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Postmortem biochemistry
Forensic Science International 165 (2007) 165–171 www.elsevier.com/locate/forsciint
Postmortem biochemistry Burkhard Madea *, Frank Musshoff Institute of Forensic Medicine, Rheinische Friedrich-Wilhelms-University Bonn, Stiftsplatz 12, D-53111 Bonn, Germany Received 30 January 2006; received in revised form 10 May 2006; accepted 10 May 2006 Available online 15 June 2006
Abstract Postmortem biochemistry may provide significant information in determining the cause of death. Due to the rapid postmortem breakdown of metabolism and active membrane transport only analytes which are stable in blood can be determined on this fluid compartment, other parameters have to be analysed on other fluid compartments like vitreous humor (VH). However, using another fluid compartment as a mirror of blood at the moment of death involves severe methodical problems. The conceptual problems of reference values in vitreous humor as a mirror of blood are addressed. Additionally, the necessary steps to be taken before calculating the discriminating values between ‘‘normal’’ and ‘‘diseased’’ are described. For all chemical determinations, a clear definition of the site of sample acquisition is necessary. Up to now chemical determinations on alternative fluids have mainly been carried out using instruments calibrated for urine or serum. Developing calibrated methods for analysis of alternative fluids is a further task for the future. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Postmortem biochemistry; Reference values; Vitreous humor; Analytical requirements
1. Introduction Clinical diagnosis is in many aspects superior to postmortem diagnosis due to the contributions of clinical chemistry and diagnostic imaging which both allow an assessment of the functional status. Clinical chemistry allows a diagnosis especially in cases where morphologic changes are missing (e.g. early myocardial infarction, diabetes mellitus, alcoholic ketoacidosis). It would of course be desirable to determine chemical abnormalities which contributed to death postmortem, too. In his last review on postmortem chemistry, Coe [8] estimates that ‘‘routine examinations of vitreous electrolytes, glucose and urea nitrogene alone will provide significant information in determining the cause of death or help in determining the time of death in >5% of all cases’’. Proper utilisation of a wide variety of chemical determinations in blood, cerebrospinal fluid (CSF), vitreous humor (VH), pericardial and other body fluids would help in solving forensic problems in nearly 10% of the routine natural deaths. However, with a postmortem breakdown of metabolism, mainly anaerobic glycolysis, active membrane transport stops and the loss of * Corresponding author. Tel.: +49 228 7383 15; fax: +49 228 738368. E-mail address: [email protected] (B. Madea). 0379-0738/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2006.05.023
selective membrane permeability and diffusion of ions and other parameters according to their concentration gradients starts. Furthermore analyte concentrations are affected by postmortem changes like redistribution or haemoconcentration. Postmortem blood specimens have been extensively studied for chemical diagnosis (e.g. carbohydrates, nitrogenous compounds, electrolytes, trace elements, lipids, proteins, enzymes, hormones). Due to the rapid breakdown of cell membranes further body fluids have been more or less extensively investigated: -
CSF, VH, pericardial fluid, joint fluid, spinal fluid.
These investigations on different analytes revealed that the cessation of energy metabolism proceeds faster in blood than in CSF, and in CSF faster than in vitreous humor. This is one of the reasons why many chemical parameters are mainly studied in VH. The diffusion process is too fast and erratic in other body fluids [21]. Furthermore, VH is a well protected fluid on the one hand and can easily be obtained on the other hand [5,6,8].
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Postmortem chemistry is mainly used as an aid in determining the cause of death in renal or liver diseases. However, renal or liver diseases usually have a morphological equivalent so that postmortem chemistry is not as essential in these cases. Postmortem chemistry might contribute essentially in determining the cause of death when morphologic alterations of the lethal disease are missing: e.g. in diabetes mellitus, alcoholic ketoacidosis or in disturbances of the electrolyte metabolism. Since carbohydrates and electrolytes which are not stable in blood are stable in vitreous humor for a comparatively longer period of time most chemical determinations are nowadays carried out in vitreous humor. It is not the aim of this paper to repeat the known facts on vitreous humor chemistry but to remind of some unsolved general problems concerning vitreous chemistry which should be kept in mind in practical casework to avoid a too schematic application of published ‘‘normal values’’, ‘‘reference intervals’’ or ‘‘combined values’’ and for planning future studies.
are the frame for interpretation (e.g. urea, creatinine). However, the site of sample taking has to be defined (e.g. blood from the femoral vein). Blind punctures of heart blood are never acceptable. Difficulties arise when the analyte is not stable postmortem and determinations are carried out on another fluid compartment than blood. Another problem is that the standard procedures of chemical analysis are calibrated for blood, serum or urine. When another fluid is used for analysis the complete analytical procedure has to be calibrated for the alternative fluid. 2.1. Determination on another fluid compartment than blood
2. General considerations
In clinical chemistry, most determinations are carried out on blood or serum. Laboratory- and methodology specific normal values or reference values exist for most parameters and chemical abnormalities can be diagnosed quite easily. When these chemical abnormalities should be diagnosed postmortem on another fluid compartment than blood or serum several questions arise [22,23].
The use of postmortem chemistry is unproblematic if the analyte remains stable postmortem and is not affected by postmortem changes like redistribution or haemoconcentration. In these cases, postmortem concentrations can be measured easily and the well established reference values for the living
- What are the ‘‘normal values’’ in vitreous humor compared to blood and serum? Frequency distributions of antemortem serum and postmortem vitreous values in normal individuals must be calculated and compared (Fig. 1). Correlations between antemortem serum and postmortem vitreous values
Fig. 1. To use vitreous humor values as mirror of antemortem serum values the following requirements must be fulfilled: frequency distributions of antemortem serum (a) and postmortem vitreous values (b) on ‘‘normal individuals’’ must be calculated and compared. Do the antemortem serum and postmortem vitreous values have the same frequency distribution? Are elevations or depressions in serum equilibrated in vitreous humor? Are the postmortem vitreous values stable (b and c: stability of the frequency distribution over the postmortem interval)?
