Legal Medicine xxx (2014) xxx–xxx

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Legal Medicine
journal homepage: www.elsevier.com/locate/legalmed

Case Report

Feasibility, diagnostic validity and limits of postmortem evaluation

of a newborn infant following an extremely prolonged freezing interval:
A thanatological case study
Jozef Krajčovič a, Martin Janík a,⇑, František Novomeský a, L’ubomír Straka a, Petr Hejna b
a
Institute of Forensic Medicine and Medicolegal Expertises, Jessenius Faculty of Medicine, Comenius University and University Hospital, Martin, Slovak Republic
b
Department of Forensic Medicine, Faculty of Medicine, Charles University and University Hospital, Hradec Kralove, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: In forensic assessment, denial and concealment of pregnancy has wide-ranging implications including
Received 15 April 2014 criminal abortions, extramural deliveries, concealment of birth, newborn infant abandonment or even
Received in revised form 1 July 2014 neonaticide. Clarification of whether a newborn was born alive is the most important factor when
Accepted 7 July 2014
evaluating an abandoned neonate or concealment of birth. Other points that need to be addressed are
Available online xxxx
determination of viability and maturity of the newborn infant, and the identity of the mother.
A prolonged postmortem interval following illegal disposal of a dead body often leads to advanced
Keywords:
decomposition, making postmortem elucidation difficult. We report an exceptionally uncommon autopsy
Frozen newborn
Decomposition
case of a well-preserved female newborn, which was accidentally found after eight years in a home
Autopsy freezer. Despite the prolonged postmortem interval, tissue preservation was sufficient for a meaningful
Immunohistochemistry autopsy including a comprehensive histopathological study. The purpose of the present investigations
was to expand our understanding of thanatological processes, as well as detectability of particular
histological findings on the remains of a newborn after extremely prolonged storage in an artificially
frozen environment. In addition, this article discusses forensically important issues regarding conceal-
ment of newborn infant under specific conditions.
Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction determining the possibility of certifying whether or a newborn

A number of important issues must be considered when assess-
ing cases of found or abandoned newborn infants, including viabil-
ity, presence of natural disease or trauma, cause and manner of
death. Other points that need to be addressed are whether the
newborn was of sufficient developmental age to survive. Unfortu-
nately, there are many uncertainties in this area, mainly caused by
decomposition. The time frame from abandonment to discovery of
a newborn may range from hours to several years [1]. Neverthe-
less, macroscopic and histopathological investigations, in particu-
larly of cardiac and pulmonic tissue, are still valuable after
several years [2]. Newborn infants may be abandoned or concealed
in a variety of ways and places. We present and discuss an excep-
tionally uncommon case of female newborn found accidentally
after eight years in a home freezer. This case study aimed at
⇑ Corresponding author. Address: Institute of Forensic Medicine and Medicolegal
Expertises, Jessenius Faculty of Medicine, Comenius University and University
Hospital, 036 59 Martin, Slovak Republic.
E-mail address: janik.mato@gmail.com (M. Janík).
has been frozen and thawed, along with the feasibility of clarifica-
tion of main medicolegal issues, including the newborn’s cause of
death, survivability, vitality, and maturity.
2. Case history
In February, 2011, in the house of a 38-year old gravida 4 para 3
mother a frozen newborn female was accidentally discovered in
the freezer compartment (36.0  35.8  37.9 cm; cca 40 L) of an
old refrigerator by moving company employees. The inside
temperature was 9 °C. The frozen infant was wrapped in newspa-
per sheets and a blood-stained towel. During thorough police
questioning, the mother admitted that she delivered the baby at
home sometime in late May, 2003. According to the mother’s
statement, she lost consciousness during delivery. After recovering,
she found the completely expelled newborn without signs of life.
She made no attempts to resuscitate the neonate. Because she
was concerned being charged of a crime, she used the freezer as
convenient hiding place for the newborńs body.

http://dx.doi.org/10.1016/j.legalmed.2014.07.004
1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Krajčovič J et al. Feasibility, diagnostic validity and limits of postmortem evaluation of a newborn infant following an
extremely prolonged freezing interval: A thanatological case study. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.07.004
2 J. Krajčovič et al. / Legal Medicine xxx (2014) xxx–xxx

