- Email: [email protected]
Immunohistochemical methods as an aid in estimating the time since death
Accepted Manuscript Title: Immunohistochemical methods as an aid in estimating the time since death Authors: Jan Ortmann, Elke Doberentz, Burkhard Madea PII: DOI: Reference:
S0379-0738(17)30049-X http://dx.doi.org/doi:10.1016/j.forsciint.2017.02.004 FSI 8753
To appear in:
FSI
Received date: Revised date: Accepted date:
27-12-2016 11-1-2017 3-2-2017
Please cite this article as: Jan Ortmann, Elke Doberentz, Burkhard Madea, Immunohistochemical methods as an aid in estimating the time since death, Forensic Science International http://dx.doi.org/10.1016/j.forsciint.2017.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immunohistochemical methods as an aid in estimating the time since death Jan Ortmann, Elke Doberentz, Burkhard Madea*
Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany *
Corresponding author: [email protected]
Highlights -
A method to estimate the time since death using immunohistochemistry was exposed. With increasing postmortem interval (pmi) the stainability of antigens decreases. This allows a rough estimation of the time since death. In this first control study the reduced stainability of antigens can be confirmed. For thyreoglobulin and calcitonin we received much earlier negative stainings.
Abstract For the estimation of time since death in the early postmortem interval, a large repertoire of valid methods is available. With increasing time since death the estimation of the postmortem interval becomes more imprecise and unreliable because validated methods are missing. Wehner et al. developed a method to estimate the time since death using immunohistochemical staining. Proteins undergo degradation after death and this reduces their stainability. Previous studies have explored the development of a method to estimate the time since death using immunohistochemical staining. A systematic analysis (Wehner et al. 1999-2002) demonstrated that calcitonin can still be stained 4 days, thyreoglobulin 5 days, glucagon 6 days, and insulin 12 days after death. After 12 days, calcitonin and thyroglobulin can no longer be stained, glucagon after 14 days, and insulin after 29 days. The aim of the present study was to test this original data on independent case material. Included in this control study were 105 cases with known time since death (between several hours and 22 days). Pancreatic tissue was stained for insulin and glucagon, and the thyroid gland for thyreoglobulin and calcitonin. The original findings could be generally confirmed, however for calcitonin and thyreoglobulin we observed earlier negative stainings. Altogether immunohistochemistry may still be useful as an additional method for estimating time since death in forensic cases. Keywords: time of death – immunohistochemistry – insulin – thyreoglobulin – glucagon – calcitonin.
1. Introduction Morphological methods for time since death estimation are thought to be of no practical value in forensic practice. Although a comprehensive evaluation of all histologically, histochemically and electron-microscopically detectable postmortem changes shows that human tissues and organs decompose and break down in a certain sequence, in practice this sequence can be difficult to recognize, or not be seen at all in some cases. Laboratory experiments with artificial autolysis have shown that under precise conditions it is possible to histologically and histochemically establish the course of the first 48 hours of this process [1]. Therefore, under laboratory conditions and after a suitable series of tests and controls, the postmortem interval could be determined from the degree of decomposition found in an organism [1].
In forensic practice the influencing factors are extremely diverse and cannot all be taken into account. According to Janssen [1], a determination of histological-histochemical autolysis and time of death estimation for forensic purposes can only be attempted when all possible influential factors are known. Valid evidence indicating the time of death can perhaps be taken from the cardiac musculature, skeletal muscles, kidneys, and liver, but none of this is conclusive. An attempt to estimate the time of death without knowledge of the environmental conditions is entirely futile and forensically unjustifiable, regardless of the combination of histological, histochemical, and biochemical methods being applied [1]. However, despite this assertion being a long-standing doctrine in forensic medicine [1-4], there is some evidence that the application of immunohistochemistry methods may still be useful, even without all the available background data. Wehner et al. [5-9] tested whether positive immunoreactions to various antigens like insulin, glucagon, thyreoglobulin, or calcitonin correlated with the time since death. The premise is that the tertiary structure of the antigen undergoes postmortem changes, and with increasing postmortem interval the protein denaturation staining efficacy decreases. For example, the colloid and the follicular cells of the thyroid gave a positive immunoreaction for thyreoglobulin until 5 days postmortem, whereas none of the cases older than 13 days showed such a reaction. This indicates that a negative reaction means more than 6 days postmortem, and a positive reaction means the postmortem interval is less than 12 days. Likewise, pancreatic -cells from up to 12-day-old corpses produced a positive immunoreaction towards insulin in all cases, whereas none of the corpses older than 30 days showed such a reaction [5-9]. This means that in a case of negative immunoreaction the time since death can be assumed to be more than 12 days before the autopsy, but a positive stain means that death occurred a maximum of 29 days earlier. Calcitonin was always detectable in c-cells of the thyroid up to 4 days postmortem, in bodies older than 13 days the staining was always negative. Meanwhile a chart was developed to give a rough estimation of time since death when using immunohistochemical methods [5] (fig.1). Until now, only the original investigations by Wehner et al. [5-9] have been published. A controlled investigation of the validity of their findings, using independent case material, has not yet been carried out. The aim of the present study was to establish a time course of the immunohistochemical staining of insulin, thyreoglobulin, calcitonin and glucagon with independent case material, and compare this with the original data.
