Journal of Forensic and Legal Medicine 61 (2019) 65–68
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine
journal homepage: www.elsevier.com/locate/yjflm
Research Paper
Comparison of post-mortem ethanol level in blood and bone marrow
∗
M. Iskierka , M. Zawadzki, P. Szpot, T. Jurek
Department of Forensic Medicine of Wroclaw Medical University, Poland
1. Introduction
Despite the dynamic development of chemical and biological sci-
ences, the contemporary legal and medical thanatology still faces a
number of challenges. One of the current problems is the search for
alternative biological matrices for toxicological studies. Although
blood, urine and the vitreous humour are usually used as a biological
material in routine toxicological tests, there are many situations in
which the availability of these biological materials is partially or
completely restricted. In order to answer the question whether the xe-
nobiotic was present in the body of the deceased at the time of death,
and if so, at what concentration and if it could become the cause of
death, it is necessary to introduce the so-called alternative biological
materials into the toxicological analysis.
Bone marrow is one of such materials1 From the point of view of
forensic toxicology, its advantages are availability and durability.
Thanks to the location of the bone marrow inside the bone, the material
is protected against the impact of external factors and the destructive
effect of bacteria and fungi; at the same time, this protection delays the
process of bone marrow decomposition in comparison with other tis-
sues. These features are particularly important when it is necessary to
take toxicological samples from decaying or exhumed bodies. In addi-
tion to standard analyses focused on ethanol and narcotic, bone marrow
can also be used for medicine analysis, e.g. opioid drugs, due to very
high vascularisation and high lipid content.2–7 Therefore, in respect of
forensic toxicology, bone marrow can be considered as a specific re-
pository of various xenobiotics.8
Only few papers on the usefulness of bone marrow in forensic tox-
icology have been published so far. These studies suggest a linear re-
lationship between the concentrations of xenobiotics in blood and bone
marrow. Their authors also point out the necessity of the evaluation of
the pre-analytical phase when interpreting the results. In the case of
bone marrow, standards for the collection, preparation and storage of
biological material as well as the analytical method used have not been
developed. Meanwhile, data from the literature informs that the loca-
tion of collection (femoral BM is fattier than rib BM), the conditions and
duration of material storage (bone marrow should be analysed as soon
as possible) influence the results of toxicology tests.9–16
The objective of the paper was to assess the degree of correlation of
∗
Corresponding author. Department of Forensic Medicine of Wroclaw Medical University, ul. Jana Mikulicza-Radeckiego 4, 50-345, Wrocław, Poland.
E-mail address: martais91@onet.eu (M. Iskierka).
https://doi.org/10.1016/j.jflm.2018.11.003
Received 3 May 2018; Received in revised form 29 October 2018; Accepted 7 November 2018
Available online 10 November 2018
1752-928X/ © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
T
ethyl alcohol concentration in two biological materials derived from
autopsies – blood and the bone marrow.
2. Materials and methods
2.1. Materials
The biological material (peripheral blood and the bone marrow)
was collected from 100 people who had a forensic and medical autopsy
performed at the Department of Forensic Medicine between 2015 and
2017. The test group consisted of cases in which the cause of death was
a suspicion of poisoning with a given xenobiotic or in which there was a
high probability of positive results of toxicology tests (noticeable smell
of alcohol, alcohol abuse). Peripheral blood samples were collected by
puncturing the femoral vein during external examinations, while the
bone marrow was collected by aspiration or curettage from the wing of
the ilium. Until the studies were performed, the material was secured in
test tubes with the addition of sodium fluoride, which were labelled
appropriately and stored in a refrigerator at 4 °C The tests were con-
ducted within a maximum of two days from the moment the biological
material was collected.
For further investigation the study group was divided into 5 sub-
groups according to age (people under 30, people between 31 and 40,
people between 41 and 50, people between 51 and 60, people over 60).
The size of the various groups is presented in Table 1.
