Comparison of modified thiel embalming and ethanol-glycerin fixation in an anatomy environment: Potentials and limitations of two complementary techniques

Authors


Abstract

Thiel-fixed specimens have outstandingly lifelike visual and haptic properties. However, the original Thiel method is expensive and requires an elaborate setup. It is therefore of principal interest to modify the Thiel method in order to make it available to a broader user group. A modified Thiel embalming method will be described in detail and compared to ethanol-glycerin fixation with the help of illustrative examples. The visual properties, haptic properties, the usability for performing histological investigations, costs and potential health aspects will be considered. Tissues fixed with the modified Thiel technique gave results similar to the original method, providing more realistic visual and haptic properties than ethanol-glycerin embalming. However, Thiel fixation is significantly more expensive and requires more precautions to minimize potential health hazards than ethanol-glycerin-fixed tissues. In contrast to ethanol-glycerin-fixed specimens, the Thiel-fixed specimens are not suitable for histological investigations. Both modes of fixation are inappropriate for biomechanical testing. Modified Thiel embalming simplifies the availability of body donors with lifelike properties and has cost-saving advantages to the original technique. Thiel-embalmed body donors are ideally suited for clinical workshops but have restrictions for student dissection courses in facilities with limited storage space, air circulation or technical staff. Vice versa, ethanol-glycerin-fixed body donors are well suited for student dissection courses in such an environment but are limited in their use for clinical workshops. Modified Thiel embalming therefore ideally complements ethanol-glycerin fixation in order to provide customized solutions for clinical workshops and student dissection courses in a wide range of applications. Anat Sci Educ 8: 74–85. © 2014 American Association of Anatomists.

INTRODUCTION

The gross anatomy course is still regarded as an integral part of human and dental medical education in Germany (Korf et al., 2008; Ochs et al., 2012). Dissection of human body donors is therefore maintained in most German anatomy departments, despite increasing pressure to cut costs on material and staff and despite the ongoing discussion concerning the suitability of body donors for medical education (Aziz et al., 2002; Lippert, 2012; Neuhuber, 2012). In addition to the dissection course carried out with undergraduate students, a variety of health care professionals use post-mortem tissues for clinical workshops, surgical training and for biomechanical investigations. Therefore, the prerequisites for using body donors in macroscopic anatomy significantly changed in recent years, regarding the appropriateness and usability of the tissues for different settings (Fasel, 2005; Drake, 2007, 2014; Joslin, 2008; Böckers et al., 2010; Sugand et al., 2010). Fixative agents have a long-standing tradition in human anatomy departments to provide durable specimens. Formaldehyde is still used ubiquitously in the 21st century (Blum, 1893, 1896; Kunz and Wilcke, 1991) due to its low cost and the wide availability. It has excellent effects on interrupting autolysis and preventing bacterial or fungal contamination (Romeis, 1989). However, formaldehyde has been classified as carcinogenic to humans (Hauptmann et al., 2009; Lunn et al., 2010), which increases the necessity for alternative fixatives.

There is a variety of fixative agents that are currently proposed to lower or abandon the use of formaldehyde, e.g. ethanol-glycerin (Hammer et al., 2011, 2012), isopropanol (Spranger, 1926), methanol (Bradbury and Hoshino, 1978; Jaung et al., 2011), phenoxyethanol (Frølich et al., 1984; Wineski and English, 1989) and commercially available fixatives (Whitehead and Savoia, 2008; Messmer et al., 2010). Another alternative is the Thiel fixation, also called Graz fixation (Jaung et al., 2011). Walter Thiel developed this method in the 1990s (Thiel, 1992; Anderhuber, 2012). Thiel-fixed specimens have outstanding visual and haptic properties close to the unfixed post-mortem condition. However, the original Thiel method requires an elaborate setup, i.e. combined intra-arterial, intra-tracheal and intra-rectal injection of the fixative agent and is expensive in use. As a consequence, the Thiel method is almost exclusively used in Europe despite the life-like quality of the tissues (Benkhadra et al., 2011b).

It might therefore be of principal interest to modify the Thiel method to the effect that it is applicable to a larger user base, which will be the focus of this descriptive article. We will report on our experience of using ethanol-glycerin fixation and Thiel fixation. We apply the ethanol-glycerin fixation annually on 40 or more body donors used for the 400-student gross anatomy course. We apply the Thiel method for simultaneously running surgical workshops for clinicians and graduate students (Löffler et al., 2009, 2013). The architectural design of our facility prohibits the installation of extensive air conditioning. Additionally, our department has very limited storing capacities for the donated bodies and we only have two members of technical staff to manage the large number of body donors. Our modified Thiel embalming technique will be described in detail and compared to ethanol-glycerin fixation, which is used in our facility since the 1960s. Visual qualities, haptic qualities, usability for histology and costs will be discussed. Additionally, literature on the biomechanical properties and on potential health effects to the exposed will be discussed. We will recommend both Thiel embalming and ethanol-glycerin fixation as complementary techniques for student and clinical workshops in a wide range of applications.

MATERIAL AND METHODS

All body donors had given their informed consent to the donation of their bodies for research and teaching purposes before their passing. All values are given for standard body donors (body height 170 cm, body weight 70 kg or less, resulting body mass index ≤ 24.2).