2. Material and methods: One hundred and five bodies were included in the examination, and the case information is summarized in Table 1. The time since death ranged from several hours up to 22 days. Cases with diabetes mellitus or diseases of the thyroid gland were excluded, as were cases with macroscopic pathologies of the pancreas and thyroid gland. Bodies found both indoors and outdoors were included in the study. External factors such as temperature, weather, and clothing varied widely from case to case. Pancreatic and thyroid tissue were fixated in formalin, then embedded in paraffin and sliced into 3-m-thick sections. Antibodies against insulin, glucagon, thyreoglobulin, and calcitonin were used for immunohistochemical staining (Kit: Universal LSAB™2 Kit/HRP, Rb/Mo, Dako K0675, antibodies: Polyclonal Guinea Pig Anti-Insulin, Dako A0564, Rabbit Monoclonal [EP3070] to Glucagon, Abcam ab92517. Kit: EnVision™+Dual Link System/HRP, Dako K4061, antibodies: Polyclonal Rabbit Anti-Human Calcitonin, Dako A0576, Polyclonal Rabbit Anti-Human Thyroglobulin, Dako A0251; all kits were sourced from Dako North America Inc., Carpinteria, CA, USA). The immunohistochemical staining protocols by Wehner et al. were used [5-8].
3. Results and Discussion Positive immunoreactions for insulin and glucagon in the pancreatic tissue, and thyreoglobulin and calcitonin in the thyroid gland are shown in Figure 2. As already shown by Wehner et al. [5-8], stainability of the pancreatic tissue as well as the thyroid gland is timedependent (Figs. 3-6). According to Wehner et al. calcitonin was always stainable for at least 4 days, thyreoglobulin until 5 days, glucagon 6 days, and insulin for 12 days, postmortem [58]. After 12 days calcitonin and thyreoglobulin are no longer stainable, extending to after 13 days for glucagon and after 29 days for insulin. Altogether, the results by Wehner et al. on a time dependent decrease of stainability can be confirmed by our findings (Figs. 3-6). Our results are compared with the previous studies by Werner et al. in the graphs in Figures 3-6. It can be easily seen that our data differ from the original work [5-9]. Especially for thyreoglobulin and calcitonin a much earlier loss of stainability was noted (already after one day instead of 5 days). For glucagon the data are in good agreement, for insulin as well (table 2). The reason for the earlier loss of stainability is not clear but must have to do with the individual cases. Of course the composition of the own study group differs from Wehner et al [5-8] according to causes of death, postmortem interval, environmental conditions etc.
Interestingly loss of stainability of one antigen, f.i. calcitonin, is not necessarily accompanied by loss of stainability of the other antigens as well (see table 1). When using the method in casework the wider limits according to the results of both studies have to be taken into account to avoid false estimation of the time since death. Immunohistochemical methods are altogether a valid contribution to the repertoire of methods for estimation of time since death. However, in cases with microscopically observable and recognized pathologies, immunohistochemical staining should not be used for time since death estimation. To improve precision in estimating the time since death, further studies would be needed, to take into account external factors, such as ambient temperature (at least mean ambient temperature) for the postmortem interval, clothing, and covering.
4. Conclusion The immunohistochemical detection of antigens allows only a rough estimation of the time since death, which may be helpful in individual cases. These methods have already been shown to give promising results in forensic practice, particularly when the question is if the postmortem interval is only a few days or more than one week. However it has to be kept in mind, that calcitonin and thyreoglobulin are the antigens becoming earlier negative than reported in the literature while the results for insulin and glucagon can be more or less confirmed.