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Swift et al. [37], Blumenfeld et al. [2], Wilkie, Bellamy [38] and several others [3,4,9,11,17,19,25,29,34]. These investigations were carried out on differing reference samples regarding age, sex, cause of death, postmortem interval, previous health status, antemortem serum values. Naumann [31] characterises his material as follows:
must be calculated. From these correlations between antemortem serum and postmortem vitreous values with upper and lower 95% confidence bands inverse prediction intervals of serum values corresponding to observed vitreous values could be derived. - Are chemical abnormalities of serum values reflected in vitreous humor and how fast are normal serum values (elevations and depressions) equilibrated in vitreous humor? - Are these values stable postmortem in vitreous humor?
‘‘Autopsies performed on 211 male bodies of an average age of 58 years examined about 9 hours after death on the average were selected on the basis of minimum changes unrelated to the metabolism of substances under investigations.’’
These questions are difficult to answer because of methodical problems:
Leahy and Farber [20]: (1) Normal values on human vitreous humor during life are not and will not be available. We have to establish our normal vitreous humor values from postmortem studies on ‘‘normal individuals’’ and compare them to vital normal serum values. Antemortem vitreous values are known from animal experiments and quite large normal ranges were observed, for instance for sodium [33]. (2) The question if and how fast serum abnormalities are equilibrated in vitreous humor cannot be answered on living humans as well.
‘‘Vitreous humor analysed in this study was obtained from both hospitalised patients and patients dead on arrival at the University of Michigan Medical Center. Necropsies were performed on all these subjects. Those with known or suspected primary ocular disease or ocular trauma were excluded. Available antemortem serum or blood levels of the measured materials were compared with postmortem vitreous levels of those same substances. The antemortem values used for comparison included only those determined within 24 hours of death.’’
There are only very few experimental animal studies on the equilibration of artificial raised serum values (urea) in vitreous humor regarding time [38]. An apparently slow rate at which serum urea equilibrated in the vitreous humor of dogs was found, more than 1 h being required for levels in vitreous humor to reach half those in serum [38]. There is even evidence that the equilibration of elevated serum levels (for instance glucose) of patients with intravenous glucose administration is continued postmortem (a second vitreous glucose level was found to be higher than the first) [5]. Studies on vitreous humor with the aim to establish normal values for several parameters began with a paper by Naumann [31], followed by Leahy and Farber [20], especially Coe [5–8],
Coe [5]: ‘‘Specimens were obtained from hospital patients whose blood chemistries were known to be normal within a few hours of death and from young individuals dying suddenly by accidental means when autopsy revealed no cause for any chemical abnormality’’. Blumenfeld et al. [2]: ‘‘Postmortem vitreous humor was aspirated from both eyes of 127 children during postmortem examination . . . Based on the cause of death the results were placed into one of the three groups:
Table 1 Standard deviations and observed ranges of different parameters in vitreous humor published as normal values in the literature VUN (mg/dl)
Naumann 1959 Leahy/Faber 1967 Coe 1969*
Creatinine (mg/dl)
n
x
Range
211 30
79
24–172
60 50 35
17 17 18
6–40 4–33 3–30
1 S.D.
2 S.D.
n
x
Range
211 29
1.2
0.3–3.0
5–29
Leahy/Faber 1967 Coe 1969
*
*
n
x
Range
144
118–154
1 S.D.
37
2 S.D.
128–158 143 143 141
n
x
Range
211
3.6
2.8–5.2 mg/dl 6–8 mg/dl
6.7 mg/dl 1.67 mmol/l
1.5–2.0 mmol/l
0.31–1.05
Cl (mmol/l)
211
60 50 35
2 S.D.
7.6 6.3 7.5
Na (mmol/l)
Naumann 1959
Calcium 1 S.D.
135–151 131–151 131–150
4.0 4.7 4.4
n
x
211
144
Range
1 S.D.
2 S.D.
89–145 108–142
121 119 118
2 S.D.
Glucose (mg/100 ml)
39 60 50 35
1 S.D.
108–132 105–132 104–130
5.9 6.1 6.8
Mean postmortem interval for taking samples: 1.75 h (0.5–2.5); 5.75 h (3–10); 17.25 h (10.5–29).
