The Journal of Plastination
The official publication of the International Society for Plastination
ISSN 2311-7761
IN THIS ISSUE:
Influence of the Temperature on the Viscosity of Different Types of Silicone – p4
A Comparison of Different De-plastination Methodologies for Preparing Histological Sections – p10
Biomechanical Analysis of The Skin and Jejunum of Dog Cadavers Subjected To A New Anatomical Preservation Technique For Surgical Teaching – p16
Bleaching of Specimens Before Dehydration in Plastination: A Small-scale Pilot Study Using Human Intestine – p24
General Issues of Safety in Plastination – p27
Volume 30 (1); July 2018 The Journal of Plastination
ISSN 2311-7761 ISSN 2311-777X online The official publication of the International Society for Plastination
Editorial Board:
Rafael Latorre Philip J. Adds Murcia, Spain Editor-in-Chief Institute of Medical and Biomedical Education Scott Lozanoff (Anatomy) Honolulu, HI USA St. George’s, University of London London, UK Ameed Raoof. Ann Arbor, MI USA Robert W. Henry Associate Editor Mircea-Constantin Sora Department of Comparative Medicine Vienna, Austria College of Veterinary Medicine Hong Jin Sui Knoxville, Tennessee, USA Dalian, China Selcuk Tunali Carlos Baptista Assistant Editor Toledo, OH USA Department of Anatomy Hacettepe University Faculty of Medicine Ankara, Turkey
Executive Committee: Rafael Latorre, President Dmitry Starchik, Vice-President Selcuk Tunali, Secretary Carlos Baptista, Treasurer
Instructions for Authors
Manuscripts and figures intended for publication in The Journal of Plastination should be sent via e-mail attachment to: [email protected]. Manuscript preparation guidelines are on the last two pages of this issue.
On the Cover: Right atrium of a plastinated human heart showing pectinate muscles and portion of the crista terminalis visualized by trans illumination. Specimen from the collection of the Liberato Didio and Peter Goldblatt Interactive Museum of Anatomy and Pathology, University of Toledo. Photography courtesy of Dr. Telma Masuko.
The Journal of Plastination 30(1):1 (2018)
Journal of Plastination Volume 30 (1); July 2018
Contents
Letter from the President, Rafael Latorre 2
Letter from the Editor, Philip J. Adds 3
Influence of the Temperature on the Viscosity of Different Types of Silicone; Athelson S. 4 Bittencourt, Yuri F. Monteiro, Laissa da S. Juvenato, et al
A Comparison of Different De-plastination Methodologies for Preparing Histological Sections 10 of Material Plastinated with Biodur® S10 / S3; M. L. Ramos, et al
Biomechanical Analysis of The Skin And Jejunum Of Dog Cadavers Subjected To A New 16 Anatomical Preservation Technique For Surgical Teaching; T.A. Rocha, C. Santos, A. Fechis, F. Oliveira, et al
Bleaching of specimens before dehydration in plastination: a small-scale pilot study using 24 human intestine; Jie-Ru Chen, Hong-Jin Sui
General Issues of Safety in Plastination, V. K. Schill 27
Instructions for Authors 37
The Journal of Plastination 30(1):2 (2018)
LETTER FROM THE
PRESIDENT
Dear Friends and Plastinators:
It is with great pleasure that I present to you Volume 30, Issue 1, of the Journal of Plastination. I would like to thank the reviewers for taking their time to review the manuscripts.
In this issue, we present remarkable papers. The first paper from Dr. Bittencourt is about the viscosity of different silicones for plastination, and the importance of considering their physicochemical characteristics and dynamic viscosity, before choosing the ideal silicone for our particular needs. The second study is about deplastination for histology studies, from Dr. Moema Lopes Ramos. It presents a very interesting result: that it is possible to produce histological sections directly from plastinated specimens, without previous deplastination, at least in the three tissues tested. The third paper, presented by Dr. Rocha, describes a new protocol Rafael Latorre, DVM, for tissue preservation, focused on surgical training applications. The fourth paper PhD of this issue is a very concise work presented by Dr. Sui, about the specific effects of bleaching before plastination on the final appearance of the specimens. The last paper, about General Issues of Safety in Plastination, by Mr. Volker Schill, is a very important paper for all of us. We all are worry if we do not have a proper vacuum pump or a good impregnation chamber for instance, however, we are not always alert about how to work in a healthy ambience in our plastination lab. This paper helps us to be aware of the potential hazards of the chemicals that we are working with every single day.
I would like to welcome all new members of the International Society for Plastination (ISP) and to invite all of you to participate in the Journal of Plastination. Please, share with us your results, and your expertise in plastination and other anatomical techniques.
With best regards from Murcia, Spain
Rafael Latorre President
The Journal of Plastination 30(1):3 (2018)
LETTER FROM Dear Colleagues, THE EDITOR Thank you to those who have submitted manuscripts to the Journal. We are pleased to publish five new papers in this issue, on topics ranging from safety in the laboratory, to technical reports on the plastination process. The world-wide reach of this technology is evidenced by the fact that the papers in this issue come from Brazil, China and Germany, with three coming from teams working in Brazil.
My main priority for next year is to get the Journal indexed. We have made changes to the journal in the light of feedback we received from the National Library of Medicine, which should help in making a strong case for getting the journal indexed on Medline in the near future.