5. References
1. Janssen W (1984) Forensic Histology. Springer, Berlin Heidelberg New York 2. Madea B (2005) Is there recent progress in the estimation of the postmortem interval by means of thanatochemistry? For Sci Int 151: 139-149 3. Madea B (2015) Immunohistochemical methods as an aid in estimating the time since death. In: Madea B (Ed.) Estimation of the Time Since Death 3rd Ed, CRC Press, Taylor & Francis, Boca Raton, London, New York. 223-226 4. Madea B (2016) Methods for determining time of death. Forensic Sci Med Pathol 12 (4): 451-485 5. Wehner F., Wehner H.-D., Schieffer M. C., Subke J., Delimination of the time since death by immunohistochemical detection of insulin in pancreatic ß-cells, Forensic Sci. International 105 (1999) 161-169 6. Wehner F., Wehner H.-D., Schieffer M. C., Subke J., Delimination of the time since death by immunohistochemical detection of thyreoglobulin, Forensic Sci. International 110 (2000) 199-206 7. Wehner F., Wehner H.-D., Subke J., Delimination of the time since death by immunohistochemical detection of calcitonin, Forensic Sci. International 122 (2001) 89-94 8. Wehner F., Wehner H.-D., Subke J., Delimination of the time since death by immunohistochemical detection of glucagon in pancreatic α-cells, Forensic Sci. International 124 (2001) 192-199 9. Wehner F., Die Eingrenzung der Leichenliegezeit im spätpostmortalen Intervall – Neue Ansätze mittels immunhistochemischen Verfahren, Med. Welt 60 (2009) 402406
Legends to figures Fig. 1 Positive immunoreaction of insulin: time since death maximal 29 days. Additional investigation of Glucagon with a negative immunoreaction: delimitation of the time since death between 7 and 29 days. Additional investigation of Thyreoglobulin with a positive immunoreaction: further delimitation of the time since death between 7 and 12 days Fig. 2 a) Pancreas tissue Anti-human insulin 200x, autopsy 8 days after death b) Pancreas tissue Anti-human glucagon 200x, autopsy 8 days after death c) Thyroid gland tissue Anti-human thyreoglobulin 400x, autopsy 8 days after death d) Thyroid gland tissue Anti-human calcitonin 400x, autopsy 11 days after death Fig. 3 Comparison of the results of the study of Wehner et al. [5] and the own study for insulin Fig. 4 Comparison of the results of the study of Wehner et al. [8] and the own study for glucagon Fig. 5 Comparison of the results of the study of Wehner et al. [6] and the own study thyreoglobulin Fig. 6 Comparison of the results of the study of Wehner et al. [7] and the own study for calcitonin
Estimation of the time since death in the later postmortem period Case:………….. Found dead:…………… Autopsy date:…………….
-
+
Insulin
- ☐ ≥ 13 days
≤ 29 days ☒ +
Glucagon
- ☒ ≥ 7
≤ 13
☐ +
Thyreoglobulin
- ☐ ≥ 6
≤ 12
☒ +
Calcitonin
- ☐ ≥ 5
≤ 12
☐ +
time since death 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
time of death between
Fig. 1
7
days and maximum
12
days
Fig. 2a
Fig. 2b
Fig. 2c
Fig. 2d
Overview of the time-dependency of the stainability of insulin (Wehner et al. For. Sci. Int. 1999)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of insulin
Fig. 3
Overview of the time-dependency of the stainability of glucagon (Wehner et al. For. Sci. Int. 2001)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of glucagon
Fig. 4
Overview of the time-dependency of the stainability of thyreoglobulin (Wehner et al. For. Sci. Int. 2001)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of thyreoglobulin
Fig. 5
Overview of the time-dependency of the stainability of calcitonin (Wehner et al. For. Sci. Int. 2001)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of calcitonin
Fig. 6
Table 1 Case material and results of the immunohistochemical investigation for 4 antigenes Time death 1 Day
2 Days
3 Days
since Place found
Clothing/Cover Time of year/month
Immunoreaction
Bathtub
unclothed
May
+
+
+
-
Home
light clothing
May
+
+
+
+
Home
light clothing
May
+
+
+
+
Hospital
clothed
April
+
+
+
+
Hospital
light clothed
March
+
+
+
+
Home
light clothed
December
+
+
+
-
Home
clothed
July
+
+
+
+
Hospital
clothed
June
+
+
+
+
Meadow
clothed
June
+
+
+
+
Carpark
clothed
March
+
+
+
+
Bicycle lane
clothed
March
+
+
+
+
Street
clothed
March
+
+
+
+
Automobile
clothed
February
+
+
+
+
Hospital
clothed
February
+
+
+
-
Home
light clothed
February
+
+
+
+
Home/Hospital
light clothed
February
+
+
+
+
Street/Automobile
clothed
January
+
+
-
-
Home/Bed
clothed
January
+
+
+
-
Home/Bed
blanket
January
+
+
-
-
Home
clothed
November
+
+
+
+
Home
clothed
November
+
+
+
+
Hospital
light clothed
November
+
+
+
+
Home
clothed
July
+
+
+
+
Hospital
light clothed
June
+
+
+
+
Ins. Glu. Thyr. Cal.
