Calculating post-mortem interval by using the morphological changes in leucocytes

Abstract

The changes in the internal environment of the body after death due to lack of oxygen result in certain morphological changes in the blood cells. This study tests the hypothesis that after we take out blood from live individuals and store it in-vitro there will also be an anoxic environment which will result in morphological changes like what happens in-vivo after death. The idea was to simulate the anoxic in-vivo condition in-vitro, by storing the blood sample taken from live persons in a vial and to look for sequential changes with respect to time and to develop a scale to see how this scale of morphological changes correlates with the time since death in post-mortem samples collected. Blood samples preserved in EDTA was taken from 186 live volunteers and 60 cadavers whose exact time of death was known. Blood smear was made and stained with Leishman stain and observed under an oil immersion microscope. Cells were classified into four stages based upon the nuclear changes – normal, pyknosis (shrunken nucleus), vacuolated and fragmented nucleus and complete lysis. The sequence of change remained same in both in-vitro stored samples and cadaver in-vivo samples. But the changes in the cadaver samples were appearing rapidly as compared to the in-vitro stored samples. It is hard to completely refute the hypothesis that in-vitro and cadaver conditions would be similar, and changes exhibited would also be similar based on this single study. Further studies with much larger sample size need to be conducted to completely refute or prove the above hypothesis. Also, other co-existing factors which can affect morphology of the cells should be considered.

Keywords

Forensic medicine forensic pathology time since death leucocytes blood cell morphology

Introduction

Estimation of the time since death is a practical task in daily forensic casework. Several methods are being used, and many novel techniques are being introduced constantly to provide an estimate which is more accurate. Despite the availability of so many techniques, the most common practical method used in most of the places especially in India is estimating time since death based on the observation of rigour mortis and hypostasis. This is mainly due to its simplicity, reliability and practicality. Such kind of simple, reliable and practical method to estimate time since death is the need of the day. The foremost motive of this study was derived from the idea that after the occurrence of death, the interior environment (milieu interieur) undergoes certain irreversible changes due to non-availability of (adequate) oxygen; accumulation of carbon dioxide; pH changes; accumulation of toxic products and so on.¹ When we store blood in-vitro at room temperature after taking it from live people the environment it gets (due to lack of oxygen) is more or less similar to the milieu interieur in the dead and the blood cells in-vitro will start degrading with time. The idea is to note these changes of blood cells sequentially, especially the nucleus with respect to time and establish a grading of these morphological changes and compare it with the morphological changes of blood cells retrieved from dead to establish a variation scale so that we could incorporate them in our calculation of time interval since death. If found suitable and successful, this method ensures developing a very simple scale and an easy practical method where in time since death can be compared and established.

Materials and methods

This was a Longitudinal Prospective Time-to-Event Study conducted in the Department of Forensic Medicine and Toxicology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh. Considering a hypothesized proportion of outcome in the study population the final sample size for the development of the scale was 186 live individuals. For the cadavers, only the number of cadavers which satisfied all the inclusion and exclusion criteria that were considered during the study period was taken. Cadavers whose time of death is already known and confirmed, with no signs of decomposition were included for the study; totally 60 cadavers were included. For live individuals, persons of adult age group were included. In both the groups, individuals with conditions which can affect the blood cell morphology like malignancy of blood cells, morbidly infectious condition, anaemia, nutritional deficiency and any chronic disorders were excluded. A 2 ml of blood sample was obtained from cubital vein in live individuals, and blood was obtained from the heart in the case of cadavers and stored in a plastic vacutainer with 0.2 ml of 10% EDTA (ethylenediaminetetraacetic acid). A blood film was constructed and stained with Leishman stain to study the morphology of neutrophils and leucocytes. The blood sample from live individuals was stored at room temperature (29–30 °C) and 13 more sub-samples were drawn from that same 2 ml blood at 6th hour, 8th hour, 10th hour, 12th hour, 14th hour, 16th hour, 18th hour, 20th hour, 22nd hour, 24th hour, 30th hour, 36th hour and 48th hour from the time of blood collection and a total of 14 blood films were prepared and stained for every individual. In the case of cadaver samples, blood films were prepared immediately after the collection and stained. The amount of time elapsed since the declaration of death and time of blood collection was taken as the time interval. There were no cases between 0 and 2 h. In the 2–4 h interval, there were totally seven cases and in 4–6 h also seven cases were present. In the 6–8 h interval, three cases were there and 10 cases were present in the 8–10 h interval. Between 10 and 12 h, six cases were there. The 12–14 h interval also had six cases. Three cases were there in the 14–16 h interval, seven cases in the 16–18 h interval, five cases in the 18–20 h interval, one case in the 20–22 h interval, three cases in the 22–24 h interval and two cases in the 24–26 h interval.