n
x
Range
211
62
17–105
1 S.D.
43 60 50 35
2 S.D.
28–89 84 66 51
37–180 27–180 18–106
40 36 28
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Hospital deaths. These 47 children (32 male, 15 female) were 1 hour to 13 years of age, and 24 were premature . . . Acute traumatic deaths. The 21 children (10 male, 11 female) were 7 weeks to 11 years of age . . . Sudden Infant Death Syndrome. These 59 children (37 male, 22 female) were 8 days to 12 months of age . . .’’ Swift et al. [37]: ‘‘80 children coming to necropsy were used. . . . The vitreous was aspirated from one eyeball as soon as possible after the child had been admitted to the mortuary and a similar specimen was taken from the other eyeball just before the child was taken from the mortuary for burial after necropsy’’. If antemortem serum values were available they were compared to postmortem levels. Naumann [31] represents in his results the range of observed values; Leahy/Faber [20] present the lower and upper second standard deviation, Coe [5] the observed range, mean value and first standard deviation (Table 1). But still the question remains what the normal range is? ‘‘Normal values’’ calculated as mean value 2 S.D. must fulfil three suppositions which are normally not given [15,16]: - Gaussean frequency distribution of values, - extensive investigations on healthy people or normal individuals, - extensive investigations on random samples with deviations and disturbances of the investigated parameter. Especially investigations on random samples with deviations (elevations and depressions) of the investigated parameter are necessary to distinguish certainly normal from certainly abnormal values [16,22,23,26] (Fig. 2). The upper and lower normal ranges are not sufficient to define a severe dysregulation. Discriminating values between normal and certainly abnormal can only be calculated after thorough and extensive
investigations have been carried out on collectives with a deviation (elevation or depression) based on independent criteria. Those investigations are missing up to now, also for dysregulation patterns in vitreous humor as dehydration pattern and low salt pattern [26]. And another point: vitreous values are used as a mirror of serum values, since due to fast autolysis serum values postmortem do not reflect the serum values at the moment of death. But do in any case the vitreous values reflect the serum values at the moment of death? The greatest material was studied by Coe [5] who compared antemortem serum with postmortem vitreous values but he has published no frequency distributions of observed values in normal and diseased individuals nor correlations. According to Coe vitreous values reflect serum values at the moment of death. This seems to be true at least for values in normal range and highly elevated values but not for low values [5]: ‘‘although abnormal vitreous values reflect abnormal serum electrolytes, the converse was frequently not true. Many hospital cases with terminal low serum sodium values were found to have vitreous sodium levels within normal limits’’. ‘‘In six instances of antemortem hyponatremia, of which the highest serum sodium value was 127 m Eq/l, the vitreous sodium concentration was within normal ranges . . . In 11 patients with antemortem hypochloremia, the vitreous chloride concentration was abnormally low in only 4 and within normal range in 7 . . . However, measurable fluctuations in these concentrations did not appear to occur with sufficient rapidity or predictability to reflect accurately agonal values of either sodium or chloride’’. From these findings and the experimental results of Wilkie and Bellamy [38], it becomes evident that the equilibration of serum values – elevated and especially depressed – with vitreous values is slow and vitreous values in normal range
Fig. 2. Statistical parameters of the distribution of values require a further verbal attribution what is certainly normal, abnormal or questionable. Therefore, frequency distributions must also be calculated on random samples with dysregulations of the investigated parameter. From the frequency distribution of values on the reference sample and the sample with the deviations discrimination values can be calculated.
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may reflect pathological serum values, especially lowered values. Correlations between antemortem serum and postmortem vitreous values are nearly completely missing in the literature. The only study in which correlations are reported is that of Choo-Kang et al. [4]. The postmortem vitreous values in this study were, however, from a pm interval from 23 to 277 h. The best correlation was found for creatinine and urea with correlation coefficients of r = 0.98 and 0.96. The correlations for sodium and chloride were with r = 0.59 and 0.43 comparatively poor. Furthermore, vitreous humor values of sodium, chloride, potassium, glucose and urea nitrogen vary due to different analytical procedures and instruments used [7]. For vitreous potassium, it has become manifest that values determined by ion-specific electrodes tend to be higher than the values obtained with flame photometry [7]. Taking into consideration all these facts it becomes obvious that the term ‘‘normal vitreous values’’ should not be used. Additionally, there are some other important reasons. The concept of ‘‘normal values’’ which is mainly based on the assumption of a Gaussean distribution and the calculation of mean value and upper and lower second standard deviation is left in clinical chemistry for several reasons [1,10,14,32,35,36]. According to Murphy ‘‘normalcy is a vestigial concept left in medicine from its unscientific era’’. Dybkaer and Gra¨sbeck (cit. according to Ref. [36]) have concluded that normality is hopeless to define in a useful way and they have suggested that the term ‘‘normal values’’ should be abolished. ‘‘Normal values’’ in vitreous humor in dead bodies are of course nonsense in another sense. What shall normal values be in dead persons? Instead the concept of reference values has been introduced to avoid many of these difficulties surrounding the term ‘‘normal values’’. The term ‘‘reference values’’ is used in the following meaning [36]: ‘‘A set of values of a certain type of quantity available from a single individual or a group of individuals corresponding to a stated description. The description must be spelled out and available if others are to use the reference values. For each type of quantity a series of reference groups will be necessary taking into consideration age, sex, race, menstruation, previous diet and exercise, posture etc.’’ (Dybkaer, cit. according to 36). Reference values for a diagnostic test in clinical chemistry or in forensic medicine should include five major categories of specification: (1) the reference population, and the way it was chosen, (2) the environmental and physiological conditions under which the specimens were obtained, (3) the technique and timing of specimen collection, transport, preparation and storage, (4) the analytical method that was used, with data regarding its accuracy, precision and quality control,
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(5) the data set that was observed and the reference intervals that were derived. The concept of reference values and relationship of recommended terms is as follows: Reference individuals comprise a reference population from which is selected a reference sample group on which are determined reference values on which is observed a reference distribution from which are calculated reference limits that may define reference intervals. There are additional factors which influence the reference values in postmortem chemistry: -