Composition of Thiel Fixation

The injection and container solutions are adapted from the original description of Walter Thiel, using the same protocol (Thiel, 1992). In a first step, 15.0 L of the Thiel fixative is injected into the arterial system. The injection fluid consists of stock solution (2.0 kg ammonium nitrate, 0.3 kg boric acid, 0.5 kg potassium nitrate and 3.0 L ethylenglycol dissolved in 10.0 L hot water; pH = 3.5), p-chlorocresol solution (0.03 kg p-chlorocresol and 0.5 L ethylenglycol dissolved in 0.5 L distilled water), 0.3 L formaldehyde and 0.7 kg sodium sulfide (Table 1). The chemicals are obtained from Applichem (Darmstadt, Germany), Merck (Hohenbrunn, Germany), Roth (Karlsruhe, Germany) and from VWR BDH Prolabo (Leuven, Belgium). Further details are given in Table 1.

Table 1. The Agents, Formulas, Characteristics, and Costs are Compared Between Ethanol-Glycerin and Modified Thiel Fixation
AgentMolecular formulaSource of supplyCharacteristicsPrice Euros [€]AmountCosts Euros [€]/donor
Modified ThielEthanol-glycerin and thymol
  1. Thiel method is significantly more expensive than ethanol-glycerin fixation.

  2. C, corrosive; F, highly flammable; N, harmful to environment; O, oxidizing; T, toxic; Xn, harmful to humans; n/a, not applicable.

Ethanol-glycerin fixation
EthanolC2H5OHBrenntag GmbHF1.04/L48.50 L 50.44
GlycerinC3H5(OH)3Chemie Vertrieb Magdeburgn/a4.31/L2.50 L 10.76
ThymolC10H14OMerckC, N27.00/kg0.30 kg 9.00
Modified Thiel method
Injection66.84 
Ammonium nitrate(NH4)(NO3)AppliChemO4.00/kg2.00 kg8.00 
Boric acidH3BO3RothT11.37/kg0.30 kg3.41 
p-ChlorocresolC7H7ClOMerckN, Xn54.40/kg0.03 kg1.63 
EthylenglycolC2H6O2AppliChemXn12.00/L3.50 L42.00 
FormaldehydeCH2ORothT9.79/L0.30 L2.94 
Potassium nitrateKNO3AppliChemO10.21/L0.50 kg5.11 
Sodium sulfideNa2SVWR BDH ProlaboC, N, T5.35/kg0.70 kg3.75 
Container solution (used for 2 donors)    725.54 
Ammonium nitrate(NH4)(NO3)AppliChemO4.00/kg20.00 kg80.00 
Boric acidH3BO3RothR,S,T11.37/kg6.00 kg68.22 
p-ChlorocresolC7H7ClOMerckN, Xn54.40/kg0.19 kg10.34 
EthylenglycolC2H6O2AppliChemXn12.00/L22.00 L264.00 
FormaldehydeCH2ORothT9.79/L4.00 L39.16 
Potassium nitrateKNO3AppliChemO10.21/L10.00 kg102.10 
Sodium sulfideNa2SVWR BDH ProlaboC, N, T5.35/kg20.00 kg107.00 
Disposal   0.24/L228.00 L54.72 
Short-term storage     7.63 
p-ChlorocresolC7H7ClOMerckN, Xn54.40/kg0.03 kg1.63 
EthylenglycolC2H6O2AppliChemXn12.00/L0.50 L6.00 
Total costs per method and donor in Euros [€] (German price list)  437.2470.20

After registration, thorough cleaning and shaving of the body donors, the femoral artery is identified within the femoral region with the same approach shown elsewhere (Hammer et al., 2012). Two cannulae are inserted cranially towards the external iliac artery and caudally towards the popliteal artery. The fixative is then injected by a pump with a maximum pressure of 0.5 bar (Ideal VA Funeralia, Würzburg, Germany). Usually, the fixative is injected in 6 to 10 cycles of 10 min each with interruptions of two hours or more to allow the fixative to distribute evenly. An injection process averages 24 hours. Longer (overnight) interruptions have little influence on fixation if the donors are covered with cotton cloths soaked in the injection fluid. The stock solution and the p-chlorocresol solution can be mixed in larger quantities and stored without effects on the fixation, but the injection solution should be mixed immediately before the injection starts. The injection is successfully finished once the keratin of the epidermis and the nails start to pull off in a stocking glove fashion (Armstrong and Erskine, 2011), which should be completed by manual preparation before the second step of fixation is started. An additional intra-thecal, intra-tracheal or intra-rectal injection of the fixative as reported by Thiel is not performed in our setup.