In the meantime, I have submitted an application to Scopus and Embase. “Over 8,500 journals are currently indexed in Embase, and each year several hundred additional journals of potential interest are screened by an editorial committee entrusted with the Philip J. Adds, MSc, FIBMS, review and quality assessment of biomedical publications for Embase.”1 SSFHEA Scopus is the largest abstract and citation database of peer-reviewed literature: scientific journals, books and conference proceedings, covering over 36,000 titles.2
The review process, however, is not fast, and it can take many months from application to actual inclusion. The acknowledgement email from Scopus advised us to allow a minimum period of 6-12 months for the review process to be completed. Yesterday I received the following message in response to my request for an update: “The title Journal of Plastination is in the final phase of the evaluation process and we are now waiting for the final decision of our Content Selection & Advisory Board (CSAB). Once the outcome of the evaluation by the CSAB is available, you will be contacted by us again. We appreciate your patience in this matter.”
I hope to be able to report more positive news in the next issue.
Best wishes,
Philip J Adds Editor-in-Chief
References
1. https://www.elsevier.com/solutions/embase-biomedical-research/journal-title- suggestion?sb=1508153513999
2. https://www.elsevier.com/en-gb/solutions/scopus
The Journal of Plastination 30 (1): 4-9 (2018)
ORIGINAL Influence of the Temperature on the Viscosity of Different RESEARCH Types of Silicone
YURI F. MONTEIRO1*, ABSTRACT: LAISSA DA S. JUVENATO1, The objective of this work was to test the influence of temperature on the viscosity of three ANA PAULA S. V. silicones of different molecular weights (Biodur® S10, Polisil® P10 and P1) commonly BITTENCOURT2,3, used in the plastination technique. For the study, the RheolabQC model rotational BRUNO M.M. SIQUEIRA2, rheometer was used to measure the dynamic viscosities of the chosen polymers at the FLÁVIO C. MONTEIRO2, following temperatures: -5, 0, 5, 10, 15, 20, 25, 30 and 35 °C. From the 9 measurements CARLOS A C BAPTISTA4, of viscosities obtained from each sample, a viscosity vs. temperature graph was ATHELSON S. constructed. The equation of the dynamic viscosity curve of each polymer was analyzed. 1,2* BITTENCOURT Polisil® P1 silicone had a much lower viscosity compared to other silicones (about 80 mPa.s at 25 °C and 550 mPa.s at -25 °C). Polisil® P10 silicone presented the highest viscosity of the polymers analyzed (approximately 1180 mPa.s at 25 °C and 3730 mPa.s Department of Morfology, at -25 °C). The Biodur®'s S10 silicone showed an intermediate viscosity (about 410 Federal University of Espirito Santo, Brazil mPa.s at 25 ° C and 1500 mPa.s at -25 °C). We conclude that Polisil® P1 silicone presented the best physico-chemical characteristics of the tested silicones for plastination, because it has high fluidity and low viscosity. It is noteworthy that the viscosity of Polisil® P1 in cold impregnation temperature (-15 °C) is still lower than the viscosity of the Biodur® S10 (control) at room temperature (20-25 °C). We also conclude that the knowledge of the intrinsic and extrinsic physicochemical characteristics of the silicone and its dynamic viscosity is helpful in choosing the ideal silicone for use in the cold or room temperature plastination techniques.
KEY WORDS: Viscosity, PDMS, temperature, silicone, plastination
* Correspondence to: Athelson S Bittencourt, Federal University of Espirito Santo Health Sciences Center, Marechal Campos Avenue, 1468 Maruipe, Vitoria- ES, Brazil Zip code: 29.043-900, Fax: +55 27 33357358, [email protected]
Introduction Silicones, technically, are polymers that can be obtained basically in three steps: synthesis of chlorosilanes, The term silicone, or polysiloxane, was created in 1901 to hydrolysis of chlorosilanes to silanols and polymerization describe mixed polymers of organic and inorganic of silanols. The first step occurs in a fluidized bed of metal materials, whose crude formula is [R2SiO]n, where R are silicon powder treated with a flow of chloromethane, organic groups such as methyl, ethyl and phenyl. These generally at temperatures of 250 to 350 °C and pressures polymers are inert, odorless, insipid and resistant to of 1 to 5 atm. A mixture of different chlorosilanes is decomposition by heat, water or oxidizing agents, besides obtained mainly containing the dimethyldichlorosilane being good electrical insulators. They exhibit good (Me2SiCl2), which represents the most important resistance to high or low temperatures (-45 to +145 °C) monomer for the subsequent steps. In the second step, and have viscosities between 10 and 100,000 millipascal polydimethylsiloxanes are obtained by the hydrolysis of second (mPa.s) (Milles et al., 1975). dimethyldichlorosilane, in the presence of excess water (Hardman, 1989). A polymer is a macromolecule formed by repetitive structural units, joined together by covalent bonds. In The products of this reaction are readily condensed, thus silicone, the repeating unit is siloxane (subgroup of silica leading to a mixture of linear and cyclic silicones. The compounds containing Si-O bonds with organic radicals linear and cyclic oligomers obtained by hydrolysis of attached to the molecule) (Carraher, 2003). dimethyldichlorosilane have still very short chains, for