age, sex; in females menstruation, pregnancy, subpopulation; race; constitution, circadian rhythm; other biological rhythms, custom of life, milieu; environmental influences, drugs, nutrition, cause of death, duration of agonal period, medical drug-, infusion therapy, environmental conditions (body in room temperature, refrigerator), - postmortem sampling time. No study in the literature on reference values in vitreous humor fulfils all these requirements. But these requirements have to be addressed in further investigations. For instance, it is very important to include no persons with resuscitation measures or intravenous fluid administration (Ringers solution, intravenous saline, sodium bicarbonate, glucose solution) prior to death into the reference group since resuscitation disturbs agonal values deeply. 2.2. Patchwork of analytes from different fluid compartments The situation becomes even more difficult when analytes from different fluid compartments are used to make a postmortem diagnosis (e.g. from blood, urine, vitreous humor). All the points listed under the section on reference values have to be kept in mind. Based on a mosaic of analytical values from different fluid compartments, only a suspicious diagnosis can be made on the cause of death after concurrent causes of death have been excluded. 3. Analytical problems Most instruments used for CSF or vitreous humor analysis are mainly used for serum determinations in daily casework. All the procedures for the different investigated analytes are calibrated for serum. However, CSF and vitreous humor have another chemical composition than serum and therefore the question arises if the methods calibrated for serum can be applied for CSF and vitreous humor analysis as well. The following items have to be considered concerning
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the pre-analysis and the analysis of CSF and vitreous humor: - Was the vitreous humor of both eyes withdrawn separately, stored and analysed separately or was it pooled? - How was the vitreous humor stored, at which temperature? - How often was the vitreous humor thawed and refrigerated before analysis? - How was the vitreous humor transported (cooled or not)? - Was the vitreous humor centrifuged before analysis? If yes, how long? - What was used for analysis (only the supernatent)? - How was the colour of the vitreous humor (clear or cloudy, brownish)? - How were the methods for the different analytes calibrated (just for serum or for vitreous humor as well)? - Were the concentrations of the different parameters within the calibrated area or was an extrapolation done? - How was the precision of the methods at that day (for vitreous humor)? - Intra- and interday precision for VH? - How were the internal quality controls at that day? - Are there any recommended guidelines for quality assurance in laboratory medicine? - Were these guidelines followed concerning vitreous humor? Beside these preanalytical aspects, the methodology used may be important for the results obtained. Coe and Apple [7] already published several years ago that vitreous humor chemical values differ according to the method applied. They compared flame photometry, colorimetry and ion-selective electrodes. With ion-selective electrodes they revealed, e.g. higher values for chloride. However, the variation of vitreous humor values is not only due to instrumentation [3,12,18,24,27,28] but the composition of vitreous humor and the preanalytical handling. The main obstacle in vitreous humor analysis is up to now that analytical methods are applied which are calibrated and validated for serum or urine but not for vitreous humor. Furthermore, differences in results obtained from left and right eyes and different results when specimens have been analysed using different instruments occur. Even the reproducibility of vitreous humor electrolyte measurement with the same analyzer is poor. These problems seem to exist due to the high viscosity of vitreous humor [13,30]. Therefore, several attempts have been made to reduce the viscosity by different sample pre-treatments as heating or enzymatic digestion by hyaluronidase. Developing a calibrated and validated method for vitreous humor analysis will be one of the tasks for the future [24]. 4. Conclusions When chemical analytes, especially vitreous chemistry is used for postmortem diagnosis and as a mirror of serum chemistry at the moment of death the scientific gaps should be kept in mind.
The correlation of serum and vitreous values was only investigated for a range of normal serum values and some values with moderate elevations or depressions but not with severe disturbances of homeostasis [26]. At the present time, we know little about the period of time needed for depressed or elevated serum levels to equilibrate within the vitreous. Furthermore we have no reference material on cases with lethal serum values comparing these to vitreous values. And lastly valid and reliable discrimination values which discriminate between certainly normal and certainly abnormal or lethal in vitreous are missing. These conceptual problems are more or less inherent in all studies on vitreous humor chemistry and have to be addressed in further systematic investigations. Some of these scientific gaps will continue for methodical problems: e.g. it will be very difficult to obtain correlations between markedly depressed or elevated antemortem serum values and postmortem vitreous values since patients with markedly disturbed values will be treated. A further task for the future will be to develop calibrated and validated methods for alternative fluids, especially vitreous humor analysis. References [1] T. Alstro¨m, R. Gradbeck, M. Hjelm, S. Skandsen, Recommendations concerning the collection of reference values in clinical chemistry and activity report, Scand. J. Clin. Lab Invest. 35 (Suppl. 144) (1975) 5–44. [2] T.A. Blumenfeld, C.H. Mantell, R.L. Catherman, W.A. Blanc, Postmortem vitreous humor chemistry in sudden infant death syndrome and in other causes of death in childhood, Am. J. Clin. Pathol. 71 (1979) 219– 223. [3] G. Bocaz-Beneventi, F. Tagliaro, F. Bortolotti, G. Manetto, J. Havel, Capillary zone electrophoresis and artificial neural networks for estimation of the post-mortem interval (PMI) using electrolyte measurements in human vitreous humor, Int. J. Legal Med. 116 (1) (2002) 5–11. [4] E. Choo-Kang, C. McKoy, C. Escoffrey, Vitreous humor analytes in assessing the postmortem interval and the antemortem clinical status, WJ Med. J. 32 (1983) 23–26. [5] J.I. Coe, Postmortem chemistries on vitreous humor, Am. J. Pathol. 51 (6) (1969) 741–750. [6] J.I. Coe, Use of chemical determinations on vitreous humor in forensic pathology, J. Forensic Sci. 17 (1972) 541–546. [7] J.I. Coe, F.S. Apple, Variations in vitreous humor chemical values as a result of instrumentation, J. Forensic Sci. 30 (3) (1985) 828–835. [8] J.I. Coe, Postmortem chemistry update—emphasis on forensic applications, Am. J. Forensic Med. Pathol. (1993) 91–117. [9] M.S. Devgun, J.A. Dunbar, Biochemical investigations of vitreus: applications in forensic medicine, especially in relation to alcohol, Forensic Sci. Int. 31 (1986) 27–34. [10] R. Dybkaer, The theory of reference values. Part 6, presentation of observed values related to reference values, J. Clin. Chem. Clin. Biochem. 20 (1982) 841–845. [11] J.G. Farmer, F. Benomran, A.A. Watson, W.A. Harland, Magnesium, potassium, sodium and calcium in postmortem vitreous humor from humans, Forensic Sci. Int. 27 (1985) 1–13. [12] K.E. Ferslew, A.N. Hagardorn, M.T. Harrison, W.F. McCormick, Capillary ion analysis of potassium concentrations in human vitreous humor, Electrophoresis 19 (1) (1998) 6–10. [13] U. Garg, R. Althahabi, V. Amirahmadi, M. Brod, C. Blanchard, T. Young, Hyaluronidase as a liquefying agent for chemical analysis of vitreous fluid, J. Forensic Sci. 49 (2004) 388–391. [14] R. Grasbeck, G. Siest, P. Wilding, G.Z. Williams, T.P. Whitehead, Provisional recommendation on the theory of reference values (1978). Part 1. The concept of reference values, J. Clin.Chem. 25 (1979) 1506–1508.
