During the investigation of accidental cases, blood samples obtained from accidental victim are submitted to forensic science laboratory for toxicological analysis. All through, ethanol analysis is performed on almost all cases of blood samples. The main problem is determining alcohol concentration in these cases is changes in Blood Alcohol Concentration (BAC) that can occur due to neoformation of ethanol. Blood samples stored under insufficient conditions or for longer times and body fluids have undergone petrifaction often contains certain amount of volatile compounds. In the light of possibility that, neoformation of ethanol in improperly stored blood might give rise to an incorrect estimation of BAC. With this in mind, to study the effect of preservative and temperature on the formation and concentration of ethanol in blood samples, we estimated the ethanol neogenesis in stored and normal blood in different time period (0, 7, 14, 21, 28, 35 and 42 days). Head Space- Gas Chromatography instrument used to measure the level of ethanol in blood sample. We found that neoformation of ethanol was observed in normal blood sample. There was no any neoformation observed in blood sample with preservative and blood stored at 4°C. Our study is supporting the studies which suggest that storage condition will affect the neoformation of ethanol in blood samples.
Keywords: Blood Alcohol Concentration; Neoformation; Head Space-Gas Chromatography; Ethanol.
Determination of alcohol concentration is of great importance in medico legal autopsy blood samples, regardless of whether civil, criminal, or insurance law is involved. Blood alcohol cases in living individuals is often performed in forensic practice (e.g. after traffic accidents). Medico legal problems are not confined to determining alcohol as either a sole or joint cause of death, such as in accidents, but also include the problem of distinguishing true alcoholic poisoning. It is known that alcohol can be produced in blood after sampling. The amount of generated ethanol depends on the species of microorganisms present, the available substrates, the temperature and time of storage and the presence of preservatives added to the specimens. Neogenesis means regeneration of biological tissue or the formation of new products.
Transformation and degradation of the well-known body structures (e.g. enzymatic digestion of cell components and membranes), together with accumulation of bacterial metabolites are likely to generate a huge range of new chemical compounds. Blood samples stored under insufficient conditions or for longer times and body fluids have undergone petrifaction often contains certain amounts of volatile compounds. A number of ubiquitous microorganisms are capable of forming
or degrading alcohol, and neogenesis of ethanol is only a
byproduct of bacterial decomposition processes. The reactions that take place are mainly influenced by temperature, pH value, concentrations of available carbohydrates, and the presence of other utilizable nutrients. Putrefaction or decomposition is the final stage produced mainly by the action of bacterial enzymes mostly anaerobic organisms. These destructive bacterial agents cause marked haemolysis, liquefaction of clots and fresh thrombi and emboli, disintegration of tissues and gas formation in blood. Bacteria produces a large variety of enzymes and these breakdown the various tissues of the body. There is a progressive alteration of proteins, carbohydrates and fats. Putrefaction begins mainly by reductive processes due to the action of endogenous and exogenous bacteria and their enzymes and decay, based on oxidative reactions. Body tissues remain bacteriologically sterile from exogenous infection for at least 20 hours after death. Whereas advanced putrefaction of a blood sample can be recognized macroscopically and by its odour, the transitional phase into putrefaction presents difficulties. As the blood decomposes its colouring matter transduce into the tissues which become uniformly red. The colour becomes darker and finally turns black. The most abundant volatiles detected during the forensic ethanol analysis are ethanol, acetaldehyde, 1-propanol, 2-propanol and acetone. These volatiles could either
be initiated in the human body after the consumption of alcoholic
beverages; or have been produced later during metabolic
processes or by microbes. Alcohol concentration often changes
in putrefying blood. These changes might be caused by either
a change in the level of ethanol or to the formation of higher
alcohols, aldehydes and ketones. Even though “fresh” samples
of blood can contain a variety of higher alcohols. Therefore, the
presence of such alcohols must be a result of neogenesis within
the corpse or the stored blood sample.