2.2. Analytical procedures
The concentrations of ethyl alcohol were determined with the use of
chromatography with a FID detector and the headspace analysis tech-
nique. The studies were carried out on the Agilent 7890A GC System
gas chromatograph with two J&W columns with different polarities:
DB- ALC1 30 m × 0.25 mm × 1.8 μm and DB- ALC2
30 m × 0.25 mm × 1.2 μm.
To determine the concentration of ethyl alcohol, the biological
material at a volume of 50 μl and 2 ml of a solution of 1-Propanol at a
concentration of 0.24 mg/ml as an internal standard were introduced
into a glass chromatography test tube. The sealed test tube was placed
in an automatic sample feeder - Agilent G1888 Network Headspace
M. Iskierka et al. Journal of Forensic and Legal Medicine 61 (2019) 65–68

Table 1 2.4. Statistics

Relationship between the level of alcohol in particular biological materials in
relation to the age of the subjects. *** statistically significant. The statistical analysis was performed using the IBM SPSS Statistics
Biological material Bone marrow 24 package. The value p < 0.05 was considered statistically sig-
nificant. With the use of Spearman correlation analysis, it was ex-
Blood < =30 years old [n = 25] 0.9*** amined whether there is a statistically significant relationship between
31–40 years old [n = 21] 0.97***
the variables studied. The analysis with the Mann-Whitney U test made
41–50 years old [n = 15] 0.98***
51–60 years old [n = 19] 0.99*** it possible to examine whether there are statistically significant differ-
> 60 years old [n = 20] 1*** ences between the two independent groups. When comparing more
than two groups, the analysis of variance was used. A relevant post-hoc
***p < 0.001. test was used in the case of statistically significant differences. The
selection was based on the homogeneity of variance in the compared
Sampler. The chromatographic separation was carried out at 40 °C groups. The arithmetic mean and standard deviation were used for
using helium with a purity of 5.0 (99.999%) as a carrier gas. quantitative variables in the statistical analysis of the results.

3. Results
2.3. Validation
The analysis of the concentration of ethyl alcohol in the biological
The method is specific for ethanol, resulting each time a well es-
material (blood and bone marrow) was carried out within a group of
tablished RT (retention time), 2.169 min (ALC1, FID-1) and 2388 min.
100 cases. 78 of them were men aged 19–78 (average - 46 years,
(ALC2, FID-2). For n-propanol the values of RT were 3581 min.(ALC1,
SD ± 17.4) and 22 were women aged 17–74 (average - 44 years,
FID-1), respectively 4278 min.(ALC2, FID-2). The calibration curves
SD ± 17.2). Positive results of ethyl alcohol content tests (above
obtained were linear, and the correlation coefficient obtained were
0.1 mg/g) were recorded in 56 cases; 41 cases were negative in terms of
0.99994 (ALC1-FID-1) and 0.99994 (ALC2-FID-2). This value is over
ethyl alcohol content in both biological materials analysed, 3 cases
0.999, indicating a relationship of direct proportionality between the
were positive only for one biological material (blood or bone marrow).
concentrations of standard solutions and the peak area.
The most common causes of death included fatal poisoning and
For precision, recovery and relative error evaluation we analysed
hanging, the average time from the moment of death to the moment of
ten times the solutions corresponding to three points from calibrating
biological material collection was 131 h, and no cases of advanced
curve, respectively with concentrations of: 0.2, 1.0 and 3.0‰. The
putrefaction were observed. The average concentration of ethyl alcohol
values for the ratio of recovery were between 99.90% and 102,6%. The
in blood was 0,68 (SD ± 1,09) mg/g, and in bone marrow 0,59
values for the ratio of precision were between 2,7% and 3,1%. The
(SD ± 0,96) mg/g.
values for the ratio of relative error were between 2,67% and 3,33%.
The next step was to examine whether individual age groups differ
The limits of quantitation (LOQ) was 0,1‰.
in a statistically significant way in terms of the concentration of ethyl
alcohol in different biological matrices. What is interesting is the fact
that in case of people over 60, a slightly higher concentration of ethyl

Fig. 1. Relationship between the concentration of alcohol in bone marrow and blood in the study group (n = 100, R = 0.97, p < 0.001).

