www.elsevier.com/locate/forsciint

Forensic Science International 165 (2007) 129–143

Sudden infant death syndrome (SIDS)—Standardised investigations

and classification: Recommendations

Thomas Bajanowski a,*, Ashild Vege b, Roger W. Byard c, Henry F. Krous d,Marianne Arnestad e, Liliana Bachs f, Jytte Banner g, Peter S. Blair h, Arne Borthne i,

Reinhard Dettmeyer j, Peter Fleming h, Peter Gaustad k, Markil Gregersen g,Jens Grøgaard l, Ellen Holter k, Christina V. Isaksen b, Jens V. Jorgensen m,Charlotte de Lange n, Burkhard Madea j, Isabella Moore o, Jorg Morland f,

Siri H. Opdal e, Petra Rasten-Almqvist p, Martin Schlaud q, Peter Sidebotham r,Kari Skullerud s, Gisela Stoltenburg-Didinger t, Arne Stray-Pedersen e,

Lisbeth Sveum e, Torleiv O. Rognum e

a Institute of Legal Medicine, University Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germanyb Section of Morphology, Department of Laboratory Medicine, Children’s and Women’s health,

Norwegian University of Science and Technology, Trondheim, Norwayc Department of Pathology, University of Adelaide & Forensic Science SA, Adelaide, Australia

d Department of Pathology, Children’s Hospital San-Diego, University of California,

San Diego School of Medicine, La Jolla, CA, USAe Institute of Forensic Medicine, University of Oslo, Norway

f Norwegian Institute of Public Health, Divison of Forensic Toxicology and Drug Abuse, Oslo, Norwayg Institute of Forensic Medicine, University of Aarhus, Denmarkh Institute of Child, Life and Health, University of Bristol, UK

i Department of Pediatric Radiology, Ulleval University Hospital, Oslo, Norwayj Institute of Legal Medicine, Rheinische Friedrich-Wilhelms-Universitat, Bonn, Germany

k Institute of Medical Microbiology, Rikshospitalet, University Hospital, Oslo, Norwayl Department of Pediatric Intensive Care, Divison of Women and Children, Ulleval University Hospital, Oslo, Norway

m Department of Pediatric Research, Rikshospitalet, University of Oslo, Norwayn Department of Radiology, Rikshospitalet, University of Oslo, Norway

o Department of Cellular Pathology, Southampton General Hospital, UKp National Board of Forensic Medicine, Solna, Sweden

q Robert-Koch-Institute, Department of Epidemiology and Health Reporting, Berlin, Germanyr University of Warick, Coventry, UK

s Department of Pathology, Divison of Neuropathology, Rikshospitalet, University hospital, University of Oslo, Norwayt Institute of Neuropathology, Charite, Berlin, Germany

Received 13 April 2006; received in revised form 20 April 2006; accepted 10 May 2006

Available online 27 June 2006

Abstract

Sudden infant death syndrome (SIDS) still accounts for considerable numbers of unexpected infant deaths in many countries. While numerous

theories have been advanced to explain these events, it is increasingly clear that this group of infant deaths results from the complex interaction of

a variety of heritable and idiosyncratic endogenous factors interacting with exogenous factors. This has been elegantly summarised in the ‘‘three

hit’’ or ‘‘triple risk’’ model. Contradictions and lack of consistencies in the literature have arisen from diverse autopsy approaches, variable

applications of diagnostic criteria and inconsistent use of definitions. An approach to sudden infant death is outlined with discussion of

appropriate tissue sampling, ancillary investigations and the use of controls in research projects. Standardisation of infant death investigations

* Corresponding author. Tel.: +49 201 723 3600; fax: +49 201 723 5940.

E-mail address: thomas.bajanowski@medizin.uni-essen.de (T. Bajanowski).

0379-0738/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2006.05.028

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143130

with the application of uniform definitions and protocols will ensure optimal investigation of individual cases and enable international

comparisons of trends.

# 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Sudden infant death; San Diego definition of SIDS; Standardised investigation; Diagnostic criteria

1. Introduction

Despite considerable decline in incidence, sudden infant

death syndrome (SIDS) is still the leading cause of death among

infants between 8 and 365 days of age [1]. In Germany, the

incidence of SIDS decreased from 602 cases in 1998 to 287 in

2004, which is a relative decline of 0.77–0.39 per 1000 live

births [2]. Similar statistics have been reported by most

industrialized countries [3]. This decline is due to a genuine

decrease, rather than as a result of changes in diagnostic

practices [4,5].

