells 15 days
16 days
Early cell infiltration 65 days
Amniotic fluid components 4.5 months
Chronic meningitis 52 days
Reticulum cell sarcoma 16 months
Coronary thrombosis and myocardial 90 days
fibrosis Expanded lung tissue in a newborn caused 4.5 months
by breathing
Bronchopneumonia 133 days/95 days
Interstitial lung fibrosis 223 days
Immunohistochemical finding with 392 days
myeloperoxidase Keratinizing squamous cell lung cancer 43 days
19 months
Brain metastasis of small-cell bronchial 73 days
Brain metastasis of lung cancer 44 days cancer
Liver metastasis of a hemangiosarcoma 27 days Immunohistochemical evidence of Negative from 14 days
Ganglion and glial cells 114 days glucagon postmortem
1,212 days Immunohistochemical evidence of Negative from 13 days
Positive evidence of iron in the tissue 1–2 years calcitonin postmortem
Fat embolism 8–10 days
(experimental data)
Table 4 Classification of pulmonary fat embolism (evaluation at
4–8 weeks
1009 magnification)
4.5 months
Coronary thrombosis 8 months Extent of Form of fat Localization of fat embolism
embolism embolism
3.5 months
3.9 months I: Mild fat Teardrop-like Scattered, but at 25-fold
96 days embolism magnification in every field of
vision
Acute myocardial infarction detected 11 days
immunohistochemically with necrosis II: Distinct Lake- or sausage- Multiple fat emboli,
6 weeks fat shaped disseminated in every field of
marker C5b-9(m) and NP57 (indicates
12 months embolism vision
neutrophil leukocytes)
63 days, 487 days III: Massive Fat emboli with Visible in huge numbers in all
128 days fat antler-like regions, no field of vision
embolism configuration without fat emboli
Liver fibrosis 4.5 months
0: No fat Punctiform where Possibly visible in isolation,
Shock liver 6 months
embolism relevant never in all fields of vision
Glomerulonephritis 3.2 months
Cervical artery dissection 20 months
Former cerebral contusion 3.5 weeks scar tissue develops (2–3 months), which exhibits decreased
Tracheitis 2 weeks cellularity and which, in some cases (usually after years) may
Epicarditis 3.75 months calcify. Although the timeline of myocardial infarction appears
Myocardial fibrosis or scarring 2.5 years well-differentiated, somewhat less precise descriptions tend to
2 years be chosen in forensic practice: fresh, no longer completely
Pulmonary amyloid bodies 3.5 years fresh, several days, weeks, months. Time data of this kind
Pneumonia 3.75 months should be compared with macroscopic findings in the heart and
Immunohistochemical detection of 1.1 year in the previous medical history (for comprehensive details, see
neutrophil granulocytes with NP57 24 months [44]).
Tuberculosis 1.5 months
10 months Grading hepatic steatosis and/or liver fibrosis
Pulmonary fat embolism 4.5 months and cirrhosis
1–2 months
1.2 months
Chronic alcohol abuse can result in increasing hepatic
steatosis together with progressive liver fibrosis and liver

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cirrhosis. In the case of detectable hepatic steatosis and
inflammation with hepatocellular single-cell necrosis, one
can assume, following exclusion of viral hepatitis and the
effects of toxins other than alcohol, that alcohol con-
sumption was continued until relatively shortly before
death, whereas hepatic steatosis is considered to be largely
reversible following prolonged abstention from alcohol.
The degree of alcoholic and non-alcoholic hepatic steatosis
can be estimated using microscopy methods (mild, mod-
erate, severe), whereby some investigators give the extent
of fatty degeneration of liver cells as a percentage [45]. By
changing from the overview magnification to a stronger
magnification, smaller fat vacuoles in hepatocyte cyto-
plasm often also become visible, thereby significantly
affecting the grade of hepatic steatosis. Thus, it is not
surprising that considerable interobserver variability may
be seen in the grading of hepatic steatosis in certain cases.
The same is true of fibrosis grading. However, it is
accepted that connected fibrous strands arising from the
portal fields and pseudolobuli formation indicate liver cir-
rhosis. Depending on the degree of activity of the process
and the size of pseudolobuli, liver function may still be
adequate. Only in advanced cases are broad fibrous zones
with bile duct proliferation and a concomitant inflamma-
tory response seen, often accompanied by fatty degenera-
tion of the remaining hepatocytes in the pseudolobuli.