In the second step of fixation and conservation, the donors are submersed in custom-made movable containers (Labecki, Leipzig, Germany; Supporting Information Fig. 1). The containers are filled with Thiel container solution consisting of 20.0 kg ammonium nitrate, 6 kg boric acid, 0.19 kg p-chlorocresol, 22.0 L ethylenglycol, 4.0 L formaldehyde, 10.0 kg potassium nitrate and 20.0 kg dissolved in 200.0 L of hot tap water (pH = 3.5; Table 1). The containers are designed for two body donors simultaneously to save costs on the container solution and on the space required for storage. A perforated sieve that is located inside the container can be lifted with a regular workshop crane. The front of the sieve can be opened with additional bayonet locks. These modifications allow the handling of the body donors by a single person. A tap at the bottom of the container eases the disposal of the container solution (not shown). The specimens are submersed in the container solution at room temperature for at least two months before initial use and between longer periods of use. Prior to use, the body donors are drained on the sieve. In case of short term intervals between specimen use of one week or less, the donors remain outside of the containers, covered with cotton cloths soaked in p-chlorocresol solution and an additional layer of polyethylene foil (Martin S. Hähn OHG Packmittel, Leipzig, Germany). For optimal results, it is essential that all ingredients, especially the chemical salts, are completely dissolved and that the solutions are cooled down before the application of the fixatives. The use of small-sized and slim body donors allows saving costs on the fixatives and eases specimen handling. A step-by-step protocol of the modified Thiel fixation is listed in Table 2.

Table 2. Step-by-Step Protocol of Modified Thiel Fixation and Conservation
ProcedureAmount of fixativesDuration, contents and concentrationsRequired conditions
  1. The amounts of fixatives, their contents and concentrations, the duration and the conditions required for each step are given.

Registration/cleaning
Cannulation of femoral artery
Fixation15.00 L of injection solution24 hrs: 6 to 10 injection cycles, each 10 min with adequate interruptions completed once epidermis and nails start to detachInjection pump
 14.20 L of stock solution 0.50 L of p-chlorocresol solution 0.30 L of formaldehyde (35% by volume)0.70 kg of potassium sulfide2.00 kg of ammonium nitrate, 0.30 kg of boric acid, 3.00 L of ethylenglycol, 0.50 kg of potassium nitrate, 9.90 L of hot water 
 0.25 L of aqua destillata, 0.25 L of ethylenglycol, 0.03 kg of p-chlorocresol 
Conservation and long-term storage228.00 L of container solutionConservation for at least 6 months before first useMetal container, storage at room temperature
 20.00 kg of ammonium nitrate 6.00 kg of boric acid 4.00 L of p-chlorocresol solution 20.00 L of ethylenglycol 4.00 L of formaldehyde (35% by volume)10.00 kg of sodium sulfide2.00 L of aqua destillata, 2.00 L of ethylenglycol, 0.19 kg of p-chlorocresol 
Short-term storage0.50 L of p-chlorocresol solutionSame contents and concentrations as used for fixationCotton cloths, polyethylene foil

Ethanol-Glycerin Fixation With Ethanol-Thymol Conservation

Parts of the methods concerning ethanol-glycerin fixation with thymol conservation will briefly be dealt with. For more details, please see (Hammer et al., 2011, 2012). A mixture of ethanol (Brenntag GmbH, Mühlheim, Germany, Table 1) and glycerin (Chemie Vertrieb Magdeburg, Germany) is injected at a ratio of 0.7 L/kg body weight in an explosion-proof room, i.e. special types of power points and light switches installed outside of the room where the fixation takes place. Glycerin is added at a ratio of 2 to 10%, depending on the physical constitution of the body donor. The injection is performed in a similar manner as stated above and takes 36 hours on the average. When the injection is completed, the donors are submersed in commercially available metal containers (Funeralia GmbH, Würzburg, Germany) for four weeks and then packed in cotton cloths and polyethylene foil for storage at 3 to 5°C. Water-diluted ethanol-thymol solution is used for the conservation of the specimens at room temperature (Table 1). Fixation of the central nervous system is either performed by means of subdural injection or by removal and immersion of water-diluted formaldehyde (1.3% by volume).

Examples

Modified Thiel fixation and ethanol-glycerin fixation were accomplished in two body donors (82-year-old female and 69-year-old male). Their forearms and hands were prepared to give comparative examples on both modes of fixation. Additionally, skeletal muscle and liver specimens were obtained for microscopy from a 91-year-old and an 84-year-old female after modified Thiel and ethanol-glycerin fixation, respectively. After removing the tissues, they were immediately fixed in phosphate-buffered formaldehyde (4% by volume, pH = 7.0), dehydrated and stained with hematoxylin and eosin according to the standard protocol (Romeis, 1989). Computed tomography (CT; Philips Healthcare MX6000 Dual, Amsterdam, The Netherlands; voxel dimensions 0.59 × 0.59 × 0.59 mm, matrix size 512 × 512 pixels) images were obtained from an 83 year-old female body donor after Thiel fixation and injection of 0.21 L of Angiofil® (Forim-X AG, Bern, Switzerland; Grabherr et al., 2008), diluted in 4.50 L of paraffin oil (Roth, Karlsruhe, Germany). On the basis of these data the circle of Willis was reconstructed virtually (OsiriX 5.7.1, Bernex, Switzerland).