66
M. Iskierka et al.
alcohol was observed in the bone marrow compared to blood as op-
posed to other age groups.
An important element of the statistical analysis of the obtained re-
sults was also the determination of correlation factors between the
concentration of ethyl alcohol in bone marrow and its concentration in
blood. Very strong, linear correlations between the concentration of
alcohol were found in blood and bone marrow (R = 0.97, p < 0.001),
the obtained data are shown in Fig. 1. It was also examined whether age
and sex have an effect on the results. The obtained data reveal that the
correlation coefficients differ slightly within the sexes (for women and
men, respectively: blood vs bone marrow R = 0.96 and 0.98), and ages.
It was observed that the strongest correlations occur within the oldest
age groups. However, these difference are not significant, which in-
dicates the usefulness of bone marrow irrespective of the age or sex of
the subjects. Table 1 presents the obtained correlation coefficients of
ethanol concentration in individual biological materials depending on
the age of the subjects.
In addition, by dividing the study group depending on the con-
centration of ethyl alcohol in blood, significant differences in correla-
tion coefficients were also observed. For blood and bone marrow, these
coefficients were: 0.6 (0.1–1.0 of ethanol in blood), 0.6 (1.01–2.0 of
ethanol in blood) and 0.92 (over 2.01 of ethanol in blood). The ob-
tained values indicate that it is necessary to try to explain this phe-
nomenon.
4. Discussion
Studies on the assessment of the degree of correlation of the in-
dividual xenobiotic concentrations in bone marrow and blood were
initiated in the 1990s by Winek et al. At that time, the studies were also
carried out on an animal model, and not only ethanol, but also iso-
propanol and pentobarbital were determined in biological materials in
a series of publications. Even at that time, the authors drew attention to
the important issue of lipid content in bone marrow.11,12 Due to higher
solubility of alcohol in water as compared to lipids, alcohol content will
be lower in bone marrow with a lipid layer.17
In subsequent studies, it was observed that the average concentra-
tion of ethanol in bone marrow is lower compared to other biological
materials. A similar relationship was also observed in our studies. In
case of blood, this phenomenon can be related to the difference in tissue
hydration, and in case of urine samples, it is probably related to the
phase of elimination of ethanol from the body at the time of death of the
deceased.
It should also be remembered that with age, a physiological con-
version of red bone marrow into yellow bone marrow occurs, and the
amount of fat component increases by approximately 7% within a
year.18,19 These data leave the authors wondering whether ethanol
concentration in bone marrow compared to other materials will depend
on age and sex. So far, no study has been published in which subgroups
would be distinguished on the basis of age or sex; however, the data
obtained by us show that sex does not significantly affect the average
ethanol concentration in individual materials. It seems that age will
also have a more significant impact on the obtained correlation coef-
ficients for bone marrow versus other biological materials rather than
on average ethanol concentration results.
The data obtained by us indicate a strong correlation between the
results achieved for bone marrow and blood. Publications in which the
correlation coefficients of ethyl alcohol concentration in blood and
bone marrow or in urine and bone marrow were determined are rare. In
their studies, Tomingawa et al. carried out a long-standing, retro-
spective analysis of more than 300 cases. The studies were conducted
over a period of over 16 years, and such a long time span allows for
differentiation of the study group in terms of sex, age, death circum-
stances, or conditions in which the corpses was kept. Thanks to the
separation of appropriate “subgroups”, it is possible to determine
whether certain circumstances of death, e.g. drowning or old age, affect
67
Journal of Forensic and Legal Medicine 61 (2019) 65–68
the correlation of the obtained results. According to the authors, the
correlation coefficient of blood versus bone marrow was at a level of
r = 0.98 and was slightly lower in case of people who drowned.14
Studies spanning for several years were also carried out in Osaka. Al-
though the study group consisted of nearly 150 cases, no ethanol was
detected in blood and bone marrow in most of them. Positive results in
terms of the presence of ethyl alcohol and a limited possibility of bone
marrow collection narrowed the study group down to only 20 cases of
autopsy, on the basis of which the correlation coefficient equal to
r = 0.983 was determined.5
In our studies, the obtained correlation coefficients were as follows:
blood vs bone marrow r = 0.97; The obtained correlation coefficients
were not differentiated depending on the age and sex of the subjects in
any of the above-mentioned studies. This paper constitutes an attempt
to perform such a differentiation and analyse the correlation coeffi-
cients in particular subgroups. This analysis shows that with age, the
obtained correlations of ethanol concentrations between blood and
bone marrow become stronger (blood vs bone marrow r = 0.91 for the
group of under 30-year-olds and r = 0.99 for the group of over 60-year-
olds).