Retained bile pigment is often the easiest to visualize using
Prussian blue staining. In forensic practice, liver cirrhosis
is a macroscopic diagnosis, while histopathological meth-
ods can help to underpin the suspicion of recent alcohol
consumption, exclude hepatocellular or cholangiocellular
liver cancer, and form conclusions relating to disease
activity. Alcoholic pancreatitis and alcoholic cardiomyop-
athy [46] are topics in the further morphologic effects of
alcohol consumption.
Diagnosing shock of varying etiology
Histopathological findings can document a shock reaction
of the organism, without permitting any conclusions to be
drawn on the cause of shock. Worthy of note here are, for
example, centrilobular necrosis in the liver, so-called
hyaline membrane formation in the lungs (possibly already
showing signs of organization), and megakaryocyte
emboli. Abundant eosinophil granulocytes and degranu-
lated mast cells point to anaphylactic shock, while the
absence of congestion in internal organs points to hemor-
rhagic-hypovolemic shock. Activation of the lymphatic
organs, particularly when bacterial pathogens are con-
comitantly detected, is suggestive of septic shock, as are
extensive fibrinous pleuritis or peritonitis. Isolated fibrin-
ous pericarditis is sometimes seen in the setting of uremic
shock. Other findings, including immunohistochemical
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Forensic Sci Med Pathol
findings, in shock fatalities have been described primarily
for septic shock [47–49].
Interpreting findings of varying characteristicness
Occasionally in routine forensic diagnosis, one sees path-
ological findings for which no precise cause can be iden-
tified. Thus, hepatic steatosis displaying fine vacuolation in
a case of suspected sudden infant death should also prompt
consideration of MCAD syndrome. Fine or coarse hepa-
tocyte vacuolation negative for Sudan-III staining, how-
ever, may be the result of chronic intoxication and has been
observed, for instance, in a case of chronic intoxication
with colchicine and beta-blockers (metoprolol). More dis-
crete findings in the liver of single-cell necrosis or mild
inflammatory responses accompanied by activated Kupffer
cells can indicate non-specific drug-induced hepatitis.
Characteristic findings may be detected in ethylene glycol
intoxication (crystalline deposits, e.g., in the renal tubules),
while extensive liver necrosis is seen in mushroom poi-
soning (in which case, the gastric contents should be
investigated microscopically, e.g., using Grocott staining).
No studies on the microscopic identification of gastric
contents, including information on the composition of the
final meal of a deceased individual, have been published in
recent years. Table 5 provides data on relatively common
and largely characteristic histopathological findings in
forensic practice.
The examples given in Table 5 demonstrate histopa-
thological findings that, in the context of an expert opinion,
need to be classified against the backdrop of further
information or findings, but which may also in themselves
prompt investigation. Common findings in general
pathology (myocardial infarction, cerebral infarction, acute
bronchial pneumonia, lobar pneumonia, its characteristic
phases, etc.) are not discussed here in detail.