RESULTS

Modified Thiel Fixation Gave Results Similar to the Original Technique

The tissues embalmed by means of the modified Thiel technique were preserved in their post-mortem condition. While the ingredients of the injection and the container solution remained unchanged from the original protocol (Thiel, 1992), our modifications on the mode of fixation and conservation can be summarized as follows:

  • The injection solution was exclusively applied via the femoral artery instead of the external iliac artery, the trachea, the rectum or the subdural space.
  • The duration of the body donors in the container solution before first use was shortened from six to two months and the body donors are returned into the container solution between periods of use.
  • Two donors were stored in one container instead of a single body.
  • The injection fluid was used to moisten the body donors in the short-term storage (one week or less) outside of the containers, and
  • For long-term storage (more than one week), the body donors were kept in the containers filled with the container solution at room temperature (due to organizational reasons in our facility).

The results of the fixation were not affected by our modification with the only exception of the central nervous system, which showed signs of incomplete penetration by the Thiel fixatives when applied only intra-arterially. The period of use for the Thiel-fixed body donors averaged ten months and varied between 2 and 12 months. Tissue properties were unaltered during the observed time: the duration in which the properties of the tissue remained in a useful state was only limited by the surgical procedures or medical treatments performed on the tissue.

Thiel Fixation Provided More Realistic Visual and Haptic Properties Than Ethanol-Glycerin-Fixation but was Costlier

In the Thiel-fixed donors, the original color of the skin was intensified after complete removal of corium and nails. The container fixation caused the skin to become tender and soft. In contrast, the injection of ethanol-glycerin bleached the skin. This effect was partially reversible by submersion in water-diluted ethanol-glycerin and was accompanied by an induration. Muscles, nerves, and vessels became overstained and softened by the Thiel fixative allowing full range of joint motion (Figs. 1A and 1B). In comparison, the ethanol-glycerin fixed muscles, nerves and vessels bleached slightly and indurated, which apparently restricted joint motion (Figs. 1C and 1D). High-resolution images will be provided in the online Supporting Information (Supporting Information Fig. 2). However, the range of joint motion of the ethanol-glycerin-fixed donors was significantly wider than in formaldehyde-fixed tissues. Bone and cartilage kept their visual and haptic properties in both modes of fixation (Figs. 2A and 2B). The only two exceptions were the elastic type cartilage of the ears and the quadrangular (hyaline) cartilage of the nasal septum which were dissolved in the Thiel-fixed donor. The viscera kept their in-vivo appearance and haptic properties after applying the Thiel fixatives (Figs. 2C and 2D). The ethanol-glycerin-fixed viscera also kept their natural appearance but parenchymatous organs such as the liver, spleen, and kidneys indurated. Anatomical fixation of the central nervous system would have been possible with an additional (intra-thecal) application of formaldehyde but was beyond the scope of this study. The odor of Thiel-fixed specimens was intrusive even after tapping, which was not the case with the ethanol-glycerin fixation. Ethanol-thymol conservation caused a thyme-like scent in the ethanol-fixed donors. Compared to standard fixation with formaldehyde (33.16 € per donor), modified Thiel was 12 times as expensive (437.24 € per donor), ethanol-glycerin is twice as expensive (70.20 €) per donor. Further details on the amounts and prices (purchase in Germany) are given in Table 3.

Figure 1.

A Thiel-fixed forearm (A) and hand of an 82-year-old female donor (B) are compared with an ethanol-glycerin-fixed forearm (C) and hand of a 69-year-old male donor (D). B and D are magnifications after removal of the palmar aponeurosis and the adjacent connective tissues of the same specimens. The palmar aspect of the right-sided arms is depicted. The properties of Thiel-fixed specimens remain close to the fresh condition, including full-range joint motion and vivid colors (A). Vessels, nerves and connective tissues maintain their flexibility (B). The ethanol-glycerin-fixed tissues are less colorful (C and D). Their vessels, nerves and connective tissues become more rigid, which is also reflected by the partially flexed elbow and interphalangeal joints. L = anular ligaments of the fingers, FR = flexor retinaculum of the hand, FT = tendons of the flexor digitorum superficialis, UA = ulnar artery, UN = ulnar nerve, arrow = bicipital aponeurosis, asterix = palmar aponeurosis; scale bar for A and C = 50 mm, and for B and D = 20 mm.

Figure 2.

Thiel-fixed specimens are ideally suited for clinical workshops due to their haptic and visual properties. Image (A) shows a hand-arthroscopy course and (B) is a snapshot of the radiocarpal joint taken in an 87 year-old female body donor during hand arthroscopy (asterix = proximal radiocarpal ligaments, groove = proximal). Image (C) and (D) show a total mesometrial resection (Höckel et al., 2005, 2009) in an 83-year-old female donor. Note the vivid colors and appearance of the subcutaneous tissue, the ileum and its mesentery (C). At a later stage of the workshop, the right-sided ovary is prepared surgically along with the suspensory ligament (D).

Table 3. The Effects of Modified Thiel Fixation and Ethanol-Glycerin Fixation are Listed
 Modified Thiel fixationEthanol-glycerin fixation
  1. The indicated characteristics refer to the unfixed post-mortem condition without signs of autolysis or rigor mortis. The visual, haptic and biomechanical properties, appearance in histology, costs and health effects are listed.

