J Punjab Acad Forensic Med Toxicol 2012;12(2)

Review article
POST MORTEM BIOCHEMISTRY- SAMPLING AND PRESERVATION
Dr. Rajanikanta Swain
Dr. Karthik Krishna
Dr. S R Singh
Dr. C. Behera, Assistant Professor, *
*Department of Forensic Medicine, All India Institute of Medical Sciences,New Delhi, India
Article history

Abstract

Received Sep30, 2012

Recd. in revised form Dec 24, 2012
Accepted on Dec 24, 2012
Available online Dec 25, 2012

Sampling and preservation of biological fluids for analysis, after

death forms an important part of post-mortem examination.
Analysis of various body fluids helps to ascertain the cause of
death as well as time since death. In developing countries like
Corresponding author
India, where there are lack of round-the-clock laboratories for
Dr C. Behera
immediate analysis, autopsy surgeons also face dilemmas
Phone: +919968320486
regarding the preservation of the body fluids collected at autopsy.
Email: drchitta75@rediffmail.com
It is hence necessary to follow a standard, practicable
methodology in collection as well as preservation of such
specimens so as to expect reliable laboratorial results. Efforts to
review the standard sampling techniques are made in this paper.
2012 JPAFMAT. All rights reserved
Keywords: Post-mortem Biochemistry; Body fluids; Preservation
Introduction
Postmortem biochemistry is an important, but
seldom practised tool in death investigations.
Biochemical analyses of different biological fluids
such as blood, vitreous humor, urine,
cerebrospinal fluid, synovial, pleural and
pericardial fluids are not only important in cases of
sudden natural deaths, but also in estimation of
time since death, in detection of various poisons,
and for better understanding of pathophysiology of
disease process[1].
Coe (1993) considered forensic biochemistry as one
of the important ancillary procedures to be
followed by a forensic pathologist [2]. The
necessity of such investigations should be decided
on case to case basis. In cases of hospital deaths,
the samples collected and investigations results
obtained during the course of treatment should be
taken into consideration. The autopsy surgeon
should have the knowledge about the selection of
sample in a particular case and at the same time
should have the knowledge of preservation and
techniques of collection of such biological samples.
It is important to follow the correct sampling
procedures as well as its proper preservation, as
reliability of laboratorical results is dependent
upon all these factors. A brief history, nature of
the sample and the preservative used should be
clearly mentioned in the laboratory request form.
The analysis report will thus provide the scientific
foundation that will help in the corroboration of
investigators theory, which can be used in
administration of justice in the court of law. Due to
lack of proper guidelines for collection of biological

samples during post-mortem examination, the

results of such investigations are often adversely
challenged in courts. Not only that many a times
the autopsy surgeon neglects to preserve the
biochemical samples, and even if he collects, there
exist faulty sampling methods or inefficient
preservation due to which vital findings are missed
or destroyed. Hence we have made an effort to
standardize the sampling method for biochemical
analysis in post-mortem cases, so that consistent
comparisons to the database can be achieved.
Collection of synovial fluid [3].
How to aspirate knee joint: Deceased in supine
posture with knee slightly flexed. After thorough
cleaning and antiseptic preparation, point of
aspiration is visually fixed. Then 18-16 gauge
aspiration needle is inserted, just superior to the
upper pole and lateral to the lateral border of the
patella at the level of patella-femoral joint. The
aspiration needle is directed horizontally and at
right angles to the long axis of the limb. The needle
should enter the joint capsule deep to the
quadriceps tendon. Fluid the joint (Most of the
synovial fluid remains in the supra-patellar pouch,
which is always in continuation with the joint
cavity) which can be easily aspirated by 20-50 ml
of syringe.
In case of difficulty in getting the fluid back, (as in
case of needle is blocked by a bit of synovium in
such a case, suction is released, the bevel moved
down or slightly in or out and tried again.
Preservation for synovial: The synovial fluid
samples should be examined immediately or