Anticoagulant is a substance that prevents blood from
clotting by suppressing the synthesis or function of various
clotting factors. The first anticoagulant preservative was
introduced by Rous and Turner in 1916. It consisted of a citrateglucose
solution in which blood from rabbits was stored for two
weeks, which prevented anaemia when transfused in another
rabbit who had suffered from blood loss. Some of the commonly
used anticoagulants are:
EDTA: Ethylen Ediaminetetra Acetic Acid as disodium or
potassium salts is used. This is a chelating agent which binds
the calcium which is needed for coagulation. It is effective at a
final concentration of 1 to 2 mg/ml of blood. More than 2mg/ml
causes shrinkage of the cells. This is the best anticoagulant for
peripheral blood smear and studies.
Drawbacks: It inhibits the activities of enzymes like alkaline
phosphatase, creatine kinase, and leucine aminopeptidase. EDTA
is not suitable for calcium and iron estimation.
Heparin: It is mucoitin polysulfuric acid available as sodium
potassium, lithium and ammonium salts. Heparin accelerate the
action of antithrombin III ——> which neutralizes thrombin
——> thus prevents the formation of ——> fibrin from
fibrinogen. Heparin is added 0.2 mg /ml of blood.
Drawback: It inhibits the acid phosphates activity. It
interferes with binding of calcium to EDTA.
Oxalate: This form insoluble complex with calcium ions.
Potassium oxalate at concentration of 1 to 2 mg/ml of blood is
used. Combination of ammonium/potassium oxalate does not
lead to shrinkage of the RBCs.
Drawbacks: If the concentration is 3 mg/ml, then there are
chances for hemolysis. Oxalates inhibit several enzymes like acid
phosphates, alkaline phosphates, amylase, LDH, and may cause
the precipitation of calcium as oxalate salt.
Sodium Fluoride: This is a weak anticoagulant but used
antiglycolytic agent to preserve the glucose. This inhibits the
system involved in glycolysis and preserves the glucose. This is
effective at a concentration of 2 mg/ml of blood along with other
anticoagulant like oxalate.
Drawback: This is also inhibitor of many enzymes and also
effect urease for the estimation of urea.
When blood is stored at 2-6°C, glycosis is reduced but does
not stop. Preservative solutions provide buffering capability
to minimize pH changes and optimize the storage period. The
lower temperature keeps the rate of glycolysis at lower limit and
minimizes the proliferation of bacteria that might have entered
the blood unit during venipuncture or from atmosphere. The
rate of diffusion of electrolytes (Na+ and K+) across the cell
membrane is also less at lower temperature.
Additive Solutions: One major benefit of the additive system is
increase in the level of ATP, and red cells viability is enhanced,
extending the shelf-life of the red cells to 42 days.
Blood sample of healthy individual was collected from blood
bank Sh. Narayan Hospital Rewari, Haryana.
Three flasks were taken having human blood (60ml) in each
flask. Chemical used: (a) Sodium fluoride (as preservative),
this inhibit the system involved in glycolysis and preserve the
glucose (b) n-propanol (for internal standard) standards are
used by which can be relate the concentration of the standard to
concentration of the peak of the ethanol. Samples were analysed
using instruments Head space - Gas Chromatography.
Blood samples were divided into three conical flasks.
a) With 500mg sodium fluoride (preservative) at room
b) Without preservative at room temperature
c) Without preservative at cold temperature.
All three flasks were kept at their respective sites up to
completion of the study from 16 February 2016 to 31 March 2016.
Two vials of 1ml each were prepared from each conical flask.
Three vials with internal standard (90ul/1ml of blood sample)
and three without internal standard. Blood was prepared for HSGC.
Three vials were subjected for qualitative study and another
three for the quantitative analysis. After instrumentation graph
was prepared and studied (Figure 1 & Table 1).