The results of our study reveal that the most linear relationships
between the levels of ethyl alcohol in bone marrow and in blood occur
when the concentration of ethanol in blood is above 2.0 mg/g. It should
be mentioned here that the exact distribution processes and other
pharmacokinetic processes occurring in bone marrow were not well
understood. Perhaps this result from the cases where despite low or
even no concentration of ethanol in blood, there was a higher con-
centration of ethanol in bone marrow, which raises the question of the
possibility of potential accumulation and neoformation.17 It appears
that the time that elapsed from the moment of death to the moment of
collecting the biological material may also be important. In case of our
studies, the average time between the moment of death and the medical
and legal autopsy was 131 h. The obtained data reveal that this time
was statistically significantly longer in case of alcohol concentration
equal to 0 in comparison to the cases where the ethanol concentration
was different than zero. The papers published so far indicate that the
stability of bone marrow samples depends on the temperature in which
the biological material was stored.20 However, the inability to estimate
the level of ethyl alcohol in bone marrow concerned the cases when the
material was stored at room temperature for more than 28 days.13 The
processes of elimination of xenobiotics in relation to bone marrow have
not been fully elucidated; however, it has been proven that even in the
case of skeletonisation or combustion of the corpse, i.e. in a situation
where it is impossible to collect routine biological materials, it is still
possible to conduct quantitative toxicology tests in the bone
marrow.17,21
In relation to determining ethanol concentration in the body at the
time of death, in addition to the determination of ethanol alone, it may
also be useful to determine ethyl glucuronide in this material, which is
formed by enzymatic conjugation of ethanol with glucuronic acid.
Schloegl et al. proved that this compound may successfully be detected
in the bone marrow. More importantly, it has been proven that this
compound can last in the bone marrow for a long time and the possi-
bility of post-mortem ethyl glucuronide synthesis has been eliminated.
These conclusions are extremely important because the simultaneous
determination of ethanol metabolites and ethanol itself can help to
verify the hypothesis of the possible formation of endogenous alcohol
and, thus, help in the interpretation of the obtained toxicological re-
sults.22
5. Conclusion
To sum up, despite many unresolved issues related to bone marrow
pharmacokinetics, this material can be used to evaluate the presence of
ethyl alcohol in the body when routine biological materials are not
available. The correlation coefficients of blood versus bone marrow
M. Iskierka et al.
indicate strong relationships between the biological materials under
discussion, which demonstrates the usefulness of bone marrow ex-
amination in forensic and medical toxicology.
Conflict of interest
Declarations of interest: none.
Acknowledgements
This work was supported by Ministry of Science and Higher
Education of Poland (grant number 0052/DIA/2014/43).
References
1. Frederick DL. Toxicology testing in alternative specimen matrices. Clin Lab Med. 2012
Sep;32(3):467–492. https://doi.org/10.1016/j.cll.2012.06.009 Epub 2012 Jul 7.
2. Wietecha-Posłuszny R, Lendor S, Garnysz M, Zawadzki M, Kościelniak P. Human
bone marrow as a tissue in post-mortem identification and determination of psy-
choactive Substances—screening methodology. J Chromatogr B Analyt Technol Biomed
Life Sci. 2017;1061:459–467.