3. Autopsy findings
A full postmortem examination was performed 5 h after the
newborn’s body was found. The thawing process was iatrogeni-
cally accelerated according to recommendations made by Kozawa
et al. [3]. This was performed by immersing the newborn, which
was virtually hermetically sealed in a vinyl pouch, in salt solution
at 37 °C. The body was completely thawed over a period of approx-
imately 105 min. External examination of the well-developed and
well-nourished (51 cm, 3450 g) female neonate showed no evi-
dence of birth-related trauma, inflicted injuries or congenital
abnormalities. The legs pointed straight up in front of the new-
born’s body, with the feet near the head (Fig. 1A1). The head cir-
cumference was 33 cm, the chest circumference was 34 cm, and
the abdominal circumference was 33 cm. The length of the foot
was 6.5 cm. Her fingernails extended slightly past her fingertips.
The vernix caseosa was present at the axillas and inguinal regions.
The skin showed no signs of maceration or severe decomposition
(Fig. 1A1 and A2). The intact umbilical cord, 59 cm in length was
relatively tightly encircled around the neck (Figs. 1A2 and 2B1).
Macroscopically, the placenta showed no structural stigmata. The
soft tissues and viscera showed moderately marked softening with
an early stage of decomposition (Fig. 2B2). The viscera were gener-
ally dark red in color. There was a small amount of bloodstained
serous liquid within the pleural and abdominal cavities. Sectioning
of the heart was unremarkable except for patent foramen ovale.
The right and left lungs weighed 25 and 20 g, respectively. The lung
parenchyma was dark purple-red in color, firm, with sharply
angled margins. The cut surfaces of the lungs showed almost an
uniform, liver-like texture with multiple ‘‘Swiss cheese’’ – like

Fig. 1. (A1) Anterior aspect of the neonate’s body. (A2) Posterior aspect of the body. Umbilical cord entangled around the neck (yellow arrow). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. (B1) Closer view of the posterior aspect of the neonate’s body. (B2) Well-preserved viscera showed moderate-marked softening with early stage of decomposition.

Please cite this article in press as: Krajčovič J et al. Feasibility, diagnostic validity and limits of postmortem evaluation of a newborn infant following an
extremely prolonged freezing interval: A thanatological case study. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.07.004
 J. Krajčovič et al. / Legal Medicine xxx (2014) xxx–xxx 3

spaces resembling to gas bubbles from putrefaction. The brain was

entirely liquefied. No structural abnormalities were grossly
identified in any visceral organs except as indicated.

4. Flotation test

The lumen of trachea was clipped before the removal of the cer-
vicothoracic block to prevent the artificial airflow. The flotation
test was initially performed by placing the entire organ complex
(lungs, trachea, heart) in the water at room temperature. Finally,
each lung was evaluated separately in addition to lung slices
(0.3–0.6 cm) from every single lobe. Initially, the flotation test
was positive. However, soon after the floating phase, the lung tis-
sue sank to the bottom. Therefore, we concluded that the floatation
test was negative. Because the slightly decomposed lung tissue
was invaded by gas-forming organisms, we assumed that the flota- Fig. 4. (D) Lung tissue with anucleated keratin lamellae exfoliated from the fetal
tion test was not reliable, despite the fact that the newborn had epidermis within the intra-alveolar spaces indicating massive amniotic fluid
aspiration (anti-cytokeratin 40; 400).
been frozen soon after death and was thawed prior to autopsy.

an antibody against keratin showed clear intraalveolar deposition

5. Histological findings
of keratin lamellae exfoliated from the fetal epidermis (Fig. 4D).
Placental microscopy showed syncytial knotting and intervillous
Organ specimens from the newborn infant taken during
fibrin deposition consistent with the third trimester. Other viscera
autopsy were fixed in buffered formalin 10% for a period of 14 days
were not microscopically compromised.
and then processed. From blocks obtained, sections thick 4–6 lm
On the basis of the circumstances surrounding death, the find-
were cut and stained with hematoxylin and eosin-H&E, Lawson
ings at autopsy, and the police reports, we conclude that the new-
staining, Gömöri reticulin staining and periodic acid Schiff-PAS.
born infant died because of intrauterinne asphyxia caused by
Besides routine histological staining, the immunohistochemical
severe respiratory distress precipitated by meconium stained
examination (CD 68, and High molecular weight antibody
amniotic fluid aspiration with the umbilical cord entanglement
34ßE12 reacting with cytokeratins 1, 5, 10 and 14; Dako, Denmark)
around the neck serving as a contributory factor.
at magnification ranging from 40 to 400 were performed for the
lung tissue. Microscopically, lung tissue displayed an alveolar stage
of development with diffuse atelectasis and signs of severe aspira- 6. Discussion
tion of amniotic fluid and multiple brownish, rugby ball shaped
meconium bodies along with the numerous decomposition-related A number of studies have explored the biological effects of
gas bubbles (Fig. 3C1 and C2). The alveolar microarchitecture, exposure of various environmental conditions on human tissue,
highlighted by Lawson and Gömöri reticulin staining, showed including taphonomical processes, such as weathering, decomposi-
partial expansion of terminal alveoli with meconium stained amni- tion, and burning. Few studies have analyzed, both microscopically
otic fluid (Fig. 3C3 and C4). Immunohistochemical staining using and radiographically, the effects of freezing temperatures on