Visual properties
Skin and subcutaneous tissueColorfastPallid, reversible after immersion
BoneIn vivo likeSlightly bleached
CartilageHyaline cartilage: in vivo like; elastic cartilage: dissolvedIn vivo like
MusclesSlightly overstainedSlightly bleached
Nerves, vesselsSlightly pallidSlightly bleached
VisceraVividColorfast
Central nervous systemn/aShrinked, browned slightly
Haptic properties
Skin and subcutaneous tissueIn vivo-likeSlight induration
BoneIn vivo likeIn vivo like
CartilageHyaline cartilage: in vivo like (except cartilage of the nasal septum); elastic cartilage: dissolvedIn vivo like
MusclesSoftenedSlight induration
Nerves, vesselsSoftened, no blood clotsSlight induration, veins filled with blood clots
VisceraIn vivo-likeflexible hollow organs, induration of parenchymatous tissues
Central nervous systemn/aSoftening
Biomechanical properties
BoneIrreversibly altered (Unger et al., 2010)Irreversibly altered (Unger et al., 2010; Hammer et al., 2014)
Ligaments, tendonsIrreversibly altered (Fessel et al., 2011)Altered but reversible (Steinke et al., 2012)
HistologyDevoid of cells blurry appearance of extracellular matrixSimilar to 4% formaldehyde fixation
OdorIntrusive (immediately after fixation) at a decreasing intensityNone or thyme-like
Health effects of componentstoxic, corrosive, carcinogenic and mutagenic (formaldehyde)None described
Costs (Germany)12× formaldehyde fixation2× formaldehyde fixation

Cells are Dissolved by the Thiel Fixative but not by Ethanol-Glycerin

Neither cell membranes nor nuclei were found in the hematoxylin-eosin-stained histology samples obtained after Thiel fixation. The tissues appeared to be washed out by the fixatives, generating blurry structural borders. These effects were similar in the tissues from liver and from skeletal muscle (Supporting Information Figs. 3A and 3B). The extracellular matrix remained, i.e. the interlobular hepatic connective tissues and the endomysium. In contrast, the histology samples obtained after ethanol-glycerin fixation revealed sharp borders between mostly intact cells and the extracellular matrix (Supporting Information Figs. 3C and 3D). Their nuclei were stained in deep blue. Despite the cell-dissolving effects of the Thiel fixative and the poor fixation of the central nervous system, no extravasation was observed in the reconstruction of the circle of Willis after CT angiography (Fig. 3; Wacker et al., 2011). In the given example, the internal and external cranial arteries are shown along with an individual variation concerning the anterior communicating and vertebral artery (Fig. 3B).

Figure 3.

Virtual reconstruction of the circle of Willis on basis of computed tomography angiography of an 84-year-old female after modified Thiel fixation; ventral (A) and dorsal view (B) after partial removal of the skull. Two variations become visible: the absence of an anterior communicating artery (arrow) and a hypoplastic left-sided vertebral artery (white triangle), compensated by a hypertrophic right-sided vertebral artery (V). AC = anterior cerebral artery, B = basilar artery, PCA = pericallosal artery, FB = frontal branch of superficial temporal artery, IC = internal carotid artery, MC = middle cerebral artery, MM = middle meningeal artery, PB = parietal branch of superficial temporal artery, PC = posterior cerebral artery, SPS = sphenoparietal sinus, ST = superficial temporal artery; cd = caudal, cr = cranial, l = left, r = right.

DISCUSSION

Modified Thiel Embalming Simplifies the Availability of Body Donors With Realistic Visual and Haptic Properties and Saves Costs

The requirements on the tissues of body donors differ, regarding their appropriateness and usability for different settings of student dissection courses and clinical workshops. A wide variety of fixatives (Spranger, 1926; Rack, 1951; Kunz and Wilcke, 1991; Thiel, 1992, 2002; Drake, 2007; Whitehead and Savoia, 2008; Messmer et al., 2010; Hammer et al., 2011, 2012; Janczyk et al., 2011b; Jaung et al., 2011) are described in literature. The most commonly used fixative, formaldehyde, causes the visual and haptic properties of biological tissues to change dramatically (Scribbans, 2011; Sharma et al., 2012; Jansen et al., 2014). Though the hardening caused by formaldehyde guarantees obtaining histology slices on a consistent level, it impairs macroscopic dissection and joint motion to the effect that the tissues often become mechanically damaged (Hammer et al., 2012). Thiel-embalmed body donors provide lifelike texture and color (Benkhadra et al., 2011a). Skin, muscle, and viscera of Thiel-fixed specimens appear even more vivid than completely unfixed tissues concerning their color and haptic properties, since the chemicals compensate for the blood-loss related paleness (Table 3). Bone and cartilage properties are quite comparable in Thiel-fixed and unfixed tissues. Two exceptions are the elastic and nasal hyaline cartilage, which become dissolved by the Thiel fixatives. Similar results have been reported using sodium chloride in a mixture with formaldehyde, phenol, glycerin and ethanol (Coleman and Kogan, 1998).