A number of theories on possible causes of SIDS have been

proposed in the past, but only a few have clearly been proven,

mostly in single cases. About 30 years ago, Naeye published his

observations in SIDS cases suggesting that seven different

findings could be suitable as tissue markers indicating hypoxia

[6]. By measuring the biochemical marker of acute hypoxia,

hypoxanthine in vitreous humour, Rognum et al. in 1988

showed [7] that a large proportion of the SIDS victims had had

significant periods of hypoxia prior to death. In the 1970s, the

sleep apnea theory [8] became the theoretical basis for the

monitoring of infants showing ‘‘atypical’’ breathing patterns. In

the late 1980s and the 1990s, SIDS research became more and

more interdisciplinary, and identical risk factors for SIDS were

identified by a number of epidemiological studies in different

countries and regions. In the second half of the 1990s,

molecular–genetic studies were performed to investigate the

genetic basis of functional disturbances. Possible cardiovas-

cular causes of SIDS, for example, include abnormal reflexes

and abnormalities of the cardiac conduction system [9,10]. In

addition, ventricular tachycardia or fibrillation, often found

without evidence of structural heart disease (so-called primary

electrical disorders), may be associated with sudden cardiac

death in neonates [11,12]. One such disorder is the long-QT

syndrome (LQTS), which is characterised by increased

sensitivity of the myocardium, with an increased propensity

to develop ventricular fibrillation. Several studies have

investigated a potential association of prolonged QT interval

and SIDS, but the results were not concordant [13–18].

Already by 1972, Wedgewood [19] had introduced a ‘‘three

hit model’’ for SIDS suggesting that sudden and unexpected

death may occur if three conditions are fulfilled simulta-

neously:

(1) A

n infant is at a vulnerable developmental stage.

(2) A

predisposing endogenous factor(s) is present.

(3) A

n exogenous trigger initiates the lethal process.

This hypothesis was later taken up and modified by Rognum

and Saugstad [20], Filiano and Kinney [21], and Kahn et al.

[22]. In particular, Rognum et al. included ‘‘genetic risk

factors’’ or ‘‘genetic make up’’ as predisposing factors [23] and

investigated the significance of mtDNA changes [24],

complement component C4 polymorphisms [25], and poly-

morphisms of the interleukin 10 gene [26]. Schneider et al. [27]

first demonstrated the link between signs of infection prior to

death in SIDS and partial deletions of the C4 gene which was

confirmed by Opdal et al. [25].

The main advantages of these ‘‘three hit theories’’ (in

contrast to others) are that most of the information from

pathology, neuropathology, microbiology, physiology, epide-

miology and pediatrics can be included in one of the three main

areas, and that these models enable integration of new

knowledge.

Currently, it is very difficult to have an overview of all of the

multiple investigations from different fields of SIDS research;

i.e. a search of PubMed using the term ‘‘SIDS’’ produces nearly

6000 publications—including 1300 from the year 2000 to 2005.

The results reported are sometimes not directly comparable as

some authors have used their own definitions for cases and

controls, and the diagnosis of SIDS has not always been

confirmed by autopsy and additional investigations, and is

therefore not in accordance with standard definitions.

2. Case definition

The investigation of cases of sudden and unexpected deaths

in all age groups is one of the major areas of forensic medicine.

The investigation of infant deaths requires the application of

special diagnostic methods and includes forensic pathology as

well as other fields of forensic medicine and medicine in

general, e.g. radiology, toxicology, microbiology, virology,

neuropathology, pediatric pathology, pediatrics, clinical chem-

istry, physiology, epidemiology and genetics. In accordance

with the internationally discussed and widely accepted new

SIDS definition (San Diego definition of SIDS [28]) the

diagnosis SIDS can be made only if an infant under 1 year of

age has died suddenly and unexpectedly, the onset of the fatal

episode was apparently associated with sleep, and a thorough

investigation of the case, including performance of a complete

autopsy and review of the circumstances of death and the

clinical history, does not provide an explanation for the death. If

these criteria are not fulfilled the cases can be termed

unclassified infant death (USID) or as sudden unexpected

death in infancy (SUDI).

USID was defined as a part of the San Diego definition of

SIDS and includes all deaths that do not meet the criteria for

SIDS but for which alternative diagnoses of natural and

unnatural conditions are equivocal, including cases for which

autopsies were not performed.

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143 131

In contrast, SUDI has been defined by Huber [29] as the part

of infant mortality where death occurs more or less suddenly

and unexpectedly. The post mortem examination, which ideally

should include a history of the gestation, delivery and postnatal

development, a death scene investigation, a family psycho-

social history, a complete autopsy, and a confidential case

conference:

- m

T

S

S

S

C

S

ay reveal changes that alone – or in combination – constitute

a sufficient cause of death (non-SIDS);

- o

r may reveal changes that even when clearly present are not

sufficient to explain the death (‘borderline’ SIDS);

- o

r may fail to demonstrate any abnormalities (SIDS).