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Postmortem biochemistry Burkhard Madea *, Frank Musshoff Institute of Forensic Medicine, Rheinische Friedrich-Wilhelms-University Bonn, Stiftsplatz 12, D-53111 Bonn, Germany Received 30 January 2006; received in revised form 10 May 2006; accepted 10 May 2006 Available online 15 June 2006
Abstract Postmortem biochemistry may provide significant information in determining the cause of death. Due to the rapid postmortem breakdown of metabolism and active membrane transport only analytes which are stable in blood can be determined on this fluid compartment, other parameters have to be analysed on other fluid compartments like vitreous humor (VH). However, using another fluid compartment as a mirror of blood at the moment of death involves severe methodical problems. The conceptual problems of reference values in vitreous humor as a mirror of blood are addressed. Additionally, the necessary steps to be taken before calculating the discriminating values between ‘‘normal’’ and ‘‘diseased’’ are described. For all chemical determinations, a clear definition of the site of sample acquisition is necessary. Up to now chemical determinations on alternative fluids have mainly been carried out using instruments calibrated for urine or serum. Developing calibrated methods for analysis of alternative fluids is a further task for the future. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Postmortem biochemistry; Reference values; Vitreous humor; Analytical requirements
1. Introduction Clinical diagnosis is in many aspects superior to postmortem diagnosis due to the contributions of clinical chemistry and diagnostic imaging which both allow an assessment of the functional status. Clinical chemistry allows a diagnosis especially in cases where morphologic changes are missing (e.g. early myocardial infarction, diabetes mellitus, alcoholic ketoacidosis). It would of course be desirable to determine chemical abnormalities which contributed to death postmortem, too. In his last review on postmortem chemistry, Coe [8] estimates that ‘‘routine examinations of vitreous electrolytes, glucose and urea nitrogene alone will provide significant information in determining the cause of death or help in determining the time of death in >5% of all cases’’. Proper utilisation of a wide variety of chemical determinations in blood, cerebrospinal fluid (CSF), vitreous humor (VH), pericardial and other body fluids would help in solving forensic problems in nearly 10% of the routine natural deaths. However, with a postmortem breakdown of metabolism, mainly anaerobic glycolysis, active membrane transport stops and the loss of * Corresponding author. Tel.: +49 228 7383 15; fax: +49 228 738368. E-mail address: [email protected] (B. Madea). 0379-0738/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2006.05.023
selective membrane permeability and diffusion of ions and other parameters according to their concentration gradients starts. Furthermore analyte concentrations are affected by postmortem changes like redistribution or haemoconcentration. Postmortem blood specimens have been extensively studied for chemical diagnosis (e.g. carbohydrates, nitrogenous compounds, electrolytes, trace elements, lipids, proteins, enzymes, hormones). Due to the rapid breakdown of cell membranes further body fluids have been more or less extensively investigated: -
CSF, VH, pericardial fluid, joint fluid, spinal fluid.
These investigations on different analytes revealed that the cessation of energy metabolism proceeds faster in blood than in CSF, and in CSF faster than in vitreous humor. This is one of the reasons why many chemical parameters are mainly studied in VH. The diffusion process is too fast and erratic in other body fluids [21]. Furthermore, VH is a well protected fluid on the one hand and can easily be obtained on the other hand [5,6,8].
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Postmortem chemistry is mainly used as an aid in determining the cause of death in renal or liver diseases. However, renal or liver diseases usually have a morphological equivalent so that postmortem chemistry is not as essential in these cases. Postmortem chemistry might contribute essentially in determining the cause of death when morphologic alterations of the lethal disease are missing: e.g. in diabetes mellitus, alcoholic ketoacidosis or in disturbances of the electrolyte metabolism. Since carbohydrates and electrolytes which are not stable in blood are stable in vitreous humor for a comparatively longer period of time most chemical determinations are nowadays carried out in vitreous humor. It is not the aim of this paper to repeat the known facts on vitreous humor chemistry but to remind of some unsolved general problems concerning vitreous chemistry which should be kept in mind in practical casework to avoid a too schematic application of published ‘‘normal values’’, ‘‘reference intervals’’ or ‘‘combined values’’ and for planning future studies.
are the frame for interpretation (e.g. urea, creatinine). However, the site of sample taking has to be defined (e.g. blood from the femoral vein). Blind punctures of heart blood are never acceptable. Difficulties arise when the analyte is not stable postmortem and determinations are carried out on another fluid compartment than blood. Another problem is that the standard procedures of chemical analysis are calibrated for blood, serum or urine. When another fluid is used for analysis the complete analytical procedure has to be calibrated for the alternative fluid. 2.1. Determination on another fluid compartment than blood
2. General considerations
In clinical chemistry, most determinations are carried out on blood or serum. Laboratory- and methodology specific normal values or reference values exist for most parameters and chemical abnormalities can be diagnosed quite easily. When these chemical abnormalities should be diagnosed postmortem on another fluid compartment than blood or serum several questions arise [22,23].
The use of postmortem chemistry is unproblematic if the analyte remains stable postmortem and is not affected by postmortem changes like redistribution or haemoconcentration. In these cases, postmortem concentrations can be measured easily and the well established reference values for the living
- What are the ‘‘normal values’’ in vitreous humor compared to blood and serum? Frequency distributions of antemortem serum and postmortem vitreous values in normal individuals must be calculated and compared (Fig. 1). Correlations between antemortem serum and postmortem vitreous values
Fig. 1. To use vitreous humor values as mirror of antemortem serum values the following requirements must be fulfilled: frequency distributions of antemortem serum (a) and postmortem vitreous values (b) on ‘‘normal individuals’’ must be calculated and compared. Do the antemortem serum and postmortem vitreous values have the same frequency distribution? Are elevations or depressions in serum equilibrated in vitreous humor? Are the postmortem vitreous values stable (b and c: stability of the frequency distribution over the postmortem interval)?