Influence of the Temperature on the Viscosity - 5 most applications. In the third step, they must be research in this area. This study aimed to show the condensed (in the case of linear ones) or polymerized (in characteristics of three silicones of different molecular the case of cyclic ones), to obtain macromolecules of weights (Biodur S10, Polisil P10 and P1), and the satisfactory lengths (Hardman, 1989). This last step is influence of the temperature in their viscosity. Knowledge decisive for the determination of the viscosity of the final obtained in this study will allow us to better understand product, since the viscosity of the silicones is directly the variables influencing the impregnation process. related to their degree of polymerization (n). Depending on the size of the polymer chain, the silicone can be Materials and Methods: produced in three forms: liquid, gel and cohesive The RheolabQC rotational rheometer, manufactured by (Hardman, 1989). the Austrian multinational Company Anton Paar, was Viscosity is a characteristic of liquids that is related to their used to test the influence of temperature on the viscosities ability to flow. The greater the viscosity of a liquid (or a of the silicones. The equipment was used with a coaxial solution), the greater the difficulty of the liquid to flow and cylinder measurement system to measure the dynamic more "viscous" the liquid is. One of the main external viscosities of the polymers Biodur® S10, Polisil® P10 and factors influencing the viscosity of a silicone is the P1 in the following temperatures: -5, 0, 5, 10, 15, 20, 25, temperature (Oliveira, Barros and Rossi, 2009). The 30, 35 °C. As recommended by the manufacturer viscosity is directly proportional to the internal friction of protocol, the samples of silicones were transferred one by the silicone, and friction originates from the pulling force one to a specific vessel for use in the rheometer with a of the silicone molecules themselves. As temperature cylindrical rod rotating within the sample, thus generating increase, this pulling force decrease, causing viscosity a shear in the fluid. The vessel was attached to the reduction. This reduction of viscosity occurs due to the measuring head of the rheometer. In addition to the increase of the intermolecular distances of the silicone by dynamic viscosity and temperature, the apparatus also the higher kinetic energy (caused by the heating), measured the shear rate and the shear stress. The reducing the attraction forces and, consequently, the apparatus has a cooling and heating system coupled to friction of molecules, allowing a faster flow (lower the samples, with the minimum and maximum viscosity) (Granjeiro et al, 2007). temperature reached varying from -5 to 80 °C.
For the rheological study of liquids, two factors are of The dynamic viscosities of the three silicone samples great importance and should always be observed: shear were measured in 9 different temperatures, and at each stress and shear rate. Shear stress is a type of stress pre-programmed temperature, the device made 100 generated by forces applied in opposite orientation, but in measurements of the actual temperature and its similar directions in the analyzed material. On the other respective viscosity, with the shear rate varying from 100 hand, shear rate or deformation rate is defined by the to 600 seconds-1 (s-1). The shear rate and stress values variation of the shear deformation in relation to the time were also measured. The data were plotted in the (Shiroma, 2012).The concepts of shear stress (applied software Start Rheoplus® by the equipment itself and force) and shear rate (velocity gradient) are used to later exported to Microsoft Excel®. describe the deformation and flow of a fluid. Fluids in The measurements obtained by the rheometer, i.e. the which the shear stress is directly proportional to the rate temperature averages and respective viscosities ± of deformation are called Newtonian fluids, and the standard deviation, were calculated and plotted. An viscosity is a constant for these fluids. However, if the equation of the dynamic viscosity curve of the different shear stress is not directly proportional to the shear rate, types of silicone was generated. the fluid is termed non-Newtonian; therefore, the viscosity varies according to the shear stress applied to the fluid Results (Schramm, 2006). Nine viscosity measurements were obtained from The main silicone used in the plastination process is each sample. The temperature averages and their polydimethylsiloxane (PDMS), which is a linear polymer respective viscosities are showed in Table 1. A viscosity whose radicals are methyl groups (Chaynes and vs. temperature graph was constructed, and the dynamic Mingotaud, 2004). A literature search on the subject of viscosity curve equation of each polymer analyzed viscosity of silicones for plastination reviewed a lack of (Graph 1).
6 - Bittencourt, Monteiro, Junvenato
Table 1. Average values of the temperatures (°C) and respective dynamic viscosities (Pa.s) of the Biodur® S10, Polisil® P10 and P1 silicones measured by the RheolabQC model rotational rheometer. SILICONE BIODUR® S10 SILICONE POLISIL® P10 SILICONE POLISIL® P1 ºC Pa.s ºC Pa.s ºC Pa.s -5,07 0,918 -5,08 2,423 -5,04 0,260 -0,08 0,791 0,27 2,110 0,09 0,213 5,91 0,677 5,27 1,846 5,89 0,174 10,33 0,593 10,20 1,630 10,35 0,147 15,11 0,515 15,46 1,451 15,22 0,118 19,93 0,458 20,22 1,301 19,80 0,098 25,37 0,404 25,48 1,168 25,40 0,080 30,29 0,361 30,47 1,055 30,42 0,068
35,40 0,323 35,27 0,958 35,09 0,059
The graphic of dispersion (XY) for each polymer from table 1 was ploted and its curve equations were calculated. The most suitable trend line that represents the viscosity points of the silicones is the exponential type (Giap, 2010; Romano et al., 2017). The use of 9 temperature points to generate the construction of a dispersion graph gave greater reliability to the results. Only three points in a scatter plot (XY) are sufficient to make a trend line, although the higher the number of measurements the more reliable the graph equation would be (Skoog, 2014). The determination of the curve equation of each silicone was done by Excel® software, through point-to-point regression. Graphs 1, 2 and 3 show the viscosity vs. temperature curves for each silicone. All graphs present decreasing of viscosity with the increasing Graph 2. Viscosity dispersion vs temperature of polymer of temperature. P10 ± standard deviation, including the curve equation (y), the value of the determination coefficient (R2).
