Enzymatic maceration of bone: a gentler technique than boiling

Introduction

Maceration of bone may be an important procedure in forensic pathology and anthropology, as tool marks, fracture patterns and impact zones in bone may be used evidentially, for instance when trying to match a special tool or weapon with lesions or when reconstructing trauma patterns.^1–4 There are several maceration techniques that remove soft tissue from bone, and some of the most common are enzymatic, treatment with warm (or boiling) water with laundry detergent, and insect consumption.^1–3,5–8 Here, we test a new enzymatic method to macerate human bone, and compare it with the current method we use, which is boiling with a laundry detergent. Enzymatic maceration may potentially be a less destructive method for removing soft tissue, and furthermore, as it does not entail heating to boiling point, the method should not impair post-maceration biomolecular analyses, for example of DNA.

To perform the enzyme maceration, a protease and a lipase in aqueous solution were used. The two enzymes have been used in another study investigating enzyme maceration on animal bones. ¹ The lipase and protease, in the correct concentration and ratio to each other, should be a fast and gentle method to remove soft tissue from bones while being easy to use, ¹ and thus should be an improvement compared with maceration using laundry detergent. Laundry detergents contain different enzymes in (not always specified) various amounts and other substances such as bleaching agents, additives and corrosion inhibitors.^9,10 The detergent used contained a combination of the above-mentioned compounds as well as protease, sulphuric acid, sodium hydroxide and glycosidase (pers. comm. Ariel Company DK). All these substances may damage the bones, making them porous or even destroying them.^1,8,10,11 The DNA may also be damaged by substances such as bleach.^2,12

Here, we present a comparison of a controlled maceration by specific enzymes to maceration by boiling with an ordinary laundry detergent for proof of concept. We purposefully asked our technical staff to execute the latter method completely, which is the method used at our institute, as per routine. Of special interest was the degree of fraying observed: fraying is important, as maceration-induced fraying may interfere with the forensic interpretation of trauma and tool marks.

Materials and methods

One rib was removed from each of six individuals at forensic autopsies. All ribs were taken from males with an age span of 22–68 years. Only ribs from bodies without fractures or severe lesions to the thorax were included. The autopsies were carried out one to three days after death; until autopsy, all bodies had been in cold storage (5℃).

Each removed rib was cut into six pieces: two pieces were macerated with enzymes, two pieces were macerated by boiling with detergent, and two pieces were contained as backup. One bone from each technique was DNA tested, and one was studied using a stereomicroscope. The pieces were prepared in such a fashion that we could directly compare the effects of the two maceration techniques on directly opposite, congruent cut surfaces (Figures 1 and 2). The macerations took place over 10 days, and the longest interval between sectioning and maceration was two days. Due to personnel and laboratory exigencies, the enzymatic maceration and boiling of the two corresponding bone samples did not occur on the same day. The bone pieces were stored in a refrigerator (5℃) between sectioning and maceration. The ribs were cut into pieces following a specific procedure using a rib cutter so that the cut edges of the macerated rib fragments, either by enzymatic action or boiling, could be matched up afterwards to allow direct comparisons of the morphology of the cut edges. The rib cutter had serrated blades, one of which was more serrated than the other. The rib cutter blades should imprint marks in the bones. OPEN IN VIEWER

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Figure 2.

Macerated bone pair: enzymatic macerated (left); boiled bone piece (right).

Enzyme maceration

The enzymes used were Lipex 100L (the lipase), which is a fat removing substance, and Savinase 16L (the protease) which removes proteins. ¹ Distilled water (750 mL) was put in a beaker and placed on a magnetic hotplate stirrer which was in a fume cupboard and warmed to about 40℃. Hereafter, 25 mL of lipase and 25 mL of protease were added to the beaker, together with the rib piece. The solution was stirred at the optimum temperature of 55 ± 5℃ until maceration was completed (Figures 3 and 4), which is the temperature where the enzymes work best. ¹ The temperature was closely monitored through the investigation. The composition of the solution was also closely observed. If the solution is greasy, there is not enough protease in the solution, while conversely a non-greasy appearance indicates that the appropriate amount of enzymes has been used. ¹ The maceration speed also depends on the right amount of enzymes, especially the protease volume. ¹ After maceration, the bones were dried by carefully pressing them with absorbent paper. No additional procedures were undertaken. OPEN IN VIEWER

Figure 3.