This means that SUDI is the most heterogeneous group that

includes all categories of sudden and unexpected infant death.

For scientific investigations, the stratified part of the San

Diego definition should be applied. There are three subtypes of

SIDS that rely on many different pieces of information from

specific investigations such as the autopsy findings (including

additional investigations), death scene investigation, and

clinical history (Table 1). This part of the definition was

introduced to:

� ‘‘

a

a

I

t

r

c

provide recommended guidelines for general case assess-

ment, classification and diagnosis;

ble 1

n Diego definition of SIDS

Clinical history (CH) Circums

DS general definition Sudden and unexpected death Unexpla

circumsUnder 1 year of age

Lethal episode associated with sleep

Death unexplained by CH

ratified definition

Category IA SIDS (1) Older than 21 days, under 9 months Scene in

and gav

(2) Normal CH Safe sle

(3) Full term pregnancy (�37 weeks) No evid

(4) Normal growth and development

(5) No similar deaths in siblings/relatives

Category IB SIDS (1–5) Criteria for category IA SIDS Scene in

undertak

Category II SIDS Differences to category I criteria: (6) age

range (0–21 days, 270–365 days)

Mechan

by over

certainty

(7) Neonatal/perinatal conditions that have

resolved by the time of death

(8) Similar deaths in siblings, near relatives

USID Criteria for category I or II SIDS are not

fulfilled

Alternat

unnatur

iteria used in the general and stratified part. USID: Unclassified sudden infant d

ience and Business Media).

� a

ta

in

tan

ve

e n

ep

en

ve

en

ica

lay

iv

al

ea

ssist pathologists by detailing steps for infant death

investigation and diagnostic categorization;

� id

entify and include cases that have been recently incorrectly

excluded from SIDS groups due to findings of bed sharing

and prone sleeping position;

� f

ormalize current practices among pathologists of separating

cases based on degrees of certainty and the comfort with

which a conclusion of SIDS could be made;

� r

educe diagnostic confusion by introducing uniform terms;

� p

rovide a framework for researchers and identify the most

typical cases for study;

� a

ssist in the evaluation of published data;

� p

rovide a readily accessible breakdown of SIDS cases based

on age groups and investigative information’’ [28].

3. Study design

Study designs depend on the hypothesis that is being tested.

In general, SIDS studies can be designated as descriptive

studies (DS), as case-control studies (CCS) or as self-controlled

case series (SCCS).

DS are suitable for comparing SIDS with control cases

for differences in, for example, morphology, immunology,

and genetics. Because cases and controls are not matched, it

has to be shown that the groups do not differ significantly

for important parameters such as age, gender, and region.

nces of death Autopsy

ed after review of the

ces

Unexplained after complete autopsy

stigation performed

o explanation

(1) No lethal pathological findings

environment (2) No unexplained trauma, abuse, neglect

or unintentional injury

ce for an accident (3) No substantial thymic stress

(4) Toxicology, microbiology, radiology,

vitreous chemistry and metabolic screening

negative

stigation was not (1–4); (5) One or more of the following

analyses were not performed: toxicology,

microbiology, radiology, vitreous chemistry

and metabolic; screening

l asphyxia or suffocation

ing not determined with

(1–5); (6) Abnormal growth and development

not thought to have contributed to death

(7) More marked inflammatory changes or

abnormalities not sufficient to cause the death

e diagnoses of natural or

death are equivocal

Autopsy has not been performed

th. Modified from Bajanowski et al. [77] (with kind permission of Springer

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143132

The number of cases in the groups should be similar. A minimal

number of five cases should be achieved as a prerequisite for

statistical tests [30].

The most suitable form of SIDS studies is at present the CCS.

This type of study can be used to evaluate, for example,

morphological variables, chemical parameters, immunological

findings, as well as epidemiological data. The ratio of cases and

controls can differ. For morphological investigations a ratio of

1:1 should be sufficient (because of the availability of suitable

controls). For epidemiological studies the findings in SIDS are

usually compared to findings in living infants who are matched

for age, gender, region, and if necessary for other variables (e.g.

time of death). A higher number of controls (cases:controls = 1:2

or 1:3) can improve the power of the investigation that makes it

possible to identify risk factors with lower relative risk.

SCCS are particularly suitable for investigating very rare

and non-recurring events [31], and were specifically developed

for vaccine safety evaluation.