Discussion
In forensic medicine, as in related fields, quality issues
concerning the processing and staining of tissue specimens
need to be considered. Some countries already have
accreditation requirements for work carried out in histo-
logical laboratories, which also cover the use of immuno-
histochemical methods. In addition to the issue of quality
in specimens for microscopic analysis, the question of
reliability of histopathological diagnoses in forensic rou-
tine in view of the strict standards of evidence, particularly
in criminal proceedings, needs to be considered. A crucial
aspect here is the number of specimens to be analyzed for
the purposes of establishing or excluding a diagnosis. Thus,
Rasten-Almqvist et al. [62] recommend that six specimens
Forensic Sci Med Pathol
Table 5 A selection of histopathological findings of particular relevance in forensic practice and in the determination of sudden unexpected
death
Finding
Peliosis hepatis [50]
Focal nodular hyperplasia
Vacuolization of the respiratory epithelium and elongation of basally
located nuclei [51]
School of fish-like pattern of nuclear elongation in basal cell layers of the
epidermis, possibly at the margins of areactive detachment of the
epidermis and homogenization in the superficial corium [52]
Multiple granulomas located in the perivascular region of the lungs with
multinucleated giant cells [53]
Substantial hemosiderin deposition in the liver
Decidual stromal reaction and Arias–Stella reaction in endometrial
epithelium
PAS-positive Armanni–Ebstein cells in the renal tubules, also readily
detected using Best’s carmine stain [21]
Birefringent crystalline deposits in the renal tubules, stained black with
von Kossa stain [54]
Immunohistochemical detection of myoglobin in the renal tubules
Extremely narrow, elongated alveolar walls due to pulmonary emphysema
with bloodless interalveolar capillaries
Fine particles of blackish-gray material at the margins of destroyed tissue
Abundant eosinophil granulocytes and (degranulated) mast cells
Yellowish pigment in the hepatic sinuses
Epithelioid-cell granulomatosis of the liver
Focal, relatively well-demarcated lympho-monocytic inflammatory
necrosis advancing toward the myocardium [55, 56]
Enlarged and hyperchromatic cell nuclei in the epithelium of the intestinal
mucosa with mitoses [57–59]
Detection of fungal conidia and fungal hyphae in gastric contents
Multiple lung granulomas with foreign body-type multinucleated giant
cells, not located in the perivascular space
Subepidermal blister formation with striated hemorrhage
Acute extensive diffuse liver tissue necrosis [60]
Accumulation of immunohistochemical IgE-positive cells in the bronchial
wall
Abundant starch granules, stained deep blue with Lugol’s solution [20]
Retinal hemorrhage [61]
Diffuse lymphocytic infiltration of thyroid tissue with inflammatory cells
between the follicular epithelium and thyroid colloid depletion
Colloid-depleted thyroid follicle and high follicular epithelium with a
papillary structure and abundant intra-colloidal absorption vacuoles
Irregular or whirlpool/storiform pattern of cardiac muscle fibers, possibly
accompanied by marked vascular wall and endothelial cell abnormalities
Forensic relevance
Uncharacteristic but common in long-term intravenous drug use
Common following use of steroids, contraceptive agents, and
anabolic drugs
Heat inhalation injury, sometimes with detection of soot particles on
the respiratory epithelium
Electrical marks, possibly with metallization
‘‘Junkie pneumopathy’’ (pulmonary granulomatosis) following
long-term i.v. drug use
‘‘Transfusions siderosis’’; an iron metabolism disorder
(hemochromatosis) should be considered in the differential
diagnosis
Evidence of previous pregnancy
Suspected diabetic coma
Suspected intoxication with ethylene glycol and oxalosis
(birefringent oxalate crystals)
Suspected muscle injury and rhabdomyolysis
Suspected emphysema aquosum
Suspected gunshot residue following gunshot wound
Suspected allergic-anaphylactic shock
Suspected intravenous use of methadone following colorant
addition (soluble colorant with yellow quinoline)
Suspected drug-induced hepatitis
Suspected drug-induced myocarditis (following use of e.g.,
clozapine, diclofenac, or other medications?)
Suspected colchicine intoxication
Status following mushroom consumption, suspected mushroom
poisoning
Status following survived aspiration of foreign material (chyme)
and foreign body-induced absorption
Suspected drug-induced skin reaction (toxic epidermal necrolysis or
Lyell’s syndrome, also referred to as burned skin syndrome)
Suspected intoxication (e.g., amanita poisoning, propane gas
inhalation)
Suspected acute allergic asthma
Starch granules in a vaginal swab following vaginal intercourse
using a condom
Shaken-baby syndrome
Suspected Hashimoto thyroiditis
Suspected hyperthyroidism due to Grave’s disease
Suspected or detected (genetic) cardiomyopathy
123

should be taken for the histological analysis of the heart,
whereas according to my own experience [63], taking eight
myocardial specimens from defined locations is advised for
the purposes of conclusively excluding myocarditis (see
also [64]). In the case of equivocal microscopic findings,
analyzing additional specimens and/or producing addi-
tional tissue sections from already selected tissue speci-
mens is recommended. In terms of ensuring that
histological findings are representative, taking fine-needle
biopsies alone (e.g., in the context of radiological diagno-
sis) in cases where no autopsy is performed, is under no
circumstances sufficient to either reliably confirm or
exclude relevant findings.