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Swift et al. [37], Blumenfeld et al. [2], Wilkie, Bellamy [38] and several others [3,4,9,11,17,19,25,29,34]. These investigations were carried out on differing reference samples regarding age, sex, cause of death, postmortem interval, previous health status, antemortem serum values. Naumann [31] characterises his material as follows:
must be calculated. From these correlations between antemortem serum and postmortem vitreous values with upper and lower 95% confidence bands inverse prediction intervals of serum values corresponding to observed vitreous values could be derived. - Are chemical abnormalities of serum values reflected in vitreous humor and how fast are normal serum values (elevations and depressions) equilibrated in vitreous humor? - Are these values stable postmortem in vitreous humor?
‘‘Autopsies performed on 211 male bodies of an average age of 58 years examined about 9 hours after death on the average were selected on the basis of minimum changes unrelated to the metabolism of substances under investigations.’’
These questions are difficult to answer because of methodical problems:
Leahy and Farber [20]: (1) Normal values on human vitreous humor during life are not and will not be available. We have to establish our normal vitreous humor values from postmortem studies on ‘‘normal individuals’’ and compare them to vital normal serum values. Antemortem vitreous values are known from animal experiments and quite large normal ranges were observed, for instance for sodium [33]. (2) The question if and how fast serum abnormalities are equilibrated in vitreous humor cannot be answered on living humans as well.
‘‘Vitreous humor analysed in this study was obtained from both hospitalised patients and patients dead on arrival at the University of Michigan Medical Center. Necropsies were performed on all these subjects. Those with known or suspected primary ocular disease or ocular trauma were excluded. Available antemortem serum or blood levels of the measured materials were compared with postmortem vitreous levels of those same substances. The antemortem values used for comparison included only those determined within 24 hours of death.’’
There are only very few experimental animal studies on the equilibration of artificial raised serum values (urea) in vitreous humor regarding time [38]. An apparently slow rate at which serum urea equilibrated in the vitreous humor of dogs was found, more than 1 h being required for levels in vitreous humor to reach half those in serum [38]. There is even evidence that the equilibration of elevated serum levels (for instance glucose) of patients with intravenous glucose administration is continued postmortem (a second vitreous glucose level was found to be higher than the first) [5]. Studies on vitreous humor with the aim to establish normal values for several parameters began with a paper by Naumann [31], followed by Leahy and Farber [20], especially Coe [5–8],
Coe [5]: ‘‘Specimens were obtained from hospital patients whose blood chemistries were known to be normal within a few hours of death and from young individuals dying suddenly by accidental means when autopsy revealed no cause for any chemical abnormality’’. Blumenfeld et al. [2]: ‘‘Postmortem vitreous humor was aspirated from both eyes of 127 children during postmortem examination . . . Based on the cause of death the results were placed into one of the three groups:
Table 1 Standard deviations and observed ranges of different parameters in vitreous humor published as normal values in the literature VUN (mg/dl)
Naumann 1959 Leahy/Faber 1967 Coe 1969*
Creatinine (mg/dl)
n
x
Range
211 30
79
24–172
60 50 35
17 17 18
6–40 4–33 3–30
1 S.D.
2 S.D.
n
x
Range
211 29
1.2
0.3–3.0
5–29
Leahy/Faber 1967 Coe 1969
*
*
n
x
Range
144
118–154
1 S.D.
37
2 S.D.
128–158 143 143 141
n
x
Range
211
3.6
2.8–5.2 mg/dl 6–8 mg/dl
6.7 mg/dl 1.67 mmol/l
1.5–2.0 mmol/l
0.31–1.05
Cl (mmol/l)
211
60 50 35
2 S.D.
7.6 6.3 7.5
Na (mmol/l)
Naumann 1959
Calcium 1 S.D.
135–151 131–151 131–150
4.0 4.7 4.4
n
x
211
144
Range
1 S.D.
2 S.D.
89–145 108–142
121 119 118
2 S.D.
Glucose (mg/100 ml)
39 60 50 35
1 S.D.
108–132 105–132 104–130
5.9 6.1 6.8
Mean postmortem interval for taking samples: 1.75 h (0.5–2.5); 5.75 h (3–10); 17.25 h (10.5–29).