An ideal study design should have the following character-

istics:

- n

one-selective, prospective case ascertainment (all cases of

sudden and unexpected infant death should be initially

included as often it is not known if the death is explained until

after the investigation),

- s

tandardised analyses of the circumstances of death by an

experienced specialist in forensic medicine and/or a police

officer,

- s

tandardised autopsies in all cases,

- f

ull information on clinical history,

- f

ull information on outcome, exposure and confounders,

- a

dequate selection of controls, and

- u

niform diagnostic criteria,

- a

ny findings should be systematically entered into the study

database for scrutiny alongside the epidemiological findings.

Prior to the beginning of any study the study region, the

study population, inclusion and exclusion criteria for cases and

controls, and the recruitment of cases and controls have to be

defined by the investigators.

4. Controls

Suitable controls for morphological, morphometric, and

chemical studies have been the subject of controversy in the

past. This is not surprising, as controls are needed for a

phenomenon with an unclear pathophysiology. Nevertheless,

there are general requirements that should be fulfilled by the

controls:

Age: Control cases should be of a similar age to SIDS cases.

This means that controls should be matched for age, or if this

is not possible, aged under 6 months.

Gender: The typical ratio of males to females should be

matched by the controls.

Region. Because of different SIDS incidences from region to

region or country to country and other possible differences

(e.g. different infant care practices), controls should have

lived in the same region as the index cases.

Ethnicity. Significant differences in the incidence of SIDS

among populations and different ethnic groups requires

matching by controls.

Cause of death. Most investigators agree that cases of

unnatural death with short clinical courses are suitable for

use as controls. Because ‘‘suffocation’’ may be a possible

factor leading to death in SIDS, other cases of unnatural

death would be better controls. If such cases are only rarely

available, natural deaths with clearly defined causes of

death, and short histories of disease could be used as controls

(e.g. fulminant pneumonia, meningitis, intussusception). It

is important that such cases are investigated separately, and

that the results are clearly separated for statistical analyses.

In addition to these controls, for genetic investigations, material

from the general population could be used.

For epidemiological CCSs, the minimal matching criteria

are age, gender and region. For each study, it has to be shown

that the groups were similar/comparable for significant

parameters, and that there was no significant re-call bias.

Proportions of response, contact and co-operation can be

calculated according to the definitions of Slattery et al. [32].

5. Circumstances of death

Investigation of the circumstances of death is important to

help to differentiate between natural and unnatural causes of

death. Furthermore, in cases of SIDS deaths the results from

death scene investigations may be suitable for generating new

hypotheses concerning risk factors. Therefore, these investiga-

tions have been included in SIDS definitions from the early

nineties as being mandatory [33]. In some studies, scoring

systems were developed and applied to standardise these

investigations and to compare the results with regard to the

cause of death [34,35]. In a pilot study, the Norwegian group

used the body position when found, the firmness of the mattress,

layers of clothing, type of duvet, room temperature and co-

sleeping as criteria [34]. A more detailed investigation has been

performed as a part of the German SIDS study, the

methodology of which has been described by Schlaud et al.

[36]. Furthermore, some investigators recommend the use of a

video recorder to document the death scene. This can help with

the case discussion held weeks later.

These investigations are usually performed by police

officers who should have had special training in this field of

investigation [34]. Specialists in forensic medicine can perform

these investigations, together with police officers, but in the

past this has been achieved in only a few scientific studies.

6. Standardised autopsy protocol (SAP)

Internationally there are certain protocols that have been

validated by different studies. These SAPs [37–39] are similar

and accord with European guidelines for medico-legal

autopsies [40] and closely reflect the International Standardised

Autopsy Protocol (ISAP, Table 2) [41] of the Global Strategy

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143 133

Table 2

International Standardised Autopsy Protocol

Table 2 (Continued )

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143134

Table 2 (Continued ) Table 2 (Continued )

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143 135

Table 2 (Continued ) Table 2 (Continued )

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143136

Table 2 (Continued )

Task Force of SIDS International. The autopsy includes a

thorough external examination, a complete internal examina-

tion, an X-ray investigation, extensive histology, full toxicol-

ogy, and microbiology (Table 2).

During the autopsy a number of measurements have to be

performed (including the body weight, body length, chest

circumference, and organ weights) to record objective criteria

for judging an infant’s development. Furthermore, all of the

material that is necessary for further investigations has to be

sampled and stored. Additional investigations have been

discussed in detail at the State of the Art Conference on the

Stratification of Sudden Unexpected Death in Infancy (Oslo,

November 2003) and at the Soria Moria Meeting on

Investigating SUDI (Oslo, October 2004) and consensus could

be reached for important parts of the investigation leading to the

recommendations discussed below.

