International J.

of Healthcare & Biomedical Research, Volume: 1, Issue: 3, April 2013, Pages 141-146

Estimation of time since death by means of changes in the eye Vitreous

humour calcium levels
*Dr. Ravindra Baliram Deokar , **Dr. Ajay Tukaram Shendarkar , ***Dr. Sachin Sudarshan Patil

*Asst. Professor & Medicolegal in-charge, Department of Forensic Medicine,

Seth G.S.Medical college & KEM Hospital, Mumbai. 400 012
**Asso. Professor, Department of Forensic Medicine,
NDMVPSs Dr. V.N.Pawar Medical college, Nashik.
***Asst. Professor, Department of Forensic Medicine,
Seth G.S.Medical college & KEM Hospital, Mumbai. 400 012
* Correspondence author mail : ravideo80@gmail.com
..
ABSTRACT:
Introduction: Time since death is a question of day to day challenge faced by teams of investigators during their course of
investigations. Vitreous humour of eye being relatively stable, less susceptible than other body fluids to rapid chemical changes and
contamination, easily accessible; thus it is suitable to estimate postmortem interval (PMI).
Methodology: The present study was carried out in Shri. Vasantrao Naik Government Medical College, Yavatmal, Maharashtra.
The dead bodies brought to the mortuary were used as material for collection of vitreous humour sample from the eyes of deceased.
The vitreous analytes have been investigated to establish their correlation ship with PMI or time since death.
Conclusion: A significant linear correlationship, at various degrees, was seen to exist between PMI and vitreous calcium can be
adjunct to vitreous potassium in estimating post mortem interval so as to reduce the error in estimating time since death.
Key words: Calcium, vitreous humour, time since death.
............................
INTRODUCTION In the present study, vitreous analytes have been
Time since death is a question of day to day investigated to establish their correlation ship with PMI or
challenge faced by teams of investigators during their time since death. A linear correlation ship, at various
course of investigations. The physical signs require a degrees, was seen to exist between PMI and vitreous
definite experience and are subject to variations in calcium. Main objectives of the present study were to
interpretations from person to person, hence are investigate the utility of vitreous calcium levels in
1
extremely subjective in nature . It was a quest for us estimating post mortem interval (PMI) and also to find the
to arrive at an objective method for determination of accuracy of vitreous calcium levels in estimating time since
time since death which would be objective and death.
simple, cheap and easily available, undeterred by MATERIALS AND METHODS
external influencing factors, providing a reliable The study was carried out in Shri. Vasantrao Naik
2
method of estimation of time since death . Vitreous Government Medical College, Yavatmal, Maharashtra. The
humour of eye being relatively stable, less susceptible dead bodies brought to the mortuary at Shri. Vasantrao Naik
than other body fluids to rapid chemical changes and Government Medical College, Yavatmal were used as
contamination, easily accessible; thus it is suitable to material for collection of vitreous humour sample from the
3
estimate postmortem interval. (PMI) 141
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 International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 3, April 2013, Pages 141-146
eyes of deceased. The information regarding exact
time of death was gathered from police inquest report,
dead body challan, clinical details from hospital
records; correlated and checked from relatives, friends
and attendants of the deceased. Cases where exact
time of death was not known were not included in this
study. Vitreous humor samples were collected from
the eyes of 152 deceased during January 2006 to May
2007 from the autopsies conducted in mortuary at
Shri. Vasantrao Naik Government Medical College,
Yavatmal. 96 subjects in all were males and 56 were
females in the age group of 07 to 80 years (Mean
SD, 35.38 years 14.37 years) and the known
postmortem interval ranges from1.6 hours to 39.2
hours (Mean SD, 13.01 7.22 hours). After sample
removal, detail postmortem examination carried out
and the general relevant findings were documented in
the proforma with details of livor mortis, rigor mortis,
external and internal sign of decomposition. The
vitreous samples drawn from the eyes of deceased at
the beginning of postmortem examination of dead
body to the mortuary, (Only the clean and clear
samples were taken, samples having any particulate
matter discarded). All the information about the
deceased i.e. age, sex, address, cause of death, exact
time of death, time of each sampling, temperature
recorded. Vitreous humour was collected at autopsy
from the posterior chamber of the eye, slowly and
gradually avoiding tearing of loose fragments of
tissues by needle aspiration through a puncture made
5-6 mm away from the limbus (sclero-corneal
junction) using 10 ml sterile syringe and 20 gauze
needle and poured in a rubber stoppered vial. Pre-
sterilized syringe was used and rubber stoppered glass
vials washed with deionised double distilled water and
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dried in hot air oven, were used for sampling. Liquid paraffin
gels were injected in the posterior chamber of eye for
cosmetic purposes. The samples after collection in a rubber
stoppered vial were labeled and transported to the
biochemistry laboratory for analysis. The vitreous samples
were centrifuged at 3000 rpm for 10 minutes; all the
biochemical analysis was carried out immediately post-
extraction. The samples were analyzed for calcium. The
analysis was carried out on the same day as collection using a
BECKMAN COULTER LX20 AUTOMATED ANALYZER.
OBSERVATIONS
The samples were taken from both eyes at same time
and evaluated separately, no significant difference was
observed. In the present study, observations were made up to
40 hours (Mean SD, 13.01 7.24) postmortem period.
During the studied postmortem period, vitreous calcium
represented a fairly linear rise with increasing PMI. The
samples were taken from both eyes at same time and
evaluated separately, no significant difference was observed.
Out of the estimated concentration in the vitreous
humor samples collected from 152 subjects the minimum
value for vitreous calcium concentration was 1.2 mmol/L and
the maximum value of 3.3 mmol/L hours with an average of
2.0691 mmol/L (Mean SD, 2.0691 0.497; SEM, 0.0403).
The minimum value for vitreous calcium
concentration noted in the cases of PMI upto 6.0 hours was
1.2 mmol/L and the maximum value of 2.8 mmol/L with an
average of 1.9 mmol/L.
The minimum value for vitreous calcium
concentration noted in the cases of PMI between 6.1 hours to
12.0 hours was 1.2 mmol/L and the maximum value of 3.1
mmol/L with an average of 2.0 mmol/L.
The minimum value for vitreous calcium
concentration noted in the cases of PMI between 12.1 hours to
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18.0 hours was 1.2 mmol/L and the maximum value of concentration noted in the cases of PMI between 18.1 hours
3.1 mmol/L with an average of 2.0 mmol/L. to 24.0 hours was 1.4 mmol/L and the maximum value of
The minimum value for vitreous calcium 3.3 mmol/L with an average of 2.3 mmol/L.