Graph 1. Graph 1. Viscosity dispersion vs temperature of polymer S10 ± standard deviation, including the curve Graph 3. Viscosity dispersion vs temperature of polymer P1 equation (y), the coefficient of determination value (R2). ± standard deviation, including the curve equation (y), the coefficient of determination (R2) value.
Influence of the Temperature on the Viscosity - 7
Equations of the curves found for the silicones S10, P10 -18° C 1254 3181 424 and P1 are y = 0,785e-0,02x, y = 2,102e-0,02x and y = -15° C 1160 2969 378 0,213e-0,03x, respectively. The degree of accuracy between the viscosity values calculated by the equation -10° C 1019 2646 312 and the actual values can be demonstrated by the - 5° C 894 2359 258 coefficient of determination (R2). R2 is a statistical real 0° C 785 2102 213 data points. An R2 of 1 indicates that the regression line perfectly fits the data.The closer R2 is to the 1, closer to 5° C 689 1874 176 real are the values calculated from the equations of each 10° C 605 1670 146 curve. The R2 values of the silicones graphs are: for P10 (0.997), S10 (0.996) and P1 (0.998). The values of R2 15° C 532 1489 121 showed a degree of accuracy of the equation higher than 20° C 467 1327 100 99.5%, a very high degree of association (Cosentino et al, 25° C 410 1183 83 2013). The standard deviation was calculated for each point of the graph, however some deviations were too 30° C 360 1054 68 small to appear in the plot, denoting a high homogeneity and precision of the measurements obtained.
The lack of linearity of the polymeric viscosity became more evident with the decrease in the viscosity of the silicone. The increase in the viscosity of the P10, S10 and P1 silicones from the maximum temperature (35 °C) to the minimum (-5 °C) measured on the rheometer was 153%, 184% and 340%, respectively.
Mathematical calculations were used to determine viscosities in temperatures outside the range of the equipment, but important for the plastination process. Plotting a value to the curve equation, an approximate value of viscosities of the silicone samples at any desired Graph 4: Comparison of the viscosity and temperature temperature is obtained. Table 2 shows the viscosity curves of the S10, P10 and P1 silicones. The viscosity values of the three tested silicones at different values were calculated from each curve equation of the temperatures, calculated from the curve equation for each tested silicones. polymer. Graph 4 shows the comparison of the viscosity curves of the silicones Biodur® S10, Polisil® P10 and P1 Discussion calculated from the curve equations of the silicones. Knowledge of the influence of temperature on the Plotting the curves of the three types of silicones on the viscosity of the silicones used in plastination is of great same graph make it possible to observe the viscosity and importance. Through a detailed study of this topic, several behavior of each silicone by changing the temperature questions can be raised, such as: 1) what is the ideal (graph 4); therefore, the viscosity values were calculated temperature of the silicone in low temperature (LT) from the curve equations of tested silicones. impregnation to decrease the retraction of the tissues and Table 2. Comparative viscosity values (mPa.s) calculated also to avoid hardening over time? 2) what is the ideal from the viscosity curve vs. temperature equation of the viscosity of the silicone for plastination at low and room silicates S10, P10 and P1 at different temperatures of temperatures? 3) what is the rheological behavior of importance in plastination. silicones with different viscosities? 4) how does the Temperature Biodur® Polisil® Polisil® viscosity of different silicones behave over time when in the reaction mixture for cold impregnation? and 5) does S10 P10 P1 the temperature increase present a linear relationship -25° C 1505 3736 552 with the viscosity increase? -20° C 1321 3330 457
8 - Bittencourt, Monteiro, Junvenato
The graphs (Graph 1, 2 & 3) for each silicone show the Silicones of lower molecular weight are more sensitive to behavior of each polymer against the temperature the temperature gradient, when compared to those of gradient. An exponential increase in the viscosity of the higher molecular weight. As expected, all graphs showed silicones was observed with each temperature decrease. a strong negative correlation, that is, the increase on the temperature variable implies in a decrease on the The shear rate and shear stress measurements made by viscosity variable. the apparatus were used to show that although the polymers are considered non-Newtonian fluids, the flow At 35 °C (maximum measured temperature), silicones curves (shear rate vs. shear stress) of the tested silicones P10 and Biodur® S10 are respectively 16.2 and 5.5 X showed that they are closer to the Newtonian fluids’ more viscous than P1. At -5 °C, P10 and Biodur® S10 are features (Orrah et al., 1988; Schneider et al., 2009). As respectively 9.3 and 3.5 X more viscous than P1. The the flow curve (rate vs shear stress) becomes more linear, lower the temperature, the lower the difference between the closer the sample will be to the behavior of Newtonian the viscosities of the silicones. fluids (Schramm, 2006). The viscosity data found are in accordance with the The rheological characteristics of the three silicones used values provided by the manufacturers (Polisil® P10 = in plastination allow us to define their dynamic viscosity 1000-1500 mPas, Biodur® S10 = 450-600 mPas and P1 as the average of hundreds of measurements made at a = maximum of 100 mPas, all at 25 °C). given temperature. Viscosities remained constant or had a minimum change, regardless of the shear stress applied The P1 Silicone appeared to be a good alternative to the to the sample at a given temperature. reference silicone in plastination (Biodur® S10), since it has a viscosity at least 4 X lower at room temperature and The different viscosities found in the tested silicones are 3 X in cold temperature, when compared to S10. The determined by the degree of polymerization. The larger lower viscosity of P1 allows the silicone to flow more the silicone chain (P10> S10> P1), the more quickly and easily into the biological tissues during forced intermolecular bonds are made with adjacent molecules, impregnation, therefore reducing shrinkage. The viscosity and thus the less fluidity of the chain. The P10 has the of P1 at cold temperature is less than the viscosity of largest chain (molecular weight), and consequently the Biodur® S10 at room temperature. Thus, plastination with highest viscosity. The different viscosities found within the the silicone P1 at low temperatures is likely to produce same silicone sample were mainly found at different equal or less shrinkage than the Biodur® S10 at room temperatures. Molecular cohesion is the dominant cause temperature. The advantages attributed to room of viscosity, and, as the temperature of the silicone temperature plastination are described in the literature increases, these cohesive forces decrease, resulting in a (Starchik and Henry, 2015b). It is known that the higher decrease in viscosity (Granjeiro et al., 2007). the viscosity of a silicone, whether by the size of the polymer chain or the reduction in silicone temperature, the The curves of viscosity vs temperature of all analyzed greater the retraction of biological tissues in the forced silicones (Graphs 1, 2 & 3) has an exponential trend, impregnation step of plastination (Starchik and Henry, showing that there is no linearity in the increase of the 2015b). The use of low molecular weight silicones may be viscosity as a function of temperature, and that these preferable when a specimen with less shrinkage is values of viscosity grow with increasing rates. Therefore, sought. Specimens more prone to shrinkage, such as the the lower the temperature, the steeper the viscosity curve brain and nervous system, will benefit from impregnation of the silicones becomes. using silicones of shorter chains (more fluid).