6.1. Histology

Histological examinations are mandatory in investigating

infant deaths, therefore an extensive sampling, cutting and

staining scheme is included in the ISAP (Table 2). Specimens

required for histology should be fixed for 24–48 h in 10%

buffered formalin before cutting. Hematoxylin and eosin

(H&E) staining is mandatory for all sections. Furthermore,

other staining methods will have to be applied for the diagnosis

of certain conditions.

Because of their significance certain organs or organ systems

will be discussed in more detail.

6.1.1. Central nervous system

Despite considerable discussion at meetings, a general

agreement on the significance of certain cerebral abnormalities

in SIDS has not been achieved. Nevertheless, guidelines for

neuropathological examinations were recommended as a result

of the State of the Art Conference on the Stratification of SUDI,

in Oslo, November 2003 [42]. Following this meeting it was

recommended that the brain should be fixed in 10% buffered

formalin for at least 6 days before cutting. For histological

investigations, samples should be taken from the hippocampus,

the frontal lobe, parietal lobe, corpus striatum thalamus,

cerebellum, mesencephalon, pons, medulla oblongata, and the

cervical part of the medullary cord [43]. All sections should be

stained with H&E. Additionally, Kluver-Barrera, Luxol fast

blue, and immunohistochemistry for myelin can be used for

myelin detection. Gliosis can be evaluated using immunohis-

tochemical detection of glial fibrillary acidic protein (GFAP).

Neurons and axons can be stained by routine Bielschowsky

stains or immunohistochemically. Microglial cells can be

stained using immunohistochemistry (CD68). Traumatic brain

injury and changes caused by ischaemia can be demonstrated

by b-amyloid precursor protein (APP) [44].

By adhering to this cutting and staining scheme, structurally

normal brains or brains with non-specific changes (e.g.

hyperemia, edema) can be differentiated from others showing

potentially fatal lesions (e.g. malformations, degenerative

disorders, hypoxia/ischemia, hydrocephalus) or from brains

with definite lethal changes (e.g. meningitis, encephalitis,

traumatic lesions).

6.1.2. Respiratory system

Inflammatory changes in the upper and lower respiratory

tract in SUDI cases are a frequent finding. In most of the cases

inflammation is characterised by only a mild cellular infiltrate

and can be judged as a ‘‘minor’’ finding without significance as

to the cause of death. On the other hand, diseases of the

respiratory tract are a common finding in sudden and

unexpected death in infancy. Up to 50–80% of SUDI cases

that can be explained after autopsy have been attributed to

respiratory tract infections [45,46]. The etiology of the

inflammation should be confirmed by microbiological inves-

tigations.

During autopsy, the lung weights must be recorded and

petechial hemorrhages noted. Because of the significance of

the respiratory tract in infant deaths a complete histological

investigation after fixation of the specimens for 24–48 h in

10% buffered formalin is mandatory. Two samples from each

lobe should be examined, one peripheral and one central. In

addition, two specimens of the lung parenchyma and one

specimen of the lower trachea/bifurcation should be frozen at

�70 8C for immunohistochemistry, PCR and genetic testing

(see below). To show which parts of the respiratory tract

should be investigated microscopically, the sampling scheme

of the German SIDS study is included (Fig. 1). As a routine,

H&E staining is recommended. Trichrome staining and iron

staining can be used to detect fresh or older hemorrhages.

Inflammatory infiltrates can be characterised immunohisto-

chemically using antibodies such as LCA, CD45R0, and

CD68.

The aim of histologic evaluation is to differentiate non-

specific lung changes (e.g. congestion, pleural haemorrhages,

mild edema), age dependent peculiarities (e.g. doubled

capillary system of alveolar walls), and pathological changes.

Based on the character and intensity of these findings the cases

can be differentiated into category I and II SIDS, USID (SUDI)

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143 137

Fig. 1. Recommended sections for non-CNS histology (modified according to Findeisen et al. [39], with kind permission of Springer Science and Business Media).

or explained infant death. The Nordic diagnostic criteria for the

exclusion of SIDS [47] were modified during the State of the

Art Conference on the Stratification of SUDI and the following

recommendations were made [48]:

Category I SIDS: Can be used when neither autopsy nor

clinical information reveals a cause of death. The upper

respiratory tract, bronchi and peribronchial tissue can show

mild to moderate lymphoid infiltrates with insignificant

numbers of neutrophils or no neutrophils within lumina.

Interstitial lung tissue may occasionally contain diffusely

scattered lymphocytes or a few moderate-sized inflamma-

tory foci. Fewer than 10 alveoli may contain a maximum of

10 neutrophils (Fig. 2).