4 Days
Home/Garden
clothed
June
+
+
+
+
Home
clothed
June
+
+
+
+
Home
clothed
May
+
+
+
+
Hospital
light clothed
May
+
+
+
+
Hospital
light clothed
May
+
+
+
+
Hospital
clothed
May
+
+
+
+
Hospital
light clothed
May
+
+
+
+
Hospital
clothed
April
+
+
+
+
Home
clothed
February
+
+
-
-
Building site
clothed
December
+
+
+
+
Home
light clothed
November
+
+
+
+
Street
clothed
October
+
+
+
+
Street
clothed
October
+
+
-
-
Hospital
clothed
September
+
+
-
-
Home
clothed
August
+
+
+
Hospital
light clothed
June
+
+
+
+
Home
clothed
June
+
+
+
+
Hospital
clothed
June
+
+
+
+
Hospital
light clothed
June
+
+
+
+
Hospital
clothed
April
+
+
+
+
Home/Hospital
clothed
April
+
+
+
+
Forest
clothed
April
+
+
+
+
Swimming pool
clothed
April
+
+
+
+
Home
light clothed
February
+
+
+
+
Hospital
light clothed
February
+
+
+
-
Outside Home
clothed
January
+
+
+
+
Street/Hospital
light clothed
January
+
+
-
-
Home
light clothed
December
+
+
+
+
Hospital
clothed
December
+
+
+
+
Time death 4 Days
5 Days
since
Street
clothed
November
+
+
+
+
Street/Hospital
clothed
October
+
+
+
+
Home
clothed
October
+
+
-
-
Hospital
clothed
October
+
+
+
+
Place found
Clothing/cover
Time year/month
of Immunoreaction Ins. Glu. Thyr. Cal.
Hospital
clothed
September
+
+
-
-
Home
clothed
September
+
+
+
+
Hospital
light clothed
September
+
+
+
-
Home
clothed
September
+
+
-
-
Home
light clothed
August
+
+
+
-
Home
clothed
August
+
+
+
+
Street
clothed
July
+
+
+
+
Home
clothed
June
+
+
+
+
Home
light clothed
June
+
+
+
-
Home
clothed
June
+
+
+
+
Street
clothed
May
+
+
+
+
Hospital
light clothed
April
+
+
+
-
Hospital
light clothed
March
+
+
+
+
Home
clothed
January
+
+
+
+
Other Home/Hospital
light clothed
January
+
+
-
-
Home
clothed
December
+
+
+
+
Hospital
clothed
December
+
+
+
+
Home
clothed
December
+
+
+
+
Hospital
clothed
November
+
+
+
-
Home
clothed
November
+
+
+
-
Home
clothed
October
+
+
+
+
Home/Hospital
clothed
October
+
+
-
-
Hospital
light clothed
October
+
+
-
-
Hospital
clothed
August
+
+
+
-
Home
clothed
January
+
(+)
+
+
Home
clothed
December
+
+
-
-
Home
clothed
December
+
+
+
+
Street
clothed
October
+
+
-
-
Hospital
Unclothed
October
+
+
-
-
Street
clothed
September
+
+
+
-
Hospital
light clothed
September
+
+
+
-
Hospital
light clothed
February
+
+
+
-
Hospital
light clothed
January
+
+
-
-
Hospital
clothed
October
+
+
+
+
Home
light clothed
October
+
+
-
-
Hospital
clothed
September
+
+
+
+
Hospital
clothed
September
+
+
-
-
Hospital
clothed
May
+
+
-
-
Home
clothed
February
+
+
+
-
Hospital
light clothed
January
+
+
-
-
Home
light clothed
December
+
+
-
-
Hospital
light clothed
October
+
+
+
-
Hospital
clothed
October
+
-
+
+
Hospital
clothed
September
+
+
-
-
11 Days
Home
clothed
November
+
+
-
-
12 Days
Hospital
clothed
May
+
+
-
-
18 Days
Hospital
light clothed
December
+
-
-
-
22 Days
Home
clothed
April
6 Days
7 Days
8 Days
-
-
-
-
Table 2 Comparison of the results of Wehner et al. for four antigens and the own study Wehner et al.