3. de Souza Santos E, Spinelli E, Vieira AA, Rodrigues SV. Postmortem analysis of
famprofazone and its metabolites, methamphetamine and amphetamine, in porcine
bone marrow. Talanta. 2019;191:545–552.
4. McIntyre LM, King CV, Boratto M, Drummer OH. Post-mortem drug analyses in bone
and bone marrow. Ther Drug Monit. 2000;22(1):79–83.
5. Maeda H, Zhu BL, Ishikawa T, et al. Evaluation of post-mortem ethanol concentra-
tions in pericardial fluid and bone marrow aspirate. Forensic Sci Int.
2006;161(2):141–143.
6. Raikos N, Tsoukali H, Njau S. Determination of opiates in postmortem bone and bone
marrow. Forensic Sci Int. 2001;123(2):140–141.
7. Travlos GS. Normal structure, function, and histology of the bone marrow. Toxicol
Pathol. 2006;34(5):548–565.
8. Guillot E, de Mazancourt P, Durigon M, Alvarez JC. Morphine and 6-acetylmorphine
68
Journal of Forensic and Legal Medicine 61 (2019) 65–68
concentrations in blood, brain, spinal cord, bone marrow and bone after lethal acute
or chronic diacetylmorphine administration to mice. Forensic Sci Int.
2007;166(2):139–144.
9. Winek CL, Pluskota M, Wahba WW. Plasma versus bone marrow flurazepam con-
centration in rabbits. Forensic Sci Int. 1982;19(2):155–163.
10. Winek CL, Westwood SE, Wahba WW. Plasma versus bone marrow desipramine: a
comparative study. Forensic Sci Int. 1990;48(1):49–57.
11. Winek CL, Morris EM, Wahba WW. The use of bone marrow in the study of post-
mortem redistribution of nortriptyline. J Anal Toxicol. 1993;17(2):93–98.
12. Winek CL, Jones T. Blood versus bone marrow ethanol concentrations in rabbits and
humans. Forensic Sci Int. 1980;16(2):101–109.
13. Winek CL, Luhanik JM. A storage study of ethanol in rabbit and human bone
marrow. Forensic Sci Int. 1981;17(2):191–196.
14. Tominaga M, Ishikawa T, Michiue T, et al. Postmortem analyses of gaseous and
volatile substances in pericardial fluid and bone marrow aspirate. J Anal Toxicol.
2013;37:147–151.
15. Bévalot F, Dujourdy L, Fanton L, et al. Statistic interpretation of meprobamate
concentrations in bone marrow, vitreous and bile. Proceedings of International Meeting
of the International Association of Forensic Toxicologists, February 18–23, Washington,
United States of America. Abstract K59. 2008; 2008:431.
16. Cartiser N, Bévalot F, Chatenay C, et al. Postmortem measurement of caffeine in bone
marrow: influence of sample location and correlation with blood concentration.
Forensic Sci Int. 2011;210(1-3):149–153.
17. Cartiser N, Bévalot F, Fanton L, Gaillard Y, Guitton J. State-of-the-art of bone marrow
analysis in forensic toxicology: a review. Int J Leg Med. 2011;125(2):181–198.
18. Travlos GS. Histopathology of bone marrow. Toxicol Pathol. 2006;34(5):566–598.
19. Liney GP, Bernard CP, Manton DJ, Turnbull LW, Langton CM. Age, gender, and
skeletal variation in bone marrow composition: a preliminary study at 3.0 Tesla. J
Magn Reson Imag. 2007;26(3):787–793.
20. Winek CL, Janssen JK. Blood versus bone marrow isopropanol concentrations in
rabbits. Forensic Sci Int. 1982;20:11–20.
21. Tattoli L, Tsokos M, Sautter J, et al. Postmortem bone marrow analysis in forensic
science: study of 73 cases and review of the literature. Forensic Sci Int.
2014;234:2–78.
22. Schloegl H, Rost T, Schmidt W, Wurst FM, Weinmann W. Distribution of ethyl glu-
curonide in rib bone marrow, other tissues and body liquids as proof of alcohol
consumption before death. Forensic Sci Int. 2006;156:213–218.