One of the bones before maceration, all covered with flesh.

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Figure 4.

The same bone as in Figure 3 after maceration, all defleshed and cleaned.

Morphological bone comparison

A Leica stereomicroscope MZ75 equipped with a DFC280 CCTV camera was used to compare the edges of the congruent bone pieces. The rib cutter used to cut the rib in pieces was grooved, so that some crushing at the cut edge should be visible. The six pairs of bone were compared for bone surface quality, tool marks and frayed/destroyed edges. To compare and quantify these variables, a post hoc scoring system was established. The scoring system was organised into four categories and scored each bone by points ranging from 1 to 3. The four categories were: cleaning of the bones, the bone edge, bone texture and tool marks. Cleaning refers to how much flesh was left on the bones after maceration (Figures 3 and 4). Bone edge appearance denotes whether there was fraying of the cut edges or destruction. The points for bone texture are allocated according to how intact the whole bones were and if they were broken (Figures 5 and 6). The tool mark category denotes how visible and well defined the tool (rib cutter) marks were. The bones were rated and scored by one person. OPEN IN VIEWER

Figure 5.

Bone 2 enzyme maceration. The texture of the bone is not intact, the bone is parted, and the circle shows this.

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Figure 6.

Bone 1 boiled. The texture of the bone is intact, and tool marks are clearly visible and well defined.

Results

The experimental results showed that enzymatic maceration was possible on human bone, and that, overall, the bones were defleshed just as thoroughly as with the current standard boiling maceration (Figures 3 and 4). After maceration, all the bones were found to be completely defleshed, so no further procedures were needed. The new method with enzymes was also much faster: the maceration time was on average three hours and five minutes (range: two hours and 40 minutes to four hours and 30 minutes), compared with the common macerations technique, with a total preparation time of approximately 24 hours. Both methods were quite simple, and none of the methods was malodorous.

The scores reflecting the qualitative differences studied with the stereomicroscope are given in Table 1. The boiling maceration technique scored lower on average in the bone edge category, as the edges were more frayed and pieces were broken off from some of the bones. Furthermore, the boiling maceration had a lower average score in the tool marks category due to the fact that the marks from the rib cutter contained less detail. Both methods defleshed the bones totally, and the bone texture was preserved equally with both methods. For each method, there was one case of the bone piece dividing longitudinally.

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Table 1.

Scoring system.

	Cleanings	Bone edge	Bone texture	Tool marks
Boiling
Bone 1	3	1	2	3
Bone 2	3	2	2	1
Bone 3	3	1	2	2
Bone 4	3	1	2	1
Bone 5	3	1	1	2
Bone 6	3	1	2	2
Total score	18	7	11	11
Average	3	1.2	1.8	1.8
Enzymatic
Bone 1	3	2	2	2
Bone 2	3	2	1	3
Bone 3	3	2	2	1
Bone 4	3	2	2	3
Bone 5	3	1	2	3
Bone 6	3	2	2	3
Total score	18	11	11	15
Average	3	1.8	1.8	2.5

Table 2 summarises the DNA yields from the bone samples. The IPC CT values from the DNA quantification showed a low level of inhibition (data not shown), indicating pure DNA. It was possible to obtain usable STR profiles from all samples, making identification possible. A DNA profile, obtained after enzymatic maceration and boiling maceration, from bone 1 is shown in Figure 7. The DNA profiles were well balanced between the different STR systems, indicating a low level of DNA degradation. OPEN IN VIEWER

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Table 2.

DNA extraction results.

	Enzymatic	Boiling
Bone 1	20.94 ± 2.40	102.54 ± 9.58
Bone 2	5.00 ± 1.11	38.21 ± 4.79
Bone 3	13.73 ± 1.49	5.23 ± 1.36
Bone 4	29.25 ± 2.26	38.37 ± 4.06
Bone 5	18.12 ± 3.62	18.47 ± 2.74
Bone 6	27.77 ± 2.17	19.35 ± 1.85

Amount of DNA per mg bone (ηg/mg) ± (SD). Elution volume was 50 μL.