own study
+
-
+
-
Insulin
29 days
13 days
18 days
22 days
Glucagon
13 days
7 days
12 days
8 days
Thyreoglobulin
12 days
6 days
8 days
1 days*
Calcitonin
12 days
5 days
8 days
1 days*
S0379-0738(17)30049-X http://dx.doi.org/doi:10.1016/j.forsciint.2017.02.004 FSI 8753
To appear in:
FSI
Received date: Revised date: Accepted date:
27-12-2016 11-1-2017 3-2-2017
Please cite this article as: Jan Ortmann, Elke Doberentz, Burkhard Madea, Immunohistochemical methods as an aid in estimating the time since death, Forensic Science International http://dx.doi.org/10.1016/j.forsciint.2017.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immunohistochemical methods as an aid in estimating the time since death Jan Ortmann, Elke Doberentz, Burkhard Madea*
Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany *
Corresponding author: [email protected]
Highlights -
A method to estimate the time since death using immunohistochemistry was exposed. With increasing postmortem interval (pmi) the stainability of antigens decreases. This allows a rough estimation of the time since death. In this first control study the reduced stainability of antigens can be confirmed. For thyreoglobulin and calcitonin we received much earlier negative stainings.
Abstract For the estimation of time since death in the early postmortem interval, a large repertoire of valid methods is available. With increasing time since death the estimation of the postmortem interval becomes more imprecise and unreliable because validated methods are missing. Wehner et al. developed a method to estimate the time since death using immunohistochemical staining. Proteins undergo degradation after death and this reduces their stainability. Previous studies have explored the development of a method to estimate the time since death using immunohistochemical staining. A systematic analysis (Wehner et al. 1999-2002) demonstrated that calcitonin can still be stained 4 days, thyreoglobulin 5 days, glucagon 6 days, and insulin 12 days after death. After 12 days, calcitonin and thyroglobulin can no longer be stained, glucagon after 14 days, and insulin after 29 days. The aim of the present study was to test this original data on independent case material. Included in this control study were 105 cases with known time since death (between several hours and 22 days). Pancreatic tissue was stained for insulin and glucagon, and the thyroid gland for thyreoglobulin and calcitonin. The original findings could be generally confirmed, however for calcitonin and thyreoglobulin we observed earlier negative stainings. Altogether immunohistochemistry may still be useful as an additional method for estimating time since death in forensic cases. Keywords: time of death – immunohistochemistry – insulin – thyreoglobulin – glucagon – calcitonin.
1. Introduction Morphological methods for time since death estimation are thought to be of no practical value in forensic practice. Although a comprehensive evaluation of all histologically, histochemically and electron-microscopically detectable postmortem changes shows that human tissues and organs decompose and break down in a certain sequence, in practice this sequence can be difficult to recognize, or not be seen at all in some cases. Laboratory experiments with artificial autolysis have shown that under precise conditions it is possible to histologically and histochemically establish the course of the first 48 hours of this process [1]. Therefore, under laboratory conditions and after a suitable series of tests and controls, the postmortem interval could be determined from the degree of decomposition found in an organism [1].
In forensic practice the influencing factors are extremely diverse and cannot all be taken into account. According to Janssen [1], a determination of histological-histochemical autolysis and time of death estimation for forensic purposes can only be attempted when all possible influential factors are known. Valid evidence indicating the time of death can perhaps be taken from the cardiac musculature, skeletal muscles, kidneys, and liver, but none of this is conclusive. An attempt to estimate the time of death without knowledge of the environmental conditions is entirely futile and forensically unjustifiable, regardless of the combination of histological, histochemical, and biochemical methods being applied [1]. However, despite this assertion being a long-standing doctrine in forensic medicine [1-4], there is some evidence that the application of immunohistochemistry methods may still be useful, even without all the available background data. Wehner et al. [5-9] tested whether positive immunoreactions to various antigens like insulin, glucagon, thyreoglobulin, or calcitonin correlated with the time since death. The premise is that the tertiary structure of the antigen undergoes postmortem changes, and with increasing postmortem interval the protein denaturation staining efficacy decreases. For example, the colloid and the follicular cells of the thyroid gave a positive immunoreaction for thyreoglobulin until 5 days postmortem, whereas none of the cases older than 13 days showed such a reaction. This indicates that a negative reaction means more than 6 days postmortem, and a positive reaction means the postmortem interval is less than 12 days. Likewise, pancreatic -cells from up to 12-day-old corpses produced a positive immunoreaction towards insulin in all cases, whereas none of the corpses older than 30 days showed such a reaction [5-9]. This means that in a case of negative immunoreaction the time since death can be assumed to be more than 12 days before the autopsy, but a positive stain means that death occurred a maximum of 29 days earlier. Calcitonin was always detectable in c-cells of the thyroid up to 4 days postmortem, in bodies older than 13 days the staining was always negative. Meanwhile a chart was developed to give a rough estimation of time since death when using immunohistochemical methods [5] (fig.1). Until now, only the original investigations by Wehner et al. [5-9] have been published. A controlled investigation of the validity of their findings, using independent case material, has not yet been carried out. The aim of the present study was to establish a time course of the immunohistochemical staining of insulin, thyreoglobulin, calcitonin and glucagon with independent case material, and compare this with the original data.