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within a few hours after arthrocentesis [4-5].If a

delay cannot be avoided, it is recommended, to
preserve the joint fluid samples in heparinized
containers at 4 degree centigrade, so as to
conserve the number of cell counts and crystal
survival. [6-9]Salinas et al. demonstrated the
advantage of EDTA as an anticoagulant to stabilize
the leukocyte number in synovial fluid samples at
24 and 48 hours[10].
Collection of Blood:
1. Femoral vein puncture: [11] Deceased in
supine position-legs extended.
Collection of blood samples are best obtained
from femoral vein by percutaneous puncture.
Femoral vein is located in inguinal canal 1 cm
medial to the midpoint of the line joining the
anterior superior iliac spine and the pubic
tubercle. A 30 or 50 ml syringe with a wide
bore needle is inserted perpendicularly at that
point and about 20 ml blood is aspirated. In
case there is stoppage or failure for obtaining
blood then aspiration is again tried after
exposing the femoral vein by dissection and
clamping or ligating it proximal to the
collection site.
2. Collection of blood from subclavian
vein [11]: Deceased in supine position. A
head support is placed in between the
scapulae and head turned to opposite side.
The index finger of non-dominant hand is
placed at the sternal notch and the thumb
of the same hand on the clavicle (Lateral
one-third and medial two-third of the
clavicle). The needle is introduced with
the bevel upwards and skin is entered 2
cm lateral to the thumb, 2 cm inferior to
the clavicle. The needle is directed by
aiming at the sternal notch, with the axes
of the needle parallel to the floor. Blood is
aspirated.
Collection of blood from the heart for toxicological
analysis is to be avoided, as substances in the
stomach and intestine can diffuse after death to the
organs in the thorax, causing false raise in the
blood level. It is also advised to strictly avoid
collection of blood from the paracolic gutter owing
to the high possibility of contamination.
Preservation of blood: 10mg per ml sodium or
potassium fluoride and 3 mg potassium oxalate
should be used for preserving the blood. Sodium
fluoride protects blood from post-mortem changes
such as bacterial production of ethanol and other
alcohol and also helps to protect other labile drugs
such as cocaine, nitrazepam, clonazepam from
degradation[12].
Vitreous humor collection method: [13]

Luna A in his review study expressed that in terms

of accessibility the vitreous humor is most easy to
collect. [14]
A sterilized 14-16G needle is introduced 4mm
lateral to limbus of each eye, the axes of the needle
parallel to the floor, till the tip of the needle
reaches the centre of eye ball. Gently aspirate 2-2.5
ml crystal clear vitreous humor without exerting
much pressure. Without displacing the needle, the
syringe is removed and same amount of
saline/water is injected back into the eyeball to
maintain the contour of the eyeball.
Preservation of Vitreous: sodium fluoride is
added if estimation of alcohol, cocaine, cyanide and
carbon monoxide is to be done.
Collection of Cerebrospinal fluid (CSF):
1. Cisternal Puncture method: This method
is ideal as well as relatively easier to
obtain CSF. A minimum of 0.5 ml is
required for glucose estimation. Deceased
is placed in prone position with head
support below the chest and neck in
flexion. The atlanto-occipital membrane is
palpated in the midline. The disposable
spinal needle is gently introduced through
the skin directed towards the bridge of the
nose. Loss of resistance is felt at a depth of
approximately 2cm where the needle
pierces the meninges and then CSF is
aspirated from that point.
2. CSF collection from lateral ventricle :
After opening the skull cavity, CSF is
aspirated directly by inserting a needle
through the corpus callosum into the
lateral ventricle
3. After Evisceration: This method is least
advisable for CSF collection due to high
rate of contamination.
CSF can be aspirated anteriorly after
evisceration by introducing a needle into
the spinal theca through the spinal
Foramina between L1 and L2.
4. Preservation of CSF: [15-16]Sodium
fluoride is added if estimation of alcohol,
cocaine, cyanide and carbon monoxide is
to be done. A revised guidelines on CSF in
suspected sub- arachnoid haemorrhage
recommended use of fluoride EDTA/oxalate vials for protein and
glucose estimation. If xanthochromia is
suspected, a minimum of 1 ml is placed in
sterile container protected from light and
sent to lab for specto-photometric scan.
Collection of urine: [17]
1. Direct puncture method: Deceased in
supine position A needle with syringe is
introduced through the skin just above the
pubic symphysis on the midline into the