Silicone P1 has a higher proportional increase in viscosity From the results presented by this research, we conclude with decreasing temperature, making its curve steeper in that the Polisil® P1 silicone presents the best physico- proportion to the others (Graph 3). However, P10 silicone chemical characteristics of the silicones tested for the has the highest viscosity and the lowest increase application in plastination, because it has high fluidity and proportional to the decrease in temperature, that is, its low viscosity. It is noteworthy that the viscosity of P1 at curve is proportionally less steep when compared to the cold impregnation temperature (-15 °C) is still lower than others (Graph 2). the viscosity of Biodur® S10 at room temperature (20 - 25 °C).
Influence of the Temperature on the Viscosity - 9
Financial support Hardman B, Torkelson A. 1989: Silicones. Kroschwitz JI, editor. Encyclopedia of Polymer Science and This research was supported by CNPq (458328/2013-8), Engineering.Silicones, 15th. John Wiley & Sons, p 204. undergraduate scholarship provided by UFES and graduate scholarship provided by CAPES. Oliveira RC, Barros STD, ROSSI RM. 2009: Aplicação da metodologia Bayesiana para o estudo reológico da polpa Acknowledgements de uva. Revista Brasileira de Produtos Agroindustriais. Special thanks to the scientific collaboration with Campina Grande, 8 p. Processing and Characterization Laboratory/LabPetro - UFES Romano MR, Cuomo F, Massarotti N, Mauro A, Salahudeen M, Costagliola C, Ambrosone L. 2017: References Temperature effect on rheological behavior of silicone oils – a model for the viscous heating. J Phys Chem B. Carraher CE. 2003: Inorganic Polymers. In: Lagowski JJ, editor. Polymer Chemistry, 6th ed. Marcel Dekker, Inc, p Schramm G. 2006: Reologia e Reometria. Artliber Editora 513-526. Ltda., Sao Paulo.
Chaynes P, Mingotaud AF. 2004: Analysis of commercial Shiroma PH. 2012: Estudo do comportamento reológico plastination agents. Surg Radiol Anat 26: 235–238. de suspensões aquosas de bentonita e CMC: influência da concentração de NaCl. Master's Dissertation. Sao Cosentino HM, Moura AAJ, Costa ACF. 2013: Estatística Paulo University. Básica para tomada de decisão. Volume 1: 1 ed. Rio de Janeiro: Editora Escola Nacional de Seguros. Skoog, West, Holler and Crouch. 2006: Amostragem, Padronização e Calibragem. Fundamentos de Química Giap SGE. 2010: The Hidden Property of Arrhenius-type Analítica, 8th Ed. Thomson, p 166-179. Relationship: Viscosity as a Function of Temperature. J Phys Sci 21(1): 29–39 (2010) Starchik D, Henry RW. 2015: Comparison of cold and room temperature silicone plastination techniques using Granjeiro AA, Queiroz AJM, Figueiredo RMF, Mata tissue core samples and a variety of plastinates. J Plast MERMC. 2007: Viscosidades de polpas concentradas de 27(2):13-19 (2015) figo-da-Índia. Revista Brasileira de Agrociência. Pelotas, 6 p.