Discussion

Sharp force trauma is one of the frequent causes of murder.^14–16 In such cases, hard tissues, such as bone and cartilage, may preserve the impression of the weapon. ¹⁶ Potentially, trauma patterns retained in hard tissues may make it possible to give statements about the weapon or tool used. ¹⁶ By securing and presenting tool marks in the bones, it may then be possible to secure sharp force trauma marks, which also will rely on a gentle method for macerating the bone. ¹⁷ In this study, a rib cutter was used because the idea was to mark/break the bones equally, so any marks left in the edges would be seen in the stereomicroscope. A rib cutter should leave more conspicuous marks (a combination of cutting, shearing and crushing) than a sharp object, such as a knife, because of its serrated blade. It would then allow a more comprehensive comparison of the two maceration techniques. A rib cutter may not be totally comparable to sharp force trauma (incision or cutting), so further studies are required to prove this.

This study shows that enzymatic maceration and maceration with boiling water and laundry detergent (the method used until now at our institute) both clean the bones and do not lead to degradation of DNA. The enzymatic method not only removed soft tissues and blood, but the study indicated that the method retained minute morphological features to a better degree and resulted in less fraying of the bone edges. The speed of the maceration method may be an issue in forensic cases, ² and our results indicated that the enzymatic method was much faster. However, in our experimental set-up, where the boiling maceration technique was applied as per routine, we had no continuous observation of this method, nor did we register exactly how long the bone pieces were kept in the detergent solution after the two hours of boiling. The bones were left over night in the solution because this is the standard maceration technique used at our institute. The fact that the boiling method had a lower average score in the qualitative differences could be a result of the many hours in the solution. The boiling method could thus be faster than our results indicate, and further investigation with standardised observations should be performed. A more standardised scoring system and more observers would also serve to analyse observation bias. This will be taken into consideration in future studies. For now, the study shows that it was possible to compare the two macerations techniques in a stereomicroscope.

Finally, we found that DNA was retained in the enzymatic macerated bones and, somewhat surprising to us, we also found that DNA was retained just as well in bone pieces which had been boiled. One would still have to assume that boiling for hours may be detrimental to DNA preservation because of the preliminary indication that excess heat may damage the DNA.^2,18,19 In the boiling method, the bones were boiled for two hours and left in the solution overnight. So the processing time in total was 24 hours, as the bones needed to be in the detergent solution overnight so all the flesh decomposed and the bones were totally defleshed. The limited time of actual boiling of only two hours may explain the DNA preservation. While DNA would probably be secured from the body at autopsy, it may potentially be of value to be able to extract DNA from bones which have been taken out and kept for further analyses such as for tool marks. While using an ordinary, commercial laundry detergent and simple boiling is less expensive than using single, special enzymes, our study, which may be seen as a proof-of-concept study, may indicate that the more delicate enzymatic maceration, especially in cases where a close scrutiny of tool marks or cut marks are of the essence, should be considered.

Conclusion

Human ribs were macerated by two different methods, one using enzymes, and one using boiling water with detergent. Both methods cleaned the bones totally, and DNA extracted from each of the two methods gave high-quality DNA profiles. The results suggest a dramatic reduction in maceration time with the use of enzymes (i.e. three hours) compared with the boiling method (i.e. 24 hours). We thus regard this as a proof of concept, and further investigation with standardised observation should be made. At the same time, the study indicates that enzymatic maceration is slightly gentler, as judged by evaluating the cut edges in a stereomicroscope. Marks made with a rib cutter seem to be retained better in the enzymatic macerated bone pieces than in the boiled bone pieces. The enzymatic macerated bones were also less frayed than the boiled ones, but both methods damaged one bone out of six. Future work will assess the applicability of the enzymatic maceration technique to larger pieces of bone and clarify the ability of the methods to retain marks.