The stained slides of both in-vitro and cadaver samples were then examined under an oil immersion in light microscope to look for any changes in the morphology of white blood cells. Two fields under a 10× objective lens, each measuring 2 mm in diameter and 3.14 mm² in area, were examined. Thirty neutrophils and 15 lymphocytes were examined in total for each slide. Based upon the morphology, white blood cells were categorized into four groups:

Stage I: This includes white blood cells showing normal morphology with no change (Figure 1).

Stage II: This includes white blood cells showing swollen cytoplasm or shrunken, pyknotic nucleus but no vacuolation or fragmentation (Figure 2).

Stage III: This includes white blood cells showing vacuolation and fragmentation of nucleus (Figure 3).

Stage IV: This includes lysed white blood cells (Figure 4).

Figure 1.

An image of peripheral blood smear with Leishman stain under oil immersion microscopy with a 100× lens showing a neutrophil with normal morphology (stage I).

Figure 2.

An image of peripheral blood smear with Leishman stain under oil immersion microscopy with a 100× lens showing a neutrophil with pyknosis (shrunken nuclei) but no vacuolation or fragmentation (stage II).

Figure 3.

An image of peripheral blood smear with Leishman stain under oil immersion microscopy with a 100× lens showing a neutrophil with vacuolation or fragmentation of nuclei (stage III) at 10 o’clock and 7 o’clock position.

Figure 4.

An image of peripheral blood smear with Leishman stain under oil immersion microscopy with a 100× lens showing a lysed neutrophil at centre and 5 o’clock position (stage IV).

In each slide, each type of cell was counted and examined. The morphological change exhibited by 50% of the total number of cells counted was noted as the category of change exhibited by that type of white blood cell at that time. Each type of white blood cell was examined in each slide and changes were noted for each cell type in the given time.

Result

In-vitro samples

For initial 6 h, both cells were in stage I. From 6 until 14 h, neutrophils were in stage II and lymphocytes were in stage I. From 14 until 18 h, neutrophils were in stage III and lymphocytes were in stage I. From 18 until 22 h, neutrophils were in stage III and lymphocytes were in stage II. From 22 until 48 h, neutrophils were in stage IV and lymphocytes were in stage II. Based upon this, a variation scale was framed (Figure 5; Tables 1 and 2).

Figure 5.

Variation scale developed using the changes observed from in-vitro samples.

Table 1.

Percentage of cases showing morphological changes in the neutrophils of in-vitro stored samples with time in hours.

Stage	Time
Stage	0 h	6 h	8 h	10 h	12 h	14 h	16 h	18 h	20 h	22 h	24 h	30 h	36 h	48 h
I	100	0	0	0	0	0	0	0	0	0	0	0	0	0
II	0	100	100	100	99.5	64	0	0	0	0	0	0	0	0
III	0	0	0	0	0.5	36	100	100	96	0	0	0	0	0
IV	0	0	0	0	0	0	0	0	4	100	100	100	100	100

Table 2.

Percentage of cases showing morphological changes in the lymphocytes of in-vitro stored samples with time.

Stage	Time
Stage	0 h	6 h	8 h	10 h	12 h	14 h	16 h	18 h	20 h	22 h	24 h	30 h	36 h	48 h
I	100	100	100	100	100	100	100	83	0	0	0	0	0	0
II	0	0	0	0	0	0	0	17	100	100	100	100	100	100
III	0	0	0	0	0	0	0	0	0	0	0	0	0	0
IV	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Cadaver samples

In the 2–4 h interval, in all cases both neutrophils and lymphocytes were in stage II. At the 4–6 h interval, in 43% cases (n = 3) neutrophils were in stage II and the remaining 57% cases (n = 4) were in stage III while the lymphocytes were in stage II in 100% cases. At the 6–8 h interval, both neutrophils and lymphocytes were in stage III and stage II respectively in 100% cases. At the 8–10 h interval, in all the cases neutrophils were in stage IV except for 10% cases (n = 1) where neutrophils showed group III change and lymphocytes were in stage II in all the cases. At the 10–12 h, 12–14 h and 14–16 h intervals in all the cases neutrophils were in stage IV and also lymphocytes were in stage II. After 16 h, blood was viscous, and slides could not be examined (Table 3).