Table-1:

Constituent n Minimum Maximum Mean

Calcium 152 1.2 3.3 2.0691

The minimum value for vitreous calcium concentration noted in the cases of PMI between 24.1 hours to
40.0 hours was 1.9 mmol/L and the maximum value of 3.2 mmol/L with an average of 2.4 mmol/L.

Table-2:The observed linear regression analyses correlation of the various vitreous analytes with PMI.

Constituent n r value p value

Calcium 152 0.2778 <0.005

The linear rise of vitreous calcium against increasing PMI is represented in Table No. 2. The
linear regression correlation of vitreous potassium and PMI was found to be highly significant (n, 152; R,
02778; P< 0.005).

The vitreous calcium concentrations were used as the dependent variable to calculate the estimated PMI.
The resulting linear regression equation in the form of y = ax + b (where, 'y' is vitreous calcium
concentration; 'x' is actual PMI in hours; 'a' is the slope of regression line and 'b' is the intercept of the
regression line) were:

y = 0.0191 x + 1.8235.

The corresponding formulae to estimate the PMI in the form of: PMI = 0 + 1 [Mean of the individual

biochemical constituent concentration] where, 0 is the estimated regression coefficient when no other

variable is included in the model and 1 is the estimated regression coefficient for vitreous calcium) was:

Estimated PMI = 1.065(calcium) - 10.8064.

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 International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 3, April 2013, Pages 141-146

Graph No 01: PMI in hours on X- axis and Ca++ concentration value in mmol on Y-axis

3.5 y = 0.019x + 1.823

3 R = 0.076
2.5
2 Series1
1.5
1 Linear
(Series1)
0.5
0
0 20 40 60

Table-3: Statistical comparison of differences between actual and estimated postmortem interval using
the derived formula.

Present n Slope mean S.D. S.E. mean

study
Using Ca++ 152 0.0191 13.00997 0.53105 0.043074

DISCUSSION There was a significant linear correlation observed for

Several authors are in agreement that there is no
relationship between calcium concentration in vitreous
4,5,14, 15
humor and time since death . Nowak and
4
Balabanova (1989) however established such a
relationship on a random sample of 19 cases suffering
from heart disease (coronary occlusion) and quote Coe
(1969, 1972) who "described also a correlation between
PMI and calcium" 6,7,8,9,10.
The vitreous calcium concentrations observed in
the present study are very similar to the concentrations
previously reported (Coe6, 1969;
4
Balabanova , 1989).
The minimum value for vitreous calcium
concentration was 1.2 mmol/L and the maximum value
of 3.3 mmol/L hours with an average of 2.0691 mmol/L.
vitreous calcium with PMI(R = 0.2778; P < 0.005). The
rate of rise of calcium concentration was 0.0191 mmol/L
per hour.
The significant correlation of vitreous calcium
and PMI observed in the present study is similar to the
findings of Nowak and Balabanova4 (1989). However, in
the study by Nowak and Balabanova4, the significant
correlation between vitreous calcium and PMI was only
noted in specific groups of death comprising of heart
disease and asphyxia.
Nowak and The present study does not support the previous
finding of a significant correlation between postmortem
vitreous calcium and PMI in deaths associated with
cardiovascular disease (Nowak and Balabanova4, 1989).
The small number of subjects with cardiovascular disease
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 International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 3, April 2013, Pages 141-146

CONCLUSION: There is a statistically significant correlation between calcium concentration in vitreous

humor and PMI (r = 0.2778) but the range of scatter of the individual calcium values over the PMI is so
great that it becomes clear that vitreous calcium can be adjunct to vitreous potassium in estimating post
mortem interval so as to reduce the error in estimating time since death.
ACKNOWLEDGEMENTS:
Special thanks to:
Dr. Keoliya Ajay, Prof & head, Dept of Forensic Medicine, IGGMC, Nagpur.
Dr. Ambade Vipul, Asso. Professor, GMC Nagpur.
Dr. Anil Batra, Asso. Professor, SVNGMC, Yavtmal.
Dr. Pathak Harish, Professor & Head, Seth GSMC & KEMH, Mumbai.
Dr. Sukhadeve Rajesh, Asso. Professor, Seth GSMC & KEMH, Mumbai.
Dr. (Mrs.) Deokar Priya & Miss Dhriti for being a continuous source of encouragement and support.
ETHICAL CLEARANCE: Approved by Institutional Ethical Committee of Shri. V N Govt. Medical
College, Yavatmal.
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Date of submission: 05 January 2013

Date of provisional acceptance: 22 January 2013
Date of Final acceptance: 13 March 2013
Date of Publication: 03 April 2013
Source of support: Nil; Conflict of Interest: Nil

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