The Journal of Plastination 30(1):10 - 15 (2018)
A Comparison of Different De-plastination Methodologies ORIGINAL RESEARCH for Preparing Histological Sections of Material Plastinated with Biodur® S10 / S3
M. L. RAMOS1,2 ABSTRACT: T. A. R. DE PAULA2, M. F. ZERLOTINI2, Objectives - The objective of this study was the evaluation of different protocols to obtain V. H. D. SILVA2, histological slides from silicone plastinated specimens. L. B. CARAZO2, Materials and Methods - Samples of pig aorta, heart, and kidney were used. Four M. F. DE PAULA2; treatments for light microscopy (LM) were compared. Treatment 1 (control): fixation in F. F. R. SILVA2 10% formalin for 48 h at room temperature; tissue samples were then processed for LM M. L. SANTANA2; histology. Treatment 2: plastinated fragments were directly embedded in paraffin wax. L. C. SILVA2; Treatment 3: plastinated tissue samples were de-plastinated by immersion in 99% ethyl L. B. C. FERREIRA2 alcohol for 24 hours, then in methylbenzene for 48 hours; samples were then processed
for LM histology. Treatment 4: plastinated samples were de-plastinated in 1,4- 1 Institute of Biological dimethylbenzene for 36 hours, and then processed for LM histology. and Health Sciences, Federal University of Results - The renal capsule was preserved intact in all treatments. The renal cortex Viçosa, campus Rio showed some damage, and the epithelium of the renal tubule had some shrinkage in Paranaíba, Brazil. treatments 3 and 4. Changes in the structure of the myocardium were visible in treatments 2, 3 and 4. It was not possible to visualize the vasa vasorum in the tunica adventitia of 2 Department of the aorta of treatments 2, 3 and 4. All treatments showed elastic lamellae relatively well Veterinary Medicine, organized following Verhoeff staining. Federal University of Conclusions - We found that de-plastination with 1,4-dimethylbenzene produced a Viçosa, Brazil. material similar in quality to de-plastination with methylbenzene, and plastinated tissue
without de-plastination produced histological material similar to de-plastinated specimens.. KEY WORDS: de-plastination; plastination; histological architecture. *Correspondence to: Moema Lopes Ramos, Institute of Biological and Health Sciences, Federal University of Viçosa, campus Rio Paranaíba, Brazil, Rodovia MG-230 – Km 7, Rio Paranaíba – MG, CEP: 38810-000. Caixa Postal 22. Tel +55 34 3855 9368; E-mail: [email protected]
Introduction the exposure of students and anatomy staff to formaldehyde (von Hagens, 1987; Latorre et al., 2007). Formaldehyde is the main fixative solution employed Plastination is based on the replacement of body fluids worldwide as a preserving solution in anatomy. It acts on and fats by a curable polymer. According to Ravi and Bhat biological tissues, preventing their degradation (Hambeli (2011), one of the most interesting, important, and et al., 2010). However, the use of formaldehyde for the potentially useful qualities of silicone plastinated tissue is preservation of bodies and parts for gross anatomy study that its microscopic structure remains intact. This implies has been discouraged. In 2004, the International Agency that the specimen can be preserved, almost indefinitely, for Research on Cancer (IARC) of the World Health in a form that is easily stored, while still retaining the full Organization (WHO), classified formaldehyde as potential for histological examination. To access the carcinogenic (group 1), tumorigenic, and teratogenic to histological structure of plastinated specimens, authors humans (INCA, 2005). have described de-plastination with sodium methoxide
(Walker et al., 1988), and methylbenzene, methylene or Thus, it has become a matter of great importance to find dichloroacetone (Ripani et al., 1996). However, the most an alternative to formaldehyde for specimen preservation in anatomy teaching. Plastination is an option to prevent
De-plastination Methodologies for Preparing Histological Sections - 11 effective substances for de-plastination, sodium forced impregnation at -25oC. The heart and aorta were methoxide and methylbenzene, are very toxic. The aim of plastinated at the Veterinary Anatomy Laboratory, this work, then, was to evaluate different protocols to Department of Anatomy and Comparative Pathological obtain histological sections from silicone plastinated Anatomy, Faculty of Veterinary Medicine, University of specimens. Murcia, Spain. The kidney was plastinated at the Anatomy Laboratory, Department of Morphology, Federal Materials and Methods University of Espírito Santo, Brazil. For this study, samples of aorta, heart, and kidney of pigs The comparative and descriptive analysis of the were used. Four different treatments were used to histological sections was performed under a conventional investigate protocols for light microscopy (LM). Treatment light microscope using a Motic® BA 410 microscope, 1: the fragments were fixed in 10% formalin for 48 hours observing any changes in the morphological structure of at room temperature. For treatments 2, 3 and 4, samples the treatments compared to the control (treatment 1). The from specimens plastinated with Biodur® S10/S3 were histological images, (magnification 40X), were captured used. Treatment 2: plastinated fragments were directly as digital images using the program Motic Images Plus embedded in paraffin wax without previous de- 2.0 ML, in the laboratory of Reproduction of Small plastination. Treatment 3: plastinated fragments were de- Animals and Wild Animals (REPAAS), at the morphology plastinated by immersion in 99% ethyl alcohol for 24 sector of the Veterinary Department, Federal University of hours, and then in methylbenzene for 48 hours. Viçosa, Brazil. Treatment 4: plastinated samples were de-plastinated in 1,4-dimethylbenzene for 36 hours. Results The samples were processed for routine histological light It was found that it is possible to make histological microscopy (LM) in a tissue processor (Leica TP 1020), sections of plastinated material, although some difficulties including dehydration with ethyl alcohol in increasing were encountered, due to the rigidity of the plastinated concentrations from 70 to 100%; clearing in xylene; material, depending on the treatment employed. paraffin wax impregnation at 58° C, and embedding. Results are presented through the histology of each organ Serial 5 μm sections were cut with a microtome, mounted in turn. Sections obtained from treatments 2, 3 and 4 were on glass slides, and stained with hematoxylin-eosin (H & compared with the control, treatment 1. In treatment 1, E) or Verhoeff’s stain. constituent elements of the renal, cardiac and aortic The plastinated fragments used in this study came from tissues preserved their morphological characteristics and complete pig heart, aorta and kidney, plastinated using histoarchitecture. the standardized Biodur® S10/S3 silicone method, with
Table 1. Treatments and main histological characteristics of the kidneys
Treatments Renal Areas of Tubular Tubular Macula densa corpuscles distortion epithelium lumen morphology
Control Preserved Absent Normal Normal Evident
Biodur Preserved Present Normal Normal Evident
De-plastinated Preserved Present Irregular Reduced Less evident methylbenzene
De-plastinated 1,4- Preserved Present Irregular Reduced Less evident dimethylbenzene
12 – Lopes Ramos et al.