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143138

Fig. 2. Lung histology from an 8-month-old boy in a case of SIDS where the autopsy and histology showed no signs of disease or malformations: (a) congestion and

edema, (b) subpleural intra-alveolar hemorrhage (H&E, magnification: a, �25; b, �100).

Fig. 3. (a) Lung histology in a 3-month-old girl with established bronchop-

neumonia showing intra-alveolar aggregates of polymorphonuclear leukocytes.

H&E, �100. (b) Lung histology in a 4-month-old boy showing interstitial

mononuclear cell infiltrates in a case of interstitial pneumonia. H&E, �40.

Category II SIDS. Congenital disorders or pre-existing

clinical symptoms, and/or post mortem findings that are not

sufficient to explain the cause of death. Mild changes are

present in different parts of the respiratory tract that are

insufficient to explain death. The upper respiratory tract,

bronchi and peribronchial tissue may have a neutrophil

infiltration in mucous membranes but insufficient inflam-

matory infiltrate is present within lumina to obstruct larger

bronchi. Lymphoid cell infiltrates form heavy cuffs around

the bronchi in more than one section. In the interstitium there

are moderate lymphoid infiltrates in several sections. The

alveoli do not show a consolidation, but at least 10 alveoli

contain each 10 or more inflammatory cells.

Explained infant death: Bronchi and peribronchial tissue are

infiltrated by a higher number of cells; purulent exudate fills

lobar bronchi or larger branches of bronchial tree; atelectasis

occurs distant to obstruction. The interstitial tissue is

characterised by widespread lymphoid infiltrates in the walls

of alveoli in all sections (interstitial pneumonia). The alveoli

may show marked, obvious pulmonary consolidation.

Furthermore, specific lung diseases (congenital, allergic,

inflammatory) can be sufficient to explain the death, as well

as established bronchiolitis (Fig. 3).

6.1.3. Heart

During autopsy the ventricular thickness and the valve

circumferences should be recorded and compared to age-

dependent reference data. Abnormalities of the heart

configuration as well as of the great vessels should be

described.

One sample of the myocardium from the left ventricle has to

be taken for immunohistochemistry, PCR and genetic testing if

indicated and frozen at �70 8C. The remaining material has to

be fixed in 10% buffered formalin before cutting [49].

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143 139

Fig. 4. Diagramatic representation of sections of the right atrium, including the

SA node, according to the NORD SIDS criteria [49] (with kind permission of

the Scand. J. Forensic Sci.).

For the histological examination, six to eight sections are

recommended (Figs. 4 and 5) [49]. All sections should be

stained with H&E. Other staining methods may be necessary to

show special changes.

The detection of myocarditis may require special methodol-

ogy, as this diagnosis may present difficulties when there are

equivocal histological findings using conventional histological

stains. When applying a comprehensive combination of molecu-

lar and immunohistochemical techniques a higher prevalence of

viral myocarditis can usually be detected. However, the detection

of myocarditis also depends on the extent of sampling.

6.1.3.1. Conventional histology. In 1986, the Dallas criteria

for the histological diagnosis of active myocarditis were

Fig. 5. Recommended sections through the myocardium. (A) Right ventricle,

(B) AV node, (C) posterior papillary muscle, (D) posterior part of the left

ventricle, (E) anterior papillary muscle, (F) anterior part of the left ventricle,

according to the NORD SIDS criteria [49] (with kind permission of the Scand. J.

Forensic Sci.).

introduced [50]. When examining endomyocardial biopsies by

using light microscopy and conventional stains, infiltrating

lymphocytes and myocytolysis were considered markers of an

active (acute) myocarditis. Without myocytolysis, a borderline

or ongoing myocarditis may not be diagnosed despite

lymphocytic infiltration. But the Dallas criteria probably

underestimate the true incidence of myocarditis [51] and the

degree of interobserver variability is large [52]. Myocarditis

can present as a pure focal inflammatory process. Therefore, a

considerable number of myocardial specimens must be

obtained at autopsy to avoid a ‘‘sampling error’’ [53] and

there are cases in which only 1 or 2 of 10 or more myocardial

samples show focal lympho-monocytic infiltrates (Fig. 6).