Fig. 3. (C1) Lung tissue changes due to early stage of putrefaction with recognizable alveolar structure (H&E 40). (C2) Note the brownish, rugby ball shaped bodies of
meconium in the intra-alveolar spaces (H&E 400). (C3 and C4) Lung tissue showing alveolar stage of development with signs of severe aspiration of amniotic fluid along
with the multiple, decomposition-related gas bubbles (Gömöri 100; 200).

Please cite this article in press as: Krajčovič J et al. Feasibility, diagnostic validity and limits of postmortem evaluation of a newborn infant following an
extremely prolonged freezing interval: A thanatological case study. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.07.004
4 J. Krajčovič et al. / Legal Medicine xxx (2014) xxx–xxx
human organ integrity, specifically in adult cardiac, hepatic, brain,
and bone tissue [4–7]. Other forensically oriented studies have
focused on the histopathological evaluation of newborn tissue after
freeze–thaw events [3,8]. The results of these studies may provide
investigators the opportunity to analyze death scene evidence to
draw more accurate conclusions about perimortem events.
Under isotonic conditions, water in tissues becomes thermody-
namically unstable at temperatures below 0 °C and tends to
crystallize [9]. Crystallization, especially extracellular, plays a
determinant role in the cryopreservation of tissues. In a previous
light microscopic study examining the influence of freezing on soft
tissues, the freeze–thaw cycle resulted in extension of extracellular
spaces and shrinkage of cells [3,5]. Scanning electron microscopy
analysis on frozen human bone shows clear cracks originating from
the Haversian system [4]. However, light microscopic observation
does not demonstrate significant differences in frozen and non-
frozen human bone. A recent biomechanical study published by
Torimitsu et al. showed that there were no significant differences
in frozen human skull specimens compared to unfrozen controls
[10].
Not all newborn infants found dead, even in highly suspicious
or concealed circumstances, are victims of neonaticide. The inci-
dence of stillbirth, even in developed countries, is estimated at 1
in 200 pregnancies and, as such, is more than five times more com-
mon than SIDS [11].
The challenge of establishing live birth is an unique factor in the
evaluation of a frozen newborn. Whether there is air in the lungs
may be assessed using the flotation test. However, the slightest
degree of decomposition immediately negates any interpretation
of this test [12]. In contrast, the results of a recent prospective
autopsy study showed reliability of the flotation test in 204 out
of 208 cases (98%) [13]. It is highly recommended, therefore, to still
perform the flotation test in every medicolegal investigation with
the uncertainty as to, if a newborn has breathed or not [13].
Determining whether a neonate was born alive or stillborn can
be ascertained histopathologically. Pulmonary interstitial emphy-
sema with distended alveolar structures has previously been
reported as an useful finding in the determination of live birth
[14]. However, Robinson and Lucas did not find this determination
to be helpful [15]. Moreover, a case of intrauterine death with
microscopically distended alveoli has been described in the litera-
ture [16]. According to Janssen, ventilation of the lungs alone
cannot be used as a certain indication of live birth [17].
Microscopic detection of amniotic fluid aspiration is particu-
larly significant when determining the cause of death in stillborns
and newborns. Aspiration of amniotic fluid and its contents plays a
much greater role in the etiology of respiratory distress in the new-
born than is generally realized [18]. Relatively large amount of
amniotic fluid may be aspirated, and when stained with meco-
nium, is highly indicative of severe respiratory distress syndrome.
In isolated cases, it may be possible that massive amniotic fluid
aspiration results in death, however, this is an exceptional event
that may mostly occur in a short period after birth [19]. The sys-
tematic examination of cases of intrapartal death in various cases
has demonstrated that intrapartal aspiration of amniotic fluid only
occurs when the function of the umbilical cord is impaired [2].
Because of amniotic fluid aspiration, the affected parts of the lung
may show unfolding of the alveoli which may simulate ventilation
of lung alveoli [17,20]. Immunohistochemical confirmation using
an antibody against keratin leads to a clear depiction of aspirated
squamous epithelial cells (as demonstrated in the present case).
Viability is the potential ability of an infant newborn to survive
after birth. The age at which a fetus becomes legally viable is
defined either by the gestational age or by the body weight, and