Chemical alterations are likely responsible for the visual and haptic properties of Thiel-fixed tissues. The Thiel fixatives are mainly composed of large amounts of salts, small percentages of formaldehyde, ethylenglyol, boric acid and p-chlorocresol (Table 1; Thiel, 1992, 2002). Generally speaking, the salts absorb water from the tissues as done when curing meat (Janczyk et al., 2011a; Weigner, 2011). Their nitrites and myoglobin form nitrosomyoglobin in muscles, staining the muscles in an intense red color. Ethylenglycol is chemically related to trivalent glycerin and therefore responsible for the soft haptics of the tissue (Thiel, 1992). Benkhadra and coworkers found that the boric acid causes fragmented muscle fibers and that tendons were mostly unaltered (Benkhadra et al., 2011a). Additionally, the boric acid and p-chlorocresol serve as powerful fixatives and disinfectants (Groscurth et al., 2001). Formaldehyde is another component of the Thiel fixation with known antiseptic properties (Romeis, 1989). It irreversibly blocks the amino groups of peptides and cross-links hydroxmethylene bridges (Werner et al., 2000; Abe et al., 2003).

The Thiel method is well known and appreciated especially in German-speaking countries. However, beyond Europe, the technique is hardly in use for a wide variety of reasons: Thiel is significantly more expensive in Germany and technically more extensive than embalming with ethanol, formaldehyde or phenol (Benkhadra et al., 2011b). Our modifications specifically addressed these issues that the costs of Thiel fixation could almost be halved. In our setup, the fixative is exclusively injected into the femoral artery instead of the external iliac artery or body cavities, as previously suggested (Groscurth et al., 2001). Using the femoral region as the injection site allows leaving the anterior body wall intact and has timesaving effects. Additional time- and cost-saving effects can be achieved by storing two body donors in the custom-made rollcontainers at room temperature and by shortening the duration of the bodies from six to two months before first use without affecting the tissue properties. A general issue of Thiel fixation which was not addressed by our modifications is the poor conservation of the central nervous system (Benkhadra et al., 2011b). Walter Thiel proposed an additional intrathekal injection if the brain is the subject matter of dissection (Thiel, 2002).

Thiel Embalmed Body Donors are Ideally Suited for Clinical Workshops but are Only Suitable for Restricted Use in Student Dissection Courses and Biomechanical Testing—Are Fresh Tissues an Alternative?

Realistic tissue properties are of principal interest for the clinicians who perform workshops and surgical training by using body donors. In many cases unfixed post-mortem tissues are used for this purpose, as most fixatives described in literature fail to provide the characteristics of vital tissues (Jansen et al., 2014). However, several unpredictable risks are involved with the use of fresh tissues: these tissues need cooled storage, are a potential source of infections and limited in the period usefulness. Taking into account the time needed for the preparation and the follow-up of clinical workshops, few interventions can be carried out on intact human corpses before autolysis and bacterial growth of human corpses will cause unacceptable conditions. The time span of useful life is additionally shortened by the rigor mortis, which is a clear benefit of Thiel-fixed tissues. One approach to solve these issues is to amputate parts of the body donors and to freeze them for separate use or to inject small amounts of fixatives (Messmer et al., 2010). Again there are some limitations as interventions can only be performed in limited regions of the body and as the body donor can hardly be positioned as in a real-life position. The positioning of body donors like “patients,” e.g. in a lithotomy position or in beach-chair position (Macksey, 2012) is greatly appreciated by the clinicians.

Thiel-fixed donors provide the visual and haptic properties that are required for clinical workshops (Alberty et al., 2002; Löffler et al., 2009; Pattanshetti and Pattanshetti, 2010; Eisma et al., 2013). Thiel is likewise applicable for arthroscopy (Figs. 3A and 3B), gynecology (Figs. 3C and 3D) and for general and visceral surgery due to the tender characteristics of the soft tissues and the natural appearance of the viscera. The considerable fragmentation of muscle proteins related to certain corrosive chemicals (e.g. boric acid) is thought to cause the flexibility of Thiel-embalmed body donors (Benkhadra et al., 2011a). The wide range of joint motion allows orthopedic and trauma surgeons to perform interventions under realistic conditions, which is not the case after ethanol and formaldehyde fixation. Thiel fixation is also suitable for training laparoscopic techniques (Pattanshetti and Pattanshetti, 2010; Prasad et al., 2012) and ultrasound-assisted interventions such as biopsies and nerve blocks (Munirama et al., 2012). Even muscle- or skin-flaps can be accomplished by head and neck (Alberty et al., 2002), plastic (Wolff et al., 2008), oral surgeons and implantologists (Hölzle et al., 2012), since the Thiel fixatives prevent blood clot formation. The limitation that cartilage becomes dissolved by the fixatives (Alberty et al., 2002) can possibly be prevented by the injection of small amounts of formaldehyde (Piechocki, 1986). Thiel-fixed specimens are also suitable for digital subtraction angiography (Wacker et al., 2011) and for CT angiography, which was shown here for the first time (Fig. 3).