Table 3.

Number of cases showing morphological changes in neutrophils (stages I to IV) with respect to time since death (TSD) in cadaver samples.

TSD	Total cases	Stage II	Stage III	Stage IV	Viscous blood
0–2 h	0	0	0	0	0
2–4 h	7	7	0	0	0
4–6 h	7	3	4	0	0
6–8 h	3	0	3	0	0
8–10 h	10	0	1	9	0
10–12 h	6	0	0	6	0
12–14 h	6	0	0	6	0
14–16 h	3	0	0	3	0
16–18 h	7	0	0	0	7
18–20 h	5	0	0	0	5
20–22 h	1	0	0	0	1
22–24 h	3	0	0	0	3
24–26 h	2	0	0	0	2

Discussion

Studies had been conducted in the past to observe the changes in the morphology of blood cells and to correlate them with post-mortem time interval (Table 4).

Table 4.

Studies comparing WBC morphological changes with time since death.

Study	Live/cadaver sample	Refrigeration status	Neutrophil	Lymphocyte
Penttilä and Laiho, 1981, Finland²	123 cadavers	4 °C	Partially disintegrated and unidentifiable by 12–24 h
Babapulle and Jayasundera, 1993, Sri Lanka¹	200 live and 30 cadavers	Room temperature	0–6 h: normal >6 h: pyknosis >12 h: nuclear vacuolation >48 h: disintegration 36 h: difficult and impossible to identify. 66 h: totally gone	3 h: normal 36 h: swollen nucleus 144 h: fragmentation of nucleus 36 h: difficult and impossible to identify. 66–84 h: totally gone
Dokgöz et al., 2001, Turkey³	10 cadavers and 40 live	Room temperature	Both samples: 0–6 h: normal >6 h: pyknosis >12 h: nuclear vacuolation >48 h: disintegration	Both samples: 0–24 h: normal 24–36 h: pyknosis >72 h: nuclear fragmentation >96 h: disintegration
Bardale and Dixit, 2006, India⁴	80 cadavers	Nil	0 h: normal 4–6 h: first degenerative change 5–15 h: cytoplasmic vacuolation >18 h: disintegrated	0 h: normal 8–10 h: first degenerative change 11–14 h: cytoplasmic vacuolation >24 h: disintegrated
Alaa El-Din et al., 2014, Egypt⁵	30 cadavers and 30 live	Not mentioned	Both samples: 0 h: normal 12 h: pyknosis >12 h: nuclear vacuolation 48–72 h: disintegration	Both samples: 0–24 h: normal 24–72 h: nucleus swelling >48 h: pyknosis >72 h: nuclear fragmentation
Kumar et al., 2014, India⁶	150 cadavers	Not mentioned	0–6 h: normal 6–12 h: slightly dysmorphic 12–24 h: grossly dysmorphic >24 h: lysed	0–6 h: normal 6–12 h: slightly dysmorphic 12–24 h: grossly dysmorphic >24 h: lysed
Jat et al., 2017, India⁷	150 cadavers	4 °C	0–6 h: normal 6–12 h: slightly dysmorphic 12–24 h: grossly dysmorphic >36 h: lysed	0–6 h: normal 6–18 h: slightly dysmorphic 18–24 h: grossly dysmorphic >24 h: lysed
Ahmed, 2017, India⁸	150 cadavers	4 °C	0–6 h: normal 6–12 h: slightly dysmorphic 12–24 h: grossly dysmorphic >36 h: lysed	0–6 h: normal 6–12 h: slightly dysmorphic 12–24 h: grossly dysmorphic >24 h: lysed
Present study, 2020, India	186 live and 60 cadavers	Nil	0–6 h: normal 6–14 h: pyknosis 14–22 h: vacuolation >22 h: lysed 2–5 h: pyknosis 5–9 h: vacuolation >9 h: lysed	0–18 h: normal 18–48 h: pyknosis 2–16 h: pyknosis