histological staining of renal tissue with H & E showed normal kidney structure, with renal corpuscles with a knot of capillaries (the glomerulus), surrounded by Bowman’s capsule (Fig 1). The tubules were observed with oval luminal morphology, and the epithelial cells showed MD MD eosinophilic cytoplasm, and central rounded nuclei (Fig. 2). The macula densa (MD) was observed in close A B proximity to the vascular pole of the renal corpuscle.
Treatments 2 and 3 revealed renal corpuscles with preserved Bowman’s space, with some distortion areas presenting silicone. Epithelial cells from the parietal layer of Bowman’s capsule were evident (Fig. 1). In treatment 2 the MD was observed in close proximity to the vascular pole, with tubular architecture preserved. In treatment 3, MD some renal tubules revealed reduced lumen, and irregular C D morphology of the tubular epithelium. In treatments 3 and Figure 1: Photomicrographs of the cortical region of pig 4, histological changes, such as some cells of the renal kidneys showing preserved renal corpuscles in all tubules with morphological alterations, reduction of the treatments and macula densa (MD). A, Control; B, tubular lumen, or capsular contour with distortion areas Plastinated; C, De-plastinated with methylbenzene; D, De- were also seen (Fig. 2). plastinated with 1,4-dimethylbenzene. H&E staining. 400X. The three experimental treatments showed preservation of the renal capsule, low affinity for hematoxylin staining, and small fragmented areas of tissue, just beneath the renal capsule.
Table 2. Treatments and main histological characteristics of the heart
Treatments Organization of Areas of Intercalated muscle bundles distortion discs A B
Control Preserved Absent Not observed
Biodur Lost Present Not observed
De-plastinated Lost Present Not observed methylbenzene
De-plastinated 1,4- Preserved Present Not observed dimethylbenzene C D Figure 2: Figure 2. Photomicrographs of the cortical region of pig kidney showing tubular cells presenting irregularities Table 2 lists the main changes observed in the in B and D that sometimes have loose epithelium in tubular histoarchitecture of the heart in all treatments. Histological lumen. A, Control; B, Plastinated; C, De-plastinated with examination of the heart sections showed clear methylbenzene; D, De-plastinated with 1,4- differences between treatment 1, the control, and the dimethylbenzene. H&E staining. 400X. Arrows indicate experimental treatments. Analysis of the myocardium in areas of distortion. treatment 1 showed cells with a single, centrally placed nucleus, well stained by hematoxylin, surrounded by Table 1 lists the main changes observed in the kidney histoarchitecture in all four treatments. In treatment 1,
De-plastination Methodologies for Preparing Histological Sections - 13 myofibrils, with blood vessels in the connective tissue. In vasorum in connective tissue was observed at random treatments 2 and 4, the central nuclei were evident in intervals. myocardial cells. Muscle bundles showed preserved architecture. Treatments 2 and 3 revealed partial loss of Table 3. Treatments and main histological characteristics of the aorta organization of the muscle bundles, and low affinity for H & E staining. In treatments 2, 3 and 4, the blood vessels Treatment Nucleus Layers of Vasa Areas of were not evident in the interstitium. In all of the endothelial elastic vasorum distortion treatments, transverse striations and intercalated discs cells membranes were seen (Fig. 3). In treatments 2, 3 and 4, areas of Control Evident Defined Present Absent distortion were seen in the slides. Slides from treatments 1 and 4 had similar staining with H & E, with both showing Biodur Little evidence Defined Not seen Present greater affinity for eosin. De-plastinated Little evidence Defined Not seen Present methylbenzene
De-plastinated Little evidence Defined Not seen Present 1,4- dimethylbenzene
A - Control B - Plastinated B
A - Control B - Plastinated
C - De-plastinated with D - De-plastinated with methylbenzene 1,4-dimethylbenzene Figure 3. Photomicrographs of left ventricular myocardium of pig, showing low affinity for H&E staining with plastinated fragments in B, and fragments after de- plastination with methylbenzene in C. A and D show standard staining with H&E. 200X. C - De-plastinated with D - – De-plastinated with methylbenzene 1,4 – dimethylbenzene Aorta Figure 4: Photomicrographs of tunica adventitia of the pig aorta showing vasa vasorum (vv) in A. Table 3 lists the main changes observed in the H&E staining. 400X. histoarchitecture of the aorta in all treatments. The histological structure of transverse sections in treatment 1 demonstrates the three layers that constitute the wall of the aorta. The tunica intima showed endothelium well stained by H & E (Fig. 4). The internal elastic membrane, however, was less obvious. The tunica media showed several layers of elastic membranes and smooth muscle cells. The elastic material was well evidenced by Verhoeff's stain (Fig. 5). The tunica adventitia appeared thinner than the tunica media, and the presence of vasa
14 – Lopes Ramos et al.
plastinated specimens. López-Albors et al. (2004)
plastinated tissue fragments with and without the curing process, and used de-plastination with sodium methoxide, according to the protocol of Walker et al. (1988). They found that the curing process influenced tissue preservation. However, the results we report here from cured samples, showed no disruption of the tissue architecture.