n
x
Range
211
62
17–105
1 S.D.
43 60 50 35
2 S.D.
28–89 84 66 51
37–180 27–180 18–106
40 36 28
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Hospital deaths. These 47 children (32 male, 15 female) were 1 hour to 13 years of age, and 24 were premature . . . Acute traumatic deaths. The 21 children (10 male, 11 female) were 7 weeks to 11 years of age . . . Sudden Infant Death Syndrome. These 59 children (37 male, 22 female) were 8 days to 12 months of age . . .’’ Swift et al. [37]: ‘‘80 children coming to necropsy were used. . . . The vitreous was aspirated from one eyeball as soon as possible after the child had been admitted to the mortuary and a similar specimen was taken from the other eyeball just before the child was taken from the mortuary for burial after necropsy’’. If antemortem serum values were available they were compared to postmortem levels. Naumann [31] represents in his results the range of observed values; Leahy/Faber [20] present the lower and upper second standard deviation, Coe [5] the observed range, mean value and first standard deviation (Table 1). But still the question remains what the normal range is? ‘‘Normal values’’ calculated as mean value 2 S.D. must fulfil three suppositions which are normally not given [15,16]: - Gaussean frequency distribution of values, - extensive investigations on healthy people or normal individuals, - extensive investigations on random samples with deviations and disturbances of the investigated parameter. Especially investigations on random samples with deviations (elevations and depressions) of the investigated parameter are necessary to distinguish certainly normal from certainly abnormal values [16,22,23,26] (Fig. 2). The upper and lower normal ranges are not sufficient to define a severe dysregulation. Discriminating values between normal and certainly abnormal can only be calculated after thorough and extensive
investigations have been carried out on collectives with a deviation (elevation or depression) based on independent criteria. Those investigations are missing up to now, also for dysregulation patterns in vitreous humor as dehydration pattern and low salt pattern [26]. And another point: vitreous values are used as a mirror of serum values, since due to fast autolysis serum values postmortem do not reflect the serum values at the moment of death. But do in any case the vitreous values reflect the serum values at the moment of death? The greatest material was studied by Coe [5] who compared antemortem serum with postmortem vitreous values but he has published no frequency distributions of observed values in normal and diseased individuals nor correlations. According to Coe vitreous values reflect serum values at the moment of death. This seems to be true at least for values in normal range and highly elevated values but not for low values [5]: ‘‘although abnormal vitreous values reflect abnormal serum electrolytes, the converse was frequently not true. Many hospital cases with terminal low serum sodium values were found to have vitreous sodium levels within normal limits’’. ‘‘In six instances of antemortem hyponatremia, of which the highest serum sodium value was 127 m Eq/l, the vitreous sodium concentration was within normal ranges . . . In 11 patients with antemortem hypochloremia, the vitreous chloride concentration was abnormally low in only 4 and within normal range in 7 . . . However, measurable fluctuations in these concentrations did not appear to occur with sufficient rapidity or predictability to reflect accurately agonal values of either sodium or chloride’’. From these findings and the experimental results of Wilkie and Bellamy [38], it becomes evident that the equilibration of serum values – elevated and especially depressed – with vitreous values is slow and vitreous values in normal range
Fig. 2. Statistical parameters of the distribution of values require a further verbal attribution what is certainly normal, abnormal or questionable. Therefore, frequency distributions must also be calculated on random samples with dysregulations of the investigated parameter. From the frequency distribution of values on the reference sample and the sample with the deviations discrimination values can be calculated.
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may reflect pathological serum values, especially lowered values. Correlations between antemortem serum and postmortem vitreous values are nearly completely missing in the literature. The only study in which correlations are reported is that of Choo-Kang et al. [4]. The postmortem vitreous values in this study were, however, from a pm interval from 23 to 277 h. The best correlation was found for creatinine and urea with correlation coefficients of r = 0.98 and 0.96. The correlations for sodium and chloride were with r = 0.59 and 0.43 comparatively poor. Furthermore, vitreous humor values of sodium, chloride, potassium, glucose and urea nitrogen vary due to different analytical procedures and instruments used [7]. For vitreous potassium, it has become manifest that values determined by ion-specific electrodes tend to be higher than the values obtained with flame photometry [7]. Taking into consideration all these facts it becomes obvious that the term ‘‘normal vitreous values’’ should not be used. Additionally, there are some other important reasons. The concept of ‘‘normal values’’ which is mainly based on the assumption of a Gaussean distribution and the calculation of mean value and upper and lower second standard deviation is left in clinical chemistry for several reasons [1,10,14,32,35,36]. According to Murphy ‘‘normalcy is a vestigial concept left in medicine from its unscientific era’’. Dybkaer and Gra¨sbeck (cit. according to Ref. [36]) have concluded that normality is hopeless to define in a useful way and they have suggested that the term ‘‘normal values’’ should be abolished. ‘‘Normal values’’ in vitreous humor in dead bodies are of course nonsense in another sense. What shall normal values be in dead persons? Instead the concept of reference values has been introduced to avoid many of these difficulties surrounding the term ‘‘normal values’’. The term ‘‘reference values’’ is used in the following meaning [36]: ‘‘A set of values of a certain type of quantity available from a single individual or a group of individuals corresponding to a stated description. The description must be spelled out and available if others are to use the reference values. For each type of quantity a series of reference groups will be necessary taking into consideration age, sex, race, menstruation, previous diet and exercise, posture etc.’’ (Dybkaer, cit. according to 36). Reference values for a diagnostic test in clinical chemistry or in forensic medicine should include five major categories of specification: (1) the reference population, and the way it was chosen, (2) the environmental and physiological conditions under which the specimens were obtained, (3) the technique and timing of specimen collection, transport, preparation and storage, (4) the analytical method that was used, with data regarding its accuracy, precision and quality control,
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(5) the data set that was observed and the reference intervals that were derived. The concept of reference values and relationship of recommended terms is as follows: Reference individuals comprise a reference population from which is selected a reference sample group on which are determined reference values on which is observed a reference distribution from which are calculated reference limits that may define reference intervals. There are additional factors which influence the reference values in postmortem chemistry: -