2. Material and methods: One hundred and five bodies were included in the examination, and the case information is summarized in Table 1. The time since death ranged from several hours up to 22 days. Cases with diabetes mellitus or diseases of the thyroid gland were excluded, as were cases with macroscopic pathologies of the pancreas and thyroid gland. Bodies found both indoors and outdoors were included in the study. External factors such as temperature, weather, and clothing varied widely from case to case. Pancreatic and thyroid tissue were fixated in formalin, then embedded in paraffin and sliced into 3-m-thick sections. Antibodies against insulin, glucagon, thyreoglobulin, and calcitonin were used for immunohistochemical staining (Kit: Universal LSAB™2 Kit/HRP, Rb/Mo, Dako K0675, antibodies: Polyclonal Guinea Pig Anti-Insulin, Dako A0564, Rabbit Monoclonal [EP3070] to Glucagon, Abcam ab92517. Kit: EnVision™+Dual Link System/HRP, Dako K4061, antibodies: Polyclonal Rabbit Anti-Human Calcitonin, Dako A0576, Polyclonal Rabbit Anti-Human Thyroglobulin, Dako A0251; all kits were sourced from Dako North America Inc., Carpinteria, CA, USA). The immunohistochemical staining protocols by Wehner et al. were used [5-8].
3. Results and Discussion Positive immunoreactions for insulin and glucagon in the pancreatic tissue, and thyreoglobulin and calcitonin in the thyroid gland are shown in Figure 2. As already shown by Wehner et al. [5-8], stainability of the pancreatic tissue as well as the thyroid gland is timedependent (Figs. 3-6). According to Wehner et al. calcitonin was always stainable for at least 4 days, thyreoglobulin until 5 days, glucagon 6 days, and insulin for 12 days, postmortem [58]. After 12 days calcitonin and thyreoglobulin are no longer stainable, extending to after 13 days for glucagon and after 29 days for insulin. Altogether, the results by Wehner et al. on a time dependent decrease of stainability can be confirmed by our findings (Figs. 3-6). Our results are compared with the previous studies by Werner et al. in the graphs in Figures 3-6. It can be easily seen that our data differ from the original work [5-9]. Especially for thyreoglobulin and calcitonin a much earlier loss of stainability was noted (already after one day instead of 5 days). For glucagon the data are in good agreement, for insulin as well (table 2). The reason for the earlier loss of stainability is not clear but must have to do with the individual cases. Of course the composition of the own study group differs from Wehner et al [5-8] according to causes of death, postmortem interval, environmental conditions etc.
Interestingly loss of stainability of one antigen, f.i. calcitonin, is not necessarily accompanied by loss of stainability of the other antigens as well (see table 1). When using the method in casework the wider limits according to the results of both studies have to be taken into account to avoid false estimation of the time since death. Immunohistochemical methods are altogether a valid contribution to the repertoire of methods for estimation of time since death. However, in cases with microscopically observable and recognized pathologies, immunohistochemical staining should not be used for time since death estimation. To improve precision in estimating the time since death, further studies would be needed, to take into account external factors, such as ambient temperature (at least mean ambient temperature) for the postmortem interval, clothing, and covering.
4. Conclusion The immunohistochemical detection of antigens allows only a rough estimation of the time since death, which may be helpful in individual cases. These methods have already been shown to give promising results in forensic practice, particularly when the question is if the postmortem interval is only a few days or more than one week. However it has to be kept in mind, that calcitonin and thyreoglobulin are the antigens becoming earlier negative than reported in the literature while the results for insulin and glucagon can be more or less confirmed.