A - Control B – Plastinated Walker et al. (1988), describe de-plastination with sodium methoxide, which achieved good quality results. Others authors report the use of methylbenzene, methylene, and bichloride acetone for the same process (Ripani et al., 1988). However, our results in plastinated kidney samples without de-plastination (Treatment 2) showed a well- preserved histological structure of tubules and renal corpuscles. Moreover, the cells of the macula densa had a similar appearance to those from the control treatment
C – De-plastinated with D – De-plastinated with 1,4 (Treatment 1). Surprisingly, the results from de- methylbenzene – dimethylbenzene plastinated kidney samples had renal tubules with Figure 5: Photomicrographs of tunica intima of pig aorta reduced lumen and thin macular densa cells (Treatment with Verhoeff staining. 400X. 3), and the capsular space was increased in some renal corpuscles, probably due to shrinkage of the glomerular In Treatments 2, 3 and 4, the tunica intima was observed, capillaries (Treatment 4). These findings for Treatments 3 however, the endothelial nuclei were not distinct; in the and 4 agree with the results of Ripani et al. (1996), who tunica adventitia it was not possible to visualize the vasa reported lesions in the renal tubule epithelium and vasorum (Fig. 5). In Treatments 2 and 4, areas of Bowman's capsule, after de-plastination with distortion in the endothelium were observed. In Treatment methylbenzene. In this study, Treatments 2, 3 and 4 4, spaces between the elastic membranes in the tunica showed some areas of distortion in the heart specimens, media were observed. In all treatments, the elastic probably due to the rigidity of the material (we used laminae were preserved in the tunica media, with few fragments of the left ventricle), and also due to the spaces between them. This was observed in both the H & hardening effect of curing, which may have caused E and Verhoeff stains. difficulty in obtaining regular histological sections, resulting in differences in the thickness of the sections, Discussion and areas of distortion, in all three treatments. In this study, we analyzed by light microscopy how the Microscopic examination of the heart in Treatment 3 histoarchitecture of different organs (kidney, heart and showed areas that were not well preserved, with loss of aorta) was preserved after plastination. As in previous organization of muscle bundles. This problem probably published studies, we used a control (Treatment 1) to occurred due to the rigidity of the cured material. It was compare and validate the different methods used to not possible visualize blood capillaries and transverse process the plastinated tissue samples (Treatments 2, 3 striations in Treatments 2, 3, and 4. In addition, the slides and 4) (Dellmann and Brown, 1982; Young and Heath, had several areas of distortion, and the sections were not 2000, Gartner and Hiatt, 2003, Junqueira and Carneiro, uniform. It is likely that this occurred due to the resistance 2008, Ross and Pawlina, 2008). of the tissue during sectioning. Our findings corroborate Manjunatha et al. (2014) compared histological sections the results presented by Patil et al. (2016), in which clear of pig organs (liver, spleen and kidneys) embedded in striations in Biodur-infiltrated cardiac tissue could not be paraffin wax, with histological sections of uncured seen after de-plastination with methylbenzene. silicone-plastinated tissues. Both were stained with H & In our findings, the tunica intima and endothelium of the E. Using light microscopy, they found that the tissue aorta were mostly preserved in Treatments 2, 3 and 4. structure was maintained, without shrinkage, in the
De-plastination Methodologies for Preparing Histological Sections - 15
The nuclei showed poor hematoxylin affinity. The tunica INCA – INSTITUTO NACIONAL DO CÂNCER – Formol media, in Treatments 2, 3 and 4, was composed of ou Formaldeído 2005. In: smooth muscle cells interspersed with the large amount
References von Hagens G, Tiedemann K, Kriz W. 1987: The current potential of plastination. Anat Embryol (Berl) 175: 411- Dellmann H, Brown EM. 1982: Histologia Veterinária. Rio 421. de Janeiro: Guanabara Koogan, 397 p. Walker AN, Jackson RL, Powell, S. 1988: Technical Gartner LP, Hiatt, JL. 2003: Tratado de Histologia em communication: routine microscopy of deplastinated Cores. 2a ed. Rio de Janeiro: Guanabara Koogan, 458 p. tissue. J Int Soc Plastination 2: 40-42.
Hambeli AT, Lombardi M, Prochownik M. 2010: Técnicas Young B, Heath J. 2000: Histologia Funcional: texto e de conservación de piezas cadavéricas. Tercera Epoca: atlas em cores. 4a ed. Rio de Janeiro: Guanabara Rev Científica de la Facultad de Ciencias Médicas 2: 1-2. Koogan. 415 p. IARC - International Agency for Research on Cancer - Summaries & Evaluations, Formaldehyde, 1995. In :
The Journal of Plastination 30(1):16 - 23 (2018)
Biomechanical Analysis of The Skin And Jejunum Of Dog ANATOMICAL Cadavers Subjected To A New Anatomical Preservation TECHNIQUE Technique For Surgical Teaching
Rocha TASS1 ABSTRACT: Yanagihara GR2 Formaldehyde is a fixative and preservative widely used in anatomy laboratories, but it is Shimano AC2 harmful to health, and poses an environmental risk. Ethyl alcohol (EtOH) has also been Rolim GS3 used for effective fixation of bird muscles, and sodium chloride has been successfully Santos CCC1 tested for the preservation of anatomical parts for more than five years. The objective of Fechis ADS1 this present study was to evaluate a new anatomical technique for teaching surgical 1 Oliveira FS techniques using dog cadavers fixed in EtOH, and