age, sex; in females menstruation, pregnancy, subpopulation; race; constitution, circadian rhythm; other biological rhythms, custom of life, milieu; environmental influences, drugs, nutrition, cause of death, duration of agonal period, medical drug-, infusion therapy, environmental conditions (body in room temperature, refrigerator), - postmortem sampling time. No study in the literature on reference values in vitreous humor fulfils all these requirements. But these requirements have to be addressed in further investigations. For instance, it is very important to include no persons with resuscitation measures or intravenous fluid administration (Ringers solution, intravenous saline, sodium bicarbonate, glucose solution) prior to death into the reference group since resuscitation disturbs agonal values deeply. 2.2. Patchwork of analytes from different fluid compartments The situation becomes even more difficult when analytes from different fluid compartments are used to make a postmortem diagnosis (e.g. from blood, urine, vitreous humor). All the points listed under the section on reference values have to be kept in mind. Based on a mosaic of analytical values from different fluid compartments, only a suspicious diagnosis can be made on the cause of death after concurrent causes of death have been excluded. 3. Analytical problems Most instruments used for CSF or vitreous humor analysis are mainly used for serum determinations in daily casework. All the procedures for the different investigated analytes are calibrated for serum. However, CSF and vitreous humor have another chemical composition than serum and therefore the question arises if the methods calibrated for serum can be applied for CSF and vitreous humor analysis as well. The following items have to be considered concerning
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the pre-analysis and the analysis of CSF and vitreous humor: - Was the vitreous humor of both eyes withdrawn separately, stored and analysed separately or was it pooled? - How was the vitreous humor stored, at which temperature? - How often was the vitreous humor thawed and refrigerated before analysis? - How was the vitreous humor transported (cooled or not)? - Was the vitreous humor centrifuged before analysis? If yes, how long? - What was used for analysis (only the supernatent)? - How was the colour of the vitreous humor (clear or cloudy, brownish)? - How were the methods for the different analytes calibrated (just for serum or for vitreous humor as well)? - Were the concentrations of the different parameters within the calibrated area or was an extrapolation done? - How was the precision of the methods at that day (for vitreous humor)? - Intra- and interday precision for VH? - How were the internal quality controls at that day? - Are there any recommended guidelines for quality assurance in laboratory medicine? - Were these guidelines followed concerning vitreous humor? Beside these preanalytical aspects, the methodology used may be important for the results obtained. Coe and Apple [7] already published several years ago that vitreous humor chemical values differ according to the method applied. They compared flame photometry, colorimetry and ion-selective electrodes. With ion-selective electrodes they revealed, e.g. higher values for chloride. However, the variation of vitreous humor values is not only due to instrumentation [3,12,18,24,27,28] but the composition of vitreous humor and the preanalytical handling. The main obstacle in vitreous humor analysis is up to now that analytical methods are applied which are calibrated and validated for serum or urine but not for vitreous humor. Furthermore, differences in results obtained from left and right eyes and different results when specimens have been analysed using different instruments occur. Even the reproducibility of vitreous humor electrolyte measurement with the same analyzer is poor. These problems seem to exist due to the high viscosity of vitreous humor [13,30]. Therefore, several attempts have been made to reduce the viscosity by different sample pre-treatments as heating or enzymatic digestion by hyaluronidase. Developing a calibrated and validated method for vitreous humor analysis will be one of the tasks for the future [24]. 4. Conclusions When chemical analytes, especially vitreous chemistry is used for postmortem diagnosis and as a mirror of serum chemistry at the moment of death the scientific gaps should be kept in mind.
The correlation of serum and vitreous values was only investigated for a range of normal serum values and some values with moderate elevations or depressions but not with severe disturbances of homeostasis [26]. At the present time, we know little about the period of time needed for depressed or elevated serum levels to equilibrate within the vitreous. Furthermore we have no reference material on cases with lethal serum values comparing these to vitreous values. And lastly valid and reliable discrimination values which discriminate between certainly normal and certainly abnormal or lethal in vitreous are missing. These conceptual problems are more or less inherent in all studies on vitreous humor chemistry and have to be addressed in further systematic investigations. Some of these scientific gaps will continue for methodical problems: e.g. it will be very difficult to obtain correlations between markedly depressed or elevated antemortem serum values and postmortem vitreous values since patients with markedly disturbed values will be treated. A further task for the future will be to develop calibrated and validated methods for alternative fluids, especially vitreous humor analysis. References [1] T. Alstro¨m, R. Gradbeck, M. Hjelm, S. Skandsen, Recommendations concerning the collection of reference values in clinical chemistry and activity report, Scand. J. Clin. Lab Invest. 35 (Suppl. 144) (1975) 5–44. [2] T.A. Blumenfeld, C.H. Mantell, R.L. Catherman, W.A. Blanc, Postmortem vitreous humor chemistry in sudden infant death syndrome and in other causes of death in childhood, Am. J. Clin. Pathol. 71 (1979) 219– 223. [3] G. Bocaz-Beneventi, F. Tagliaro, F. Bortolotti, G. Manetto, J. Havel, Capillary zone electrophoresis and artificial neural networks for estimation of the post-mortem interval (PMI) using electrolyte measurements in human vitreous humor, Int. J. Legal Med. 116 (1) (2002) 5–11. [4] E. Choo-Kang, C. McKoy, C. Escoffrey, Vitreous humor analytes in assessing the postmortem interval and the antemortem clinical status, WJ Med. J. 32 (1983) 23–26. [5] J.I. Coe, Postmortem chemistries on vitreous humor, Am. J. Pathol. 51 (6) (1969) 741–750. [6] J.I. Coe, Use of chemical determinations on vitreous humor in forensic pathology, J. Forensic Sci. 17 (1972) 541–546. [7] J.I. Coe, F.S. Apple, Variations in vitreous humor chemical values as a result of instrumentation, J. Forensic Sci. 30 (3) (1985) 828–835. [8] J.I. Coe, Postmortem chemistry update—emphasis on forensic applications, Am. J. Forensic Med. Pathol. (1993) 91–117. [9] M.S. Devgun, J.A. Dunbar, Biochemical investigations of vitreus: applications in forensic medicine, especially in relation to alcohol, Forensic Sci. Int. 31 (1986) 27–34. [10] R. Dybkaer, The theory of reference values. Part 6, presentation of observed values related to reference values, J. Clin. Chem. Clin. Biochem. 20 (1982) 841–845. [11] J.G. Farmer, F. Benomran, A.A. Watson, W.A. Harland, Magnesium, potassium, sodium and calcium in postmortem vitreous humor from humans, Forensic Sci. Int. 27 (1985) 1–13. [12] K.E. Ferslew, A.N. Hagardorn, M.T. Harrison, W.F. McCormick, Capillary ion analysis of potassium concentrations in human vitreous humor, Electrophoresis 19 (1) (1998) 6–10. [13] U. Garg, R. Althahabi, V. Amirahmadi, M. Brod, C. Blanchard, T. Young, Hyaluronidase as a liquefying agent for chemical analysis of vitreous fluid, J. Forensic Sci. 49 (2004) 388–391. [14] R. Grasbeck, G. Siest, P. Wilding, G.Z. Williams, T.P. Whitehead, Provisional recommendation on the theory of reference values (1978). Part 1. The concept of reference values, J. Clin.Chem. 25 (1979) 1506–1508.
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