varies from country to country. An accurate estimation of gesta-
tional age should be made, and then this estimation should be used
Please cite this article in press as: Krajčovič J et al. Feasibility, diagnostic validity and limits of postmortem evaluation of a newborn infant following an
extremely prolonged freezing interval: A thanatological case study. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.07.004
to make an assessment of intrauterine growth. Measurements of
crown-rump, crown-heel, and foot length, together with whole
body weight and organ weights are the best starting points. An
assessment of organ maturation, especially the stage of lung devel-
opment, is highly recommended. Fixation of the lungs by intratra-
cheal introduction of fixing solution under low pressure similarly
distends the alveolar ducts and alveoli, and permits visualization
of alveolar microarchitecture [21]. This technique allows for
evaluation of the extent of alveolar development, the thickness of
alveolar septa, and the location of abnormal infiltration of cells,
which is often impossible in lungs handled by the usual methods.
For studying the lungs, it is desirable to inject one lung, leaving the
other in its natural state. The presence and quantity of extramed-
ullary hematopoiesis in the liver and the ratio of stroma to paren-
chyma in the pancreas may be also useful [22]. In cases when
advanced decomposition has taken place, measurements of the
basilar part of the occipital bone can be applied for estimating fetal
and post-natal age [23].
The most common cause of death in forensic neonatal and peri-
natal cases is asphyxiation, followed by cerebral injury and intra-
uterine asphyxia [1,24]. However, in approximately half of the
cases the cause of death, secondary to severe decomposition or
skeletonization, remains unknown. Surprisingly, even preservation
of the newborn’s body by freezing may cause difficulty in estab-
lishing the cause and manner of death. Kozawa et al. failed to prove
the cause of death on the basis of autopsy findings, although a
police investigation revealed that the infants were smothered by
occlusion of the mouth and nose, and then frozen [3]. Tabata
et al. conducted a case study on a frozen newborn infant who
was found in a freezer at 18 °C [8]. They found froth within the
upper respiratory tract and evidence of vitality in pulmonary
tissue. This case showed that froth persisted in the internal air pas-
sages for a long time as a result of freezing. Moreover, froth in the
air passages, along with the findings of the lungs, demonstrated
that the newborn infant was born alive.
In our case, a well preserved female newborn was found after
8 years in an artificially frozen environment. Based on the position
of the body prior to the thawing process and the absence of birth
trauma, such as caput succedaneum or subperiosteal hemorrhages,
we assume delivery in breech presentation. In contrast, the preva-
lence of caput succedaneum, according to medicolegally oriented
study, was only 33.9% [25]. Moreover, the abovementioned study
found a correlation between the number of previous pregnancies
and the presence of birth-associated head injuries. Breech presenta-
tion is still associated with statistically significant perinatal
mortality.
Finally, from the jurisdictional viewpoint, the mother was found
not guilty of concealing her newborn’s death. In most jurisdictions,
concealment of birth is a relatively minor crime and is basically an
offence against registration, in that the body of a newborn is not
reported to the authorities or accorded a proper burial or other
form of disposal [12].
In conclusion, examination of a newborn infant following an
extremely prolonged freezing interval is presented. Despite the
fact that death was 8 years ago, tissue preservation was adequate
for answering forensically important questions. To investigate
pathological findings potentially associated with freezing, we also
conducted a study from a histological, as well as immunohisto-
chemical approach. The following points should be noted:
 It is important to be completely familiar with the location
and circumstances of the concealment of the body prior to
autopsy.
 Although prolonged freezing interval neither macroscopically
nor histologically noticable changes associated with freezing
are often presented.
 J. Krajčovič et al. / Legal Medicine xxx (2014) xxx–xxx
 Detectability of particular histological findings such as amniotic
fluid aspiration in lungs after freeze–thaw process remains
immunohistochemically possible when conventional H&E
staining no longer shows results.
 In cases of frozen neonatal remains, accurate estimation of via-
bility, survivability, and cause of death is possible.
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