Like all the studies mentioned above, in the present study also the blood sample from live individuals was kept at room temperature and sequentially examined. The sequence of morphological changes in the present study was similar to other studies. Also, lymphocytes remained resistant to changes for a long time than other type of white blood cells which was in accordance with the above-mentioned studies. The time of shift from one stage to other in the present study was similar to the studies mentioned above except for the appearance of stage IV. In the present study, the neutrophils started to lyse by 22 h while other studies mentioned that disintegration started by 48 h. This could have been due to varying ambient room temperature, variable degeneration of cells and demographic conditions.

Another finding worth noting is the appearance of pyknotic changes in the 6th hour sample is important in the context of blood cell morphology examination in clinical settings. Microscopic examination of blood cell morphology is a crucial diagnostic method in certain blood cell disorders. Generally, EDTA is the anti-coagulant of choice for haematological analysis of whole blood since it prevents clotting by acting with the calcium ions and preserves the blood cell morphology much better than other anti-coagulants like heparin. But here the neutrophils show morphological changes by 6th hour in the blood samples stored at room temperature. This finding can have an applied aspect in clinical settings that when blood samples are collected and taken for analysis in laboratories there may be a delay. And if the blood samples remain at room temperature without proper refrigeration, these alteration in the morphology can happen. These changes can cause diagnostic difficulties and confusions. Duration between collection and analysis and the temperature at which the samples are being maintained should be taken into consideration while evaluating these samples.

The similar kind of studies conducted on cadavers can be mainly divided into two groups. One group where the cadavers were kept in cold storage prior to autopsy and other group where the cadavers were maintained at a room temperature prior to autopsy and not subjected to any refrigeration.

In the present study, on comparing the changes in cadaver samples with that of in-vitro samples, there are differences. The appearance of degenerative changes in the post-mortem sample was earlier than in the in-vitro stored samples. The appearance of degenerative changes started by 2 h itself in the neutrophils of cadaver blood while it started only by 6 h in the in-vitro stored samples. While by 9 h itself the neutrophils started to lyse in cadaver sample it happened only after 22 h in the in-vitro stored sample. The similar kind of rapid changes were noted in lymphocytes as well; even when both lymphocyte and neutrophil changes were considered together the changes of the cadaver samples were not correlating with the blood cell variation scale. Even though the sequence of morphological change of leucocytes was similar in both groups of samples, the time of appearance is different. This was in accordance with the study by Babapulle and Jayasundera¹ who also found rapid degeneration in the case of cadaver samples compared to in-vitro stored samples. The other two studies by Dokgöz et al.³ and Alaa El-Din et al.⁵ studied both in-vitro stored samples and cadaver samples and mentioned that the sequence and time of appearance is the same. The reason for this rapid degeneration in cadaver samples could be many. The anoxic environment of the cadaver samples is perhaps accelerated by the process of decomposition which provides more free radicals, toxic metabolites, increased carbon dioxide accumulation and pH alteration. All of these occur partially in parallel and successively after death. All these changes can result in further degeneration of blood and blood cells in the dead. Also, all the in-vitro stored samples were kept at room temperature adequately preserved from any insect activity, spillage, air and so on. All these pre-made setups result in a constant and secured environment. This would be lacking and missed out for a cadaver which we normally get to autopsy for medicolegal purposes and the resultant degeneration might not be equally and effectively simulated in-vitro. This could have been the reason why the changes appear earlier in cadaver samples. Along with this, during post-mortem period there is a rapid increase in haemoconcentration. This is mainly facilitated by the cessation of circulation. The cessation of the circulation induces clotting processes because the fibrinogen within the post-mortem serum as well as the platelets are still capable of functioning for a variable post-mortem period.⁹ This rapid increase in the haematocrit values of the blood after death is mainly due to the loss of the liquid phase of the blood into the surrounding tissues rather than to the increase in the volume of red cells. Penttilä and Laiho reported a highly significant increase in haematocrit values between 12 and 24 h and continued during longer postmortem intervals.² The similar kind of reduced fluidity of blood was observed in our cadaver blood samples after 16 h while the in-vitro stored samples were still fluid. And because of this rise in the viscosity of the blood, blood cells could not be examined beyond 16 h. All these changes illustrate the stark difference in the internal environment of cadavers and in-vitro stored blood. The in-vitro samples were stored in a secured room with constant room temperature which is not the same for cadaver samples, this could have affected the intensity of autolysis and time of appearance of changes. This is also apparent when we compare the time of appearance of changes in refrigerated and non-refrigerated cadavers. The changes appeared earlier in the case of non-refrigerated when compared to refrigerated cadavers. The demography, environmental condition, storage and refrigeration of samples were all different between our study and other studies. The few differences in the time of appearance of degenerative changes between our study and other similar kind of studies could also have been due to these factors.