Dispensing with histological investigations can some-
times lead to allegations of failure to duly exclude com-
peting causes of death. In other cases, the decision to
dispense with additional histological findings needs to be
supported in the expert opinion. Naturally, histopatholo-
gical findings are of less relevance when establishing cause
of death in cases of obvious homicide (fatal gunshot
wound, death due to stab wounds, death due to ligature or
manual strangulation, and obvious suicide). In this context,
their relevance lies in determining vitality and the length of
time a victim survived their injuries, as well as in
answering questions about previous history, e.g., additional
alcohol-related liver damage, histopathological signs of
drug use, and pre-existing diseases, such as malignant
cancer as a motive for suicide, etc.
Ultimately, it is up to the individual expert to decide the
extent of diagnostic measures he/she deems necessary to for-
mulate an expert opinion and is prepared to support vis-a-vis the
public prosecutor’s office and court. In the case of homicides
where cause of death is clear, it may be reasonable to dispense
with microscopic investigations; on the other hand, histological
investigations are mandatory when dealing with medical mal-
practice claims and pre-existing disease and possible known
multimorbidity in the deceased. If cause of death cannot be
established macroscopically, histological diagnosis needs to be
considered alongside toxicological investigations. Indeed,
there are cases of a supposedly unequivocal diagnosis that need
to be revised following microscopic investigations; examples
include the macroscopic diagnosis of meningitis that proves to
be meningeal lymphoma, or supposed lethal coronary sclerosis
that turns out on histology to be cardiovascular amyloidosis.
Other examples similarly serve to underpin the value of autopsy
and histological investigations, such as sudden death due to
undiagnosed malaria [65] or undiagnosed pheochromocytoma.
Moreover, sudden deaths due to, for example, infectious
mononucleosis [66] or cardiac sarcoidosis [67], can only be
explained with the help of histological diagnosis. However, it is
always necessary to view fatalities in the context of all available
information. Thus, increased heart weight and histological
evidence of myocardial hypertrophy and hepatic focal nodular
123
Forensic Sci Med Pathol
hyperplasia in a relatively young, muscular man should prompt
consideration of anabolic steroid misuse, without each indi-
vidual (histo-) pathological finding being conclusive alone; in
such cases, previous medical history and toxicological findings
should be included in an expert opinion. Other questions posed
in forensic medicine relate to sudden unexpected death in
patients with pre-existing diseases such as trisomy 21 or Marfan
syndrome [68], among other case groups. Lastly, detecting and
determining the age of neurological lesions or cerebral injuries
can be helpful when formulating an expert opinion [69–72], as
can determining the (approximate) age of skin wounds [73–76],
subdural hematomas, and fractures [77].
Although histopathological investigations alone are often
not able to establish cause of death, they can provide addi-
tional information crucial to the interpretation of findings as a
whole, for example, in overwhelming postsplenectomy
infection syndrome or to confirm inhalation of volatile toxic
substances [78]. Particular experience in microscopy is
required to diagnose neuropathological lesions of forensic
relevance, e.g., the detection of hypoxic cerebral tissue injury
using immunohistochemical methods [7, 79]. The spectrum
of primarily forensic histopathological diagnostic methods
discussed here is not exhaustive; for forensic appraisals in
particular, other conventional histological and, more partic-
ularly, immunohistochemical studies are required to gain new
information and ensure the reliability of forensic expert
opinions by histological means in specific cases.
Key points
1. Forensic histopathology is an integral part of diagnosis
and has been since the inception of forensic medicine.
2. In forensic medicine, histopathological evidence of
vitality at the time of an event (e.g., infliction of a
wound or aspiration of foreign material) is of impor-
tance as is histopathological determination of the time
interval since a finding was caused (e.g., determining
the age of myocardial infarction or a thrombus).
3. Histopathological investigations can give further infor-
mation about toxin-induced changes in several organs,
e.g., alcoholic liver cirrhosis or a so called ‘‘junkie
pneumopathy.’’
4. For forensic purposes, gradings and classifications are
helpful, e.g., grading of a steatototic hepatis and
quantifying pulmonary fat embolism.
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