However, the Thiel fixation has several disadvantages such as its use for student dissection courses. The costs and the workload related to moving the Thiel-fixed body donors between the dissection tables and the containers with the fixation fluid keep us from using these donors for student education. In our dissection facilities with only limited air conditioning, a large number of students would be faced with a large number of (fully) uncovered body donors, potentially causing high indoor air concentrations of the fixatives. In contrast, only a small number of body donors are used in clinical workshops and these donors are mostly covered with surgical drapes in an operating-room like manner to the effect that evaporation is of minor interest here. Another aspect that has to be taken into account is the poor quality of the histology specimens from Thiel-fixed tissues (Supporting Information Figs. 3A and 3B), e.g. if the entity of a tumor is of further interest in a student teaching setting. However, the primary goal of the Thiel fixation is to provide tissues with a macroscopically lifelike appearance (Thiel, 1992).

Concerning biomechanical testing, Thiel-fixed tissues should not be used to obtain material properties or implant behavior, if data close to the vital condition is of interest. Boric acid and formaldehyde as ingredients of Thiel embalming have been shown to alter the elastic modulus and ultimate stress of tendons (Fessel et al., 2011) and ligaments (Steinke et al., 2012), as compared to the unfixed fresh condition. In bone specimens, Thiel fixation causes the elastic modulus to become lowered, though plastic absorption and ultimate stress increased significantly (Unger et al., 2010). Boric acid and formaldehyde again seem to be partially responsible for the change of bone biomechanics (Currey et al., 1995; Ohman et al., 2008; van Haaren et al., 2008; Hammer et al., 2014) in spite of an unaltered bone mineral density (Unger et al., 2010). Shrinkage artifacts (Unger et al., 2010) and the decellularization (Supporting Information Fig. 3A; Benkhadra et al., 2011a) resulting from the Thiel fixatives might additionally impair tissue biomechanics and morphometry. However, these issues are controversially discussed and will need further clarification in future studies.

Ethanol-Glycerin-Fixed Body Donors are Well Suited for Student Dissection Courses but are Limited in Use for Clinical Workshops

The requirements of human tissues for the dissection course differ from clinical workshops and surgical training, regarding visual and haptic properties. In order to facilitate the dissection of anatomical structures, a certain amount of rigidity may be required. The slight induration caused by the ethanol-glycerin fixative facilitates the separation of the skin from the subcutaneous tissue, muscles from their surrounding fascia or viscera from their capsules, which is hardly done in a clinical setting. These effects are of interest e.g. when visualizing the arterial arches of the hand, as shown in Figures 1B and 1D. The slight bleaching related to ethanol-glycerin contributes to the aesthetic appearance of the tissues (Hammer et al., 2011, 2012). Though the range of joint motion is significantly smaller in ethanol-glycerin-fixed donors than in the Thiel-fixed or fresh ones, it allows dissecting the extremities in an abducted position without damaging adjacent muscles. However, rigidity, color fastness and the range of joint motion of ethanol-glycerin-fixed specimens are superior to formaldehyde-fixed tissues (Hammer et al., 2011, 2012). The odors related to ethanol-glycerin fixation are less intrusive, as compared to Thiel or formaldehyde embalming. In contrast, thyme-like odors related to the application of ethanol-thymol might even be considered as pleasant. Conclusively, ethanol-glycerin fixation is well suited for large-scale settings of body donors in student dissection courses. This is also the case for rooms without air ventilation, possibly due to regulations relating to the preservation of historic buildings as in our case, or to financial considerations.

Histology specimens can be obtained and stained successfully from ethanol-glycerin fixed tissues. The results are similar to tissues initially fixed with formaldehyde, as shown here (Supporting Information Figs. 3C and 3D) and elsewhere (Benkhadra et al., 2011a; Hammer et al., 2012). For biomechanical purposes, similar recommendations apply for ethanol-glycerin fixed tissues as for the formaldehyde- or Thiel-fixed ones. Material properties obtained from uniaxial testing of soft tissues such as tendons or ligaments are strongly altered by ethanol, which is likely caused by denaturation of the tertiary structures of the proteins (Steinke et al., 2012). However, this alteration seems to be reversible after rinsing (Steinke et al., 2012). In contrast, bone specimens are irreversibly altered by ethanol (Sedlin, 1965; Linde and Sørensen, 1993; Unger et al., 2010; Anderssohn, 2011), likely caused by changes of the bone's organic components (Hammer et al., 2014). Material testing of anatomically unfixed tissues therefore remains the gold standard if material properties similar to the vital condition are of interest.

Financial Aspects and Health Issues Related to Thiel Fixation

Thiel is much costlier than the standard techniques with ethanol-glycerin (Hammer et al., 2011, 2012), formaldehyde (Blum, 1893, 1896) and phenol (Murray et al., 2007) in Germany. However, the financial efforts have to be weighed against the tissue characteristics resulting from the fixation agents. As shown in our study and by others (Thiel, 1992, 2002; Wolff et al., 2008; Benkhadra et al., 2009; Pattanshetti and Pattanshetti, 2010; Jaung et al., 2011; Hölzle et al., 2012; Munirama et al., 2012; Prasad et al., 2012; Eisma et al., 2013), Thiel-fixed donors provide outstanding visual and haptic properties. Our modifications may likely aid saving costs in order to make the Thiel technique available to a broader user group (Benkhadra et al., 2011b).

Health effects of the Thiel-fixatives have hardly been investigated until today. Anatomists, embalmers, medical students are well known to be exposed to high peak concentrations of the fixatives (Ryan et al., 2003; Shiraishi, 2006; Vimercati et al., 2007).