A finding which is worth mentioning for practical reasons and reasons related to research is variable degeneration, that is, even at a single point of time the cells observed on the slide belong to different stages of degeneration. The similar kind of variable degenerating pattern was observed by Babapulle et al. also. Figure 6 shows a slide of in-vitro stored sample at 8th hour. Here, one neutrophil which is on the right is showing pyknotic nuclei which is a stage II change while the other one at the left top corner is showing vacuolation which is a stage III change, while the one below is having vacuolation on cytoplasm. These two different appearances of the neutrophil at a particular time suggests variable degenerative changes. This is why the method that was being utilized to categorize the cells into stages plays an important role. In other studies, conducted in this regard, the method of categorization of cells into groups when variable degeneration is observed has not been mentioned or clarified. In those studies, change of even a single cell has been considered. This kind of evaluation cannot be entirely reliable given that there is a probability for cells to show any change at any time and a completely lysed cell can be observed even at 0 h because of apoptotic changes. So, relying on a single cell change and to conclude can be disrupting. Also, the area viewed under a microscope for evaluation has not mentioned in the studies except for Dokgöz et al.³ and Alaa El-Din et al.⁵ To bring in objectivity, the morphology of cells needs to be examined under a microscope with a pre-determined criterion, as similar to something used in the present study. In the present study, the stage of change shown by 50% and more of the cells was taken as the stage of change for that particular time. And, the number of neutrophils and lymphocytes counted were also fixed at 30 and 15 respectively. To incorporate the tests and verify its findings it is important to know the methodology that was followed. So proper mentioning of the methodology is a necessary step in all research studies.

Figure 6.

Variable degeneration – peripheral blood smear slide of in-vitro stored sample at 8th hour. Here one neutrophil which is on the right is showing pyknotic nuclei which is a stage II change while the other one at left top corner is showing vacuolation which is a stage III change, while the one below is having vacuolation on cytoplasm.

Limitation

Observer bias could be one important limitation when we consider these kinds of studies. This study needs to be elaborated with a large number of non-refrigerated cadavers with varying post-mortem interval to observe further changes and variations. Application of this method in later post-mortem intervals is not feasible since after 16 h the blood is becoming viscous and unfit for further examination, and this cannot be applied for refrigerated cadavers. It is also important to identify artefacts which can be observed during smear preparation should be differentiated from actual pathology/change. The whole process of decomposition can be affected by the environmental conditions which also applies to this group of cells and can result in multiple variations.

Conclusion

Practical methods that can be applied easily and at all places without any need for sophistication or equipment are highly needed for calculation of post-mortem interval and the present study was an attempt to achieve this objective. In our study, we found the sequence of changes in the in-vitro blood and in-vivo blood to be the same, but the time of appearance is rapid in cadavers thus making it hard to apply the variation scale for estimation of time since death. However, it is hard to completely refute the hypothesis that in-vitro and cadaver conditions would be similar and changes exhibited would also be similar on the basis of this single study. Further studies with much larger sample size need to be conducted for completely refute or prove the above hypothesis. But also, other co-existing factors which can affect the morphology of the leucocytes should also be taken into consideration. The appearance of variable degeneration and its causes should be explored further.

Footnotes

ORCID iDs

Mahaluxmi Saravanamurugan

Jayanthi Yadav

Ethical considerations

This study was approved by Research Review Board (RRB) and Institutional Human Ethical Committee (IHEC) vide LOP letter no IHEC-LOP/2019/MD0063.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

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