5. References
1. Janssen W (1984) Forensic Histology. Springer, Berlin Heidelberg New York 2. Madea B (2005) Is there recent progress in the estimation of the postmortem interval by means of thanatochemistry? For Sci Int 151: 139-149 3. Madea B (2015) Immunohistochemical methods as an aid in estimating the time since death. In: Madea B (Ed.) Estimation of the Time Since Death 3rd Ed, CRC Press, Taylor & Francis, Boca Raton, London, New York. 223-226 4. Madea B (2016) Methods for determining time of death. Forensic Sci Med Pathol 12 (4): 451-485 5. Wehner F., Wehner H.-D., Schieffer M. C., Subke J., Delimination of the time since death by immunohistochemical detection of insulin in pancreatic ß-cells, Forensic Sci. International 105 (1999) 161-169 6. Wehner F., Wehner H.-D., Schieffer M. C., Subke J., Delimination of the time since death by immunohistochemical detection of thyreoglobulin, Forensic Sci. International 110 (2000) 199-206 7. Wehner F., Wehner H.-D., Subke J., Delimination of the time since death by immunohistochemical detection of calcitonin, Forensic Sci. International 122 (2001) 89-94 8. Wehner F., Wehner H.-D., Subke J., Delimination of the time since death by immunohistochemical detection of glucagon in pancreatic α-cells, Forensic Sci. International 124 (2001) 192-199 9. Wehner F., Die Eingrenzung der Leichenliegezeit im spätpostmortalen Intervall – Neue Ansätze mittels immunhistochemischen Verfahren, Med. Welt 60 (2009) 402406
Legends to figures Fig. 1 Positive immunoreaction of insulin: time since death maximal 29 days. Additional investigation of Glucagon with a negative immunoreaction: delimitation of the time since death between 7 and 29 days. Additional investigation of Thyreoglobulin with a positive immunoreaction: further delimitation of the time since death between 7 and 12 days Fig. 2 a) Pancreas tissue Anti-human insulin 200x, autopsy 8 days after death b) Pancreas tissue Anti-human glucagon 200x, autopsy 8 days after death c) Thyroid gland tissue Anti-human thyreoglobulin 400x, autopsy 8 days after death d) Thyroid gland tissue Anti-human calcitonin 400x, autopsy 11 days after death Fig. 3 Comparison of the results of the study of Wehner et al. [5] and the own study for insulin Fig. 4 Comparison of the results of the study of Wehner et al. [8] and the own study for glucagon Fig. 5 Comparison of the results of the study of Wehner et al. [6] and the own study thyreoglobulin Fig. 6 Comparison of the results of the study of Wehner et al. [7] and the own study for calcitonin
Estimation of the time since death in the later postmortem period Case:………….. Found dead:…………… Autopsy date:…………….
-
+
Insulin
- ☐ ≥ 13 days
≤ 29 days ☒ +
Glucagon
- ☒ ≥ 7
≤ 13
☐ +
Thyreoglobulin
- ☐ ≥ 6
≤ 12
☒ +
Calcitonin
- ☐ ≥ 5
≤ 12
☐ +
time since death 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
time of death between
Fig. 1
7
days and maximum
12
days
Fig. 2a
Fig. 2b
Fig. 2c
Fig. 2d
Overview of the time-dependency of the stainability of insulin (Wehner et al. For. Sci. Int. 1999)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of insulin
Fig. 3
Overview of the time-dependency of the stainability of glucagon (Wehner et al. For. Sci. Int. 2001)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of glucagon
Fig. 4
Overview of the time-dependency of the stainability of thyreoglobulin (Wehner et al. For. Sci. Int. 2001)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of thyreoglobulin
Fig. 5
Overview of the time-dependency of the stainability of calcitonin (Wehner et al. For. Sci. Int. 2001)
positive immunoreaction
negative immunoreaction
Overview of the time-dependency of the stainability of calcitonin
Fig. 6
Table 1 Case material and results of the immunohistochemical investigation for 4 antigenes Time death 1 Day
2 Days
3 Days
since Place found
Clothing/Cover Time of year/month
Immunoreaction
Bathtub
unclothed
May
+
+
+
-
Home
light clothing
May
+
+
+
+
Home
light clothing
May
+
+
+
+
Hospital
clothed
April
+
+
+
+
Hospital
light clothed
March
+
+
+
+
Home
light clothed
December
+
+
+
-
Home
clothed
July
+
+
+
+
Hospital
clothed
June
+
+
+
+
Meadow
clothed
June
+
+
+
+
Carpark
clothed
March
+
+
+
+
Bicycle lane
clothed
March
+
+
+
+
Street
clothed
March
+
+
+
+
Automobile
clothed
February
+
+
+
+
Hospital
clothed
February
+
+
+
-
Home
light clothed
February
+
+
+
+
Home/Hospital
light clothed
February
+
+
+
+
Street/Automobile
clothed
January
+
+
-
-
Home/Bed
clothed
January
+
+
+
-
Home/Bed
blanket
January
+
+
-
-
Home
clothed
November
+
+
+
+
Home
clothed
November
+
+
+
+
Hospital
light clothed
November
+
+
+
+
Home
clothed
July
+
+
+
+
Hospital
light clothed
June
+
+
+
+
Ins. Glu. Thyr. Cal.