The Thiel fixatives are known to be corrosive, irritating and oxidizing (Table 1). Boric acid is toxic to the reproductive tract of mammals (Chapin and Ku, 1994; Roth, 2012). Formaldehyde as another component of the Thiel fixatives is classified as a Group 1 human carcinogen (Hauptmann et al., 2009; Lunn et al., 2010). A number of malignancies have been described (Coggon et al., 1984; Hauptmann et al., 2009; Dreyfuss, 2010; Goldstein, 2011) especially when formaldehyde is used as an exclusive fixation agent. When done so, health effects such as nose, eye, skin and airway irritation (Chia et al., 1992; Kriebel et al., 2001; Tanaka et al., 2003; Lakchayapakorn and Watchalayarn, 2010; Wolkoff and Nielsen, 2010) additionally impact respiratory function (Akbar-Khanzadeh et al., 1994), though dosage-response relationships are still poorly understood (Akbar-Khanzadeh et al., 1997). Formaldehyde-fixation related personal exposure was repeatedly reported to be higher than indoor concentrations (Ohmichi et al., 2006; Vohra, 2011) and it varied dramatically depending on the anatomical site that is being dissected (Perkins and Kimbrough, 1985; Takayanagi et al., 2007). The existing indoor air guidelines are hard to comply with (van Gemert, 2003; Wolkoff and Nielsen, 2010). High-performance air extraction systems (Keil et al., 2001; Kurose et al., 2004), combined with protective devices (Kurose et al., 2004; Lakchayapakorn and Watchalayarn, 2010) may help reduce peak values of the vapors rising from the tissues with related personal exposure and to meet the indoor guidelines. However, such architectural changes are impossible in our facility due to the strict German regulations on the preservation of historic buildings. Since the final concentration of the formaldehyde in the Thiel fixative is approximately 0.8% (Groscurth et al., 2001), the indoor air concentrations will likely be lower than those stated in the guidelines, especially if the body donors are covered with surgical drapes and if only a limited number of body donors is used at once. Consequently, the formaldehyde concentration must not exceed the values given in the original description of the Thiel fixative in order to circumvent formaldehyde-related health risks. However, the use of formaldehyde in Thiel-fixed tissues or in ethanol-glycerin fixed tissues in case of progressed autolysis (Hammer et al., 2012) should be an issue to be addressed in further studies.

CONCLUSIONS

Life-like conditions are consistently reported when using the Thiel technique and can be maintained with cost and room savings by our modifications. However, the agents and methods used in anatomy departments are also influenced by internal historical experiences and preferences. In our setting, with a large number of body donors, limited staff and storing space, Thiel embalming ideally complements ethanol-glycerin fixation in order to provide customized solutions for clinical workshops and student dissection courses, respectively. The parallel use of either one of the techniques therefore provides anatomical specimens in a wide range of applications.

ACKNOWLEDGMENTS

The authors thank Matthias Oehme and Uwe Deubel for their support with the body donors and Angela Ehrlich for her help with histology. Christine Auste took the pictures of the specimens. Stefan Schleifenbaum made the design drawing of the custom-made container. Lina Woydt and Thomas Doberstein prepared the forearms for the comparison of the fixation techniques. Franziska Eplinius obtained the CT images. Dr. Peter Haensel demonstrated the radiocarpal arthroscopy in the body donors and Prof. Dr. Dr. Michael Höckel the gynecological surgery shown in this article.

NOTES ON CONTRIBUTORS

NIELS HAMMER, M.D., is an anatomist at the Institute of Anatomy at the University of Leipzig, Faculty of Medicine, Leipzig, Germany. He teaches anatomy and histology to undergraduate students and clinical anatomy to graduate students. His research is focused on biomechanics of the pelvis and on the anatomy of the subthalamus.

SABINE LÖFFLER, M.D., M.M.E., is a prosector at the Institute of Anatomy at the University of Leipzig, Faculty of Medicine, Leipzig, Germany. She teaches anatomy and histology to first- and second-year medical students and clinical anatomy to graduate students.

INGO BECHMANN, M.D., is a professor and the Director of the Institute of Anatomy at the University of Leipzig, Faculty of Medicine, Leipzig, Germany. He teaches anatomy and histology to undergraduate students and clinical anatomy to graduate students. His research is focused on multiple sclerosis and Alzheimer's disease.

HANNO STEINKE, Ph.D., is an anatomist in the Institute of Anatomy at the University of Leipzig, Faculty of Medicine, Leipzig, Germany. He teaches anatomy and histology to undergraduate students and clinical anatomy to graduate students. His research is focused on the biomechanics of the pelvis and the lower extremities.

CARSTEN HÄDRICH, M.D., is a prosector at the Institute of Legal Medicine at the University of Leipzig, Faculty of Medicine, Leipzig, Germany. He teaches forensic medicine to medical students and to graduate students.

CHRISTINE FEJA, Ing. (F.H.), is an anatomist at the Institute of Anatomy at the University of Leipzig, Faculty of Medicine, Leipzig, Germany. She teaches anatomy and histology to undergraduate students and clinical anatomy to graduate students.

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