6.1.3.2. Immunohistochemistry. The use of immunohisto-

chemical methods for the diagnosis of acute myocarditis has

been described in previous studies [54–56]. The immunohis-

tochemistry given below is advised for research purposes: As a

fixative, neutral phosphate-buffered formaldehyde (pH 7.0)

should be used for not longer than 48 h. The de novo expression

of antigens such as the major histocompatibility complex (MHC)

may indicate immune system activation (Fig. 7) as a result of

lympho-monocytic viral myocarditis associated with an

increased number of LCA+-leucocytes, CD45R0+-T-lympho-

cytes and CD68+-macrophages. T-lymphocytes are known to

react with foreign antigens when present on cell surfaces in

conjunction with MHC antigens [57]. Additionally, the expres-

sion of cell adhesion molecules such as E-selectine, vascular cell

adhesion molecule-1, and intercellular adhesion molecule-1, as

well as cytokines (e.g. IL-1, IL-2, tumour necrosis factor—TNF,

perforin), is increased in cases of viral myocarditis [58–61].

In the past, the finding of a mean value of more than 5 T-

lymphocytes per high-power field (HPF), when investigating 20

visual fields at 400-fold magnification, has been regarded as a

sign of active myocarditis in adults [62]. In the younger age

groups, perhaps more rigorous criteria should be applied to

avoid false positive diagnoses of myocarditis [63]. The finding

of more than 15 T-lymphocytes per HPF could be interpreted as

a reliable sign of active myocarditis, in younger age groups.

Fig. 6. Focal lympho-monocytic infiltration in one out of eight myocardial

sections using conventional H&E stains in a 4-month-old boy who had a viremia

due to cytomegalovirus detected by PCR (magnification �400).

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143140

Fig. 7. Marked expression of MHC-class-II-molecules on the endothelium of

small vessels and on interstitial leucocytes (same case as Fig. 6). The case was

suspected as being SIDS according to the death certification and at the time of

autopsy.

Cases with 5–10 T-lymphocytes per HPF can be regarded as

‘‘suspicious’’, as should cases with more than 10 macrophages

per HPF. The significance of cases with an increased number of

macrophages remains unclear at the moment, however, it may

indicate a late inflammatory process. Qualification and

quantification of interstitial leucocytes, T-lymphocytes, and

macrophages may help to identify cases with a high index of

suspicion of myocarditis.

6.1.3.3. Molecular pathological methods. In adults, virus

detection by reverse transcriptase (rt)-PCR and in situ

hybridization has revealed an association between myocarditis

and virus infection, especially enterovirus infection with

coxsackie viruses group B, may occur. These viruses are one

of the most frequently identified infectious agents responsible

for acute myocardial infections and outbreaks have been

described [64]. Several studies have used molecular patholo-

gical methods to detect viral genome in cases of suspected

SIDS [65]. Over the past few years, numerous reports based

mainly on PCR-derived molecular data have shown an

association between other viral infection and myocarditis:

e.g. adenoviruses, Epstein-Barr-virus, cytomegaloviruses,

parvovirus B 19. Modern immunohistochemical techniques

and molecular pathological methods have provided powerful

tools to investigate lethal myocarditis in cases devoid of

traditional histological findings according to the Dallas criteria.

With regard to the time-dependent course of viral myocarditis,

early virus-induced myocardial damage may already have

taken place before histological and immunohistochemical signs

of myocarditis can be observed [66]. Combined investigations

using molecular pathological techniques and immunohisto-

chemical methods may assist in establishing more reliable

diagnoses of myocarditis in certain pediatric fatalities.

6.2. Diagnostic criteria

(1) Category I SIDS: Cases without any significant

pathological changes or with less than 15 lymphocytes in

one section. (2) Category II SIDS: Cases showing changes that

might be associated with functional disturbances, but that

cannot explain death (e.g. focal hemorrhages, patent ductus

arteriosus after the first month of age, minor ventricular septal

defect (VSD) or atrial septal defect (ASD) without significant

influence on cardiac morphology, a family history of sudden

death). (3) Non-SIDS: Changes that are undoubtedly fatal, or

that could be fatal in combination with alterations in other

organs [5].

6.3. Metabolic/genetic investigation

It is recognised that the risk of unexpected infant death is

increased in those families where one infant has died.

Nevertheless, the presence of multiple deaths in one family

should raise concerns. This may reflect the persistence of

underlying recognised risk factors, or may indicate a specific

cause of death including inherited disorders and homicide. In

the past diagnostic tools were limited, but newer methods, such

as tandem mass spectrometry and PCR have opened new

possibilities.

During the State of the Art Conference on the Stratification

of SUDI, Oslo, November 2003 minimal requirements for

metabolic and genetic investigations were discussed [67]. It

was stressed that a detailed family history was necessary in

addition to a full autopsy, including frozen sections of the liver,

the heart and the muscles for fat staining. Metabolic screening

is indicated in all cases. For this reason, material has to be taken

during the autopsy for further testing: blood and spleen for

molecular–genetic investigations, fibroblast cultures for chro-

mosomal analysis, and blood or bile spots for metabolic

screening, as well as urine when possible.