Headspace GC is used for the analysis of volatile and semivolatile
organics in solid, liquid and gas samples. The headspace
method is especially suitable for the very fast separation of volatile
components (alcohols, acetone, aldehydes) in complex biological
matrices especially blood in mass-liquor and prohibition law
related cases. This method has the advantage of avoiding the
risk of contamination of non-volatile components, which may be
eliminated due to on-line analysis by gas chromatography. The
principle underlying the headspace analysis is that in a sealed
vial at constant temperature, equilibrium is established between
the volatile components of a liquid sample and the gas phase
above it (the head space). After allowing the time for equilibrium
a portion of the headspace may be withdrawn one by one from
vials using a gas-tight syringe and injected to GC for on-line
analysis (Table 2).
Blood samples stored under insufficient conditions or for
longer times have undergone putrefaction often contains certain
amounts of volatile compounds . The increase of ethanol and
higher alcohols in putrefying blood is often recognized, for which
first and foremost bacteria’s are reasonable . Therefore, ethanol
is only a byproduct of bacterial decomposition processes. The
reactions that take place are mainly influenced by temperature,
pH value, concentrations of available carbohydrates, and the
presence of other utilizable nutrients .
Comparing blood samples will recognize that the qualitative
and quantitative; concentration of alcohols can be quite different.
In this study we examined the alcohol concentration qualitatively
and quantitatively using head space-gas chromatography at the
interval of successive seventh day from 16 Feb 2016 to 31 March
2016. The chromatogram of three different samples at different
time periods is shown in following figures. Putrefaction of a
blood sample was recognized by its odour and color [4,5]. The
predominating compound of alcohol formation in anaerobic
putrefaction is ethanol. Additional formation of methanol,
acetone was also observed only in trace amounts . But this
study was only focused on the formation of Ethanol . The
concentration of ethyl alcohol varies. However no alcohol
was detected at 4°C. Graphs of preservative, cold, and normal
condition is showing ethanol neogenesis in blood (Figure 2-12
& Table 3).
Neo-formation of ethanol is due to microbial action
either post-mortem in the body or from improper storage or
preservation of the biological samples is a common problem
in forensic toxicology. Whether the victim consumed ethanol
ante-mortem or whether the ethanol formed due to putrefactive
processes is an important forensic issue and much research
has been made into this area. Various indicators have been
proposed to identify ethanol neo-formation. In this project
report neo formation of ethanol has been checked by using
different temperature conditions and preservative. The alcohol
concentration qualitatively and quantitatively using head spacegas
chromatography at the interval of successive seventh day
from 16 Feb 2016 to 31 March 2016. The chromatogram of three
different samples at different time periods is shown in figures
given above. At normal room temperature i.e. 20-28°C without
preservation the decomposition of the blood start (putrifaction)
leads to formation of Ethyl alcohol. The concentration of ethyl
alcohol varies (as shown in Table 3). However no alcohol was
detected at 4°C. It was found that the concentration of ethyl
alcohol have varied pattern followed by a slight concentration
decrease after 4th day. Thereafter, ethanol concentration remains
more or less constant. The interpretation of postmortem alcohol
levels is still problematic in the forensic pathological setting and
in the field of legal medicine.
I express my sincere and profound gratitude and deep
regards to prof. Dr. R.K Sharma, Chairperson, Department of
Zoology Kurukshetra University Kurukshtra for allowing me to
carry out the present study. I am highly obliged to Dr. R.K Sarin,
Director Forensic Science Laboratory, NewDelhi – Rohini) for
giving me the opportunity to undergo training course at their
estimated institute. However, it would not have been possible
without the kind support and help of many individuals and
organizations. I would like to extend my sincere thanks to all of
them. I am highly indebted to Mr. Loveleen Kumar Katyal for their
guidance, ideas and constant supervision as well as for providing
necessary information regarding the project and also for their
support in completing the project. I would like to express my
gratitude towards Dr. Jogender Tanwar (maternal uncle) and all member of Forensic Science Laboratory, New Delhi (Rohini) for
their kind co-operation and encouragement which help me in
completion of this project. My thanks and appreciations also go
to my colleague my friend Ritu Malik in developing the project
and people who have willingly helped me out with their abilities.