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bladder with the direction of needle

towards umbilicus at a 100 to 200 angle
from the perpendicular at midline. Gently
aspirate while introducing the needle. If
no urine is aspirated, withdraw the needle
to the subcutaneous space and re-advance
in a slightly different direction, 100 caudad
or cephalad and aspirate again.
In case the above method fails to obtain urine,
then abdomen is opened first, exposing the dome
of urinary bladder and then it is punctured with a
sterile needle and syringe through the dome and
urine is aspirated.
2. Catheterization of the bladder: Insert a
urinary catheter through the urethra into
the bladder before the start of autopsy and
collect the urine in a sterile container.
Preservation of Urine: [18] One ml of
concentrated Hydrochloric acid or 0.1 g of Thymol
is added to 100ml of urine. Toluene is considered
as a better preservative. It is also suggested that
urine can be preserved for a short period in
refrigerator even without preservatives at a
temperature of 40 C.
Collection of Bile: Deceased in supine positionAbdomen is opened by conventional midline
incision. Gall bladder is exposed by carefully
raising the inferior surface of the liver. A sterile
needle with syringe is gently introduced into the
gall bladder through the fundus and bile is
aspirated. Many a times due to high viscosity of the
contents it is difficult to obtain the bile through
aspiration, in such cases a small nick can be given
at the fundus of gall bladder and contents can be
gently squeezed. [18]In case of cholecystectomy
patient the bile may still be obtained from the
common bile duct by aspiration.
Preservation: It is not routinely preserved, but
only in selected cases. It is preserved in 30 ml glass
screw capped container and transferred to lab for
analysis.
Collection of pericardial fluids: [19]Body in
supine position. Thoracic cavity is opened by
standard midline incision by cutting the ribs away
from the heart. Once the chest is exposed, the
pericardial sac is opened near the apex of heart
using a scissor. The heart is lifted up gently and the
content of the sac inspected and measured.
Normally the pericardial sac contains about 30-50
ml of fluid. Using a sterile syringe and needle,
about 10ml of pericardial fluid is aspirated.
For pleural fluid: [19] The chest cavity is opened
in the above mentioned manner and the pleural
sacs are inspected by lifting the lower lobe of lungs
gently out of the chest cavity. Normally at autopsy,
pleural cavity does not contain any appreciable
quantity of fluid. If it is present it must be collected
with the help of sterile needle and syringe and

measured before dispatching to biochemical

analysis.
Preservation of pericardial/ pleural fluids:
[20]For most chemical examination, no additive is
used. The samples are to be analysed immediately,
if not possible then it is suggested that it is
maintained in a common refrigerator (4-8 C) and
not in a freezer until the delivery to the laboratory.
Conclusion
We emphasize that postmortem sampling should
be an essential practice especially in cases of
sudden deaths, poisoning, drug overdoses and
hypersensitivity. Efforts should be taken towards
collection of adequate amount of samples, so that
all required analyses can be performed.
Biochemical studies along with other laboratory
and postmortem findings should be considered
together, to arrive at a logistic conclusion. Instead
of being over-reliable on a single test, we suggest
that, the results of multiple biochemical tests
should be considered in conjunction for accuracy.
Conflict of Interest
None Declared
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This article can be cited as:

Swain R, Krishna K, Singh S, Behera C. Post mortem biochemistry- sampling and preservation. J Punjab
Acad Forensic Med Toxicol 2012;12(2):121-4.

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