The increasing knowledge of genetically determined

disorders associated with sudden death in infancy and

childhood makes it difficult to recommend a defined set of

analyses or investigations. If there is any indication of the

presence of a genetically determined disturbance, for example

disorders of cardiac function or metabolism, specific testing

must be pursued.

6.4. Bacteriology and virology

Bacteriological and virological investigations are manda-

tory in cases of sudden death in infancy and childhood. In

cases of histologically diagnosed infection these investi-

gations may prove the etiology. A discussion of all of

the findings may be necessary to enable correct interpreta-

tion of the findings. Important questions to be answered are

[68]:

- C

ould the bacteria/virus be a cause of death or a contributor to

death or not have any significance as to the death?

- D

o other findings support the diagnosis of infection (e.g.

previous history, histology)?

- D

o the potentially pathogenic bacteria detected belong to the

normal post mortem flora? Are the viruses detected a

reflection of asymptomatic carriage?

T. Bajanowski et al. / Forensic Science International 165 (2007) 129–143 141

7. Radiology

Radiological investigation is mandatory as part of the post

mortem investigation of infant death. In particular, older

traumatic lesions, minor changes, and skeletal abnormalities

and diseases may be overlooked if these investigations are not

performed. Radiology should be performed prior to the autopsy

to define which parts of the skeleton have to be removed for

subsequent investigations, e.g. histology. From the literature, it is

known, that radiology contributes to the diagnosis in about 3–4%

of infant deaths [69] and in about 25% of unnatural deaths [70].

As a first approach the use of high quality radiographs is

recommended [71]. The standard projections are: antero-

posterior, as well as lateral views of the appendicular and axial

skeleton and thorax/abdomen, and two oblique views of the ribs

[72]. These standard views can be completed by additional

projections if necessary. New techniques (multislice computed

tomography, magnetic resonance imaging) can be used for the

detection of specific lesions [73,74], but are not routinely used

in the post mortem investigation of SUDI at present [71].

8. Toxicology

The frequency of poisoning as a cause of death in infancy

has varied between 2 and 4% of all sudden und unexpected

infant deaths [75] and reached about 10–12% in unnatural

deaths [70]. Therefore, toxicological analyses are mandatory in

investigating SUDI.

Although blood from the femoral vein is the most suitable

material to perform toxicological analyses on, blood from the

cardiac ventricles has to be used in most cases because it is

often impossible to obtain sufficient amounts of femoral vein

blood from infants at autopsy. Bachs et al. [76] suggest that the

primary screening could be done using cardiac blood, and that

positive results should be confirmed by investigating femoral

vein blood. Furthermore, cerebrospinal fluid, vitreous humour,

urine, stomach contents, and liver tissue should also be taken

and retained for possible future analyses.

Toxicological screening should include alcohols, narcotics

and common prescription and illicit drugs [76]. as a minimum.

If positive results are obtained the significance of the findings

has to be established, i.e. is this within the therapeutic range, or

is it toxic or even lethal? To answer these questions detailed

knowledge of the drug history, clinical circumstances

(resuscitation attempts), circumstances of death, drug distribu-

tion in infants, and metabolic capacity in this age group, is

necessary. Therefore, Bachs et al. recommend cooperation

among pediatricians, pathologists/forensic pathologists and

toxicologist in interpreting such results [76]. It is recognised,

however, that the interpretation of toxicologic findings in

infants may be difficult, and that therapeutic, toxic and lethal

levels in adults may not be the same in infants.

9. Final diagnosis

Finally, it is essential that all information available should

be shared at a multiprofessional meeting where the case is

thoroughly discussed, so that the members of the case

conference may contribute to the classification. The task of

solving these cases requires the knowledge of all the

professionals involved, and should not be left to a single

person, whatever the field of expertise.

10. Conclusions

During the previous 2 years discussions on the investigation

of sudden and unexpected deaths in infancy have led to detailed

recommendations for the examination of different organs and

organ systems as well as for additional investigations.

Furthermore, consensus at various meetings has been achieved

for diagnostic criteria for SIDS, and for the exclusion of SIDS

with regards to cardiac pathology, lung pathology, metabolic/

genetic investigations, toxicology, microbiology/virology, and

radiology. Nevertheless, the new San Diego definition of SIDS

can only be successfully used if further progress in the

standardisation of diagnostic criteria can be achieved. The

recommendations above can be used as a guide for such further

standardisation.

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