4 Days
Home/Garden
clothed
June
+
+
+
+
Home
clothed
June
+
+
+
+
Home
clothed
May
+
+
+
+
Hospital
light clothed
May
+
+
+
+
Hospital
light clothed
May
+
+
+
+
Hospital
clothed
May
+
+
+
+
Hospital
light clothed
May
+
+
+
+
Hospital
clothed
April
+
+
+
+
Home
clothed
February
+
+
-
-
Building site
clothed
December
+
+
+
+
Home
light clothed
November
+
+
+
+
Street
clothed
October
+
+
+
+
Street
clothed
October
+
+
-
-
Hospital
clothed
September
+
+
-
-
Home
clothed
August
+
+
+
Hospital
light clothed
June
+
+
+
+
Home
clothed
June
+
+
+
+
Hospital
clothed
June
+
+
+
+
Hospital
light clothed
June
+
+
+
+
Hospital
clothed
April
+
+
+
+
Home/Hospital
clothed
April
+
+
+
+
Forest
clothed
April
+
+
+
+
Swimming pool
clothed
April
+
+
+
+
Home
light clothed
February
+
+
+
+
Hospital
light clothed
February
+
+
+
-
Outside Home
clothed
January
+
+
+
+
Street/Hospital
light clothed
January
+
+
-
-
Home
light clothed
December
+
+
+
+
Hospital
clothed
December
+
+
+
+
Time death 4 Days
5 Days
since
Street
clothed
November
+
+
+
+
Street/Hospital
clothed
October
+
+
+
+
Home
clothed
October
+
+
-
-
Hospital
clothed
October
+
+
+
+
Place found
Clothing/cover
Time year/month
of Immunoreaction Ins. Glu. Thyr. Cal.
Hospital
clothed
September
+
+
-
-
Home
clothed
September
+
+
+
+
Hospital
light clothed
September
+
+
+
-
Home
clothed
September
+
+
-
-
Home
light clothed
August
+
+
+
-
Home
clothed
August
+
+
+
+
Street
clothed
July
+
+
+
+
Home
clothed
June
+
+
+
+
Home
light clothed
June
+
+
+
-
Home
clothed
June
+
+
+
+
Street
clothed
May
+
+
+
+
Hospital
light clothed
April
+
+
+
-
Hospital
light clothed
March
+
+
+
+
Home
clothed
January
+
+
+
+
Other Home/Hospital
light clothed
January
+
+
-
-
Home
clothed
December
+
+
+
+
Hospital
clothed
December
+
+
+
+
Home
clothed
December
+
+
+
+
Hospital
clothed
November
+
+
+
-
Home
clothed
November
+
+
+
-
Home
clothed
October
+
+
+
+
Home/Hospital
clothed
October
+
+
-
-
Hospital
light clothed
October
+
+
-
-
Hospital
clothed
August
+
+
+
-
Home
clothed
January
+
(+)
+
+
Home
clothed
December
+
+
-
-
Home
clothed
December
+
+
+
+
Street
clothed
October
+
+
-
-
Hospital
Unclothed
October
+
+
-
-
Street
clothed
September
+
+
+
-
Hospital
light clothed
September
+
+
+
-
Hospital
light clothed
February
+
+
+
-
Hospital
light clothed
January
+
+
-
-
Hospital
clothed
October
+
+
+
+
Home
light clothed
October
+
+
-
-
Hospital
clothed
September
+
+
+
+
Hospital
clothed
September
+
+
-
-
Hospital
clothed
May
+
+
-
-
Home
clothed
February
+
+
+
-
Hospital
light clothed
January
+
+
-
-
Home
light clothed
December
+
+
-
-
Hospital
light clothed
October
+
+
+
-
Hospital
clothed
October
+
-
+
+
Hospital
clothed
September
+
+
-
-
11 Days
Home
clothed
November
+
+
-
-
12 Days
Hospital
clothed
May
+
+
-
-
18 Days
Hospital
light clothed
December
+
-
-
-
22 Days
Home
clothed
April
6 Days
7 Days
8 Days
-
-
-
-
Table 2 Comparison of the results of Wehner et al. for four antigens and the own study Wehner et al.
own study
+
-
+
-
Insulin
29 days
13 days
18 days
22 days
Glucagon
13 days
7 days
12 days
8 days
Thyreoglobulin
12 days
6 days
8 days
1 days*
Calcitonin
12 days
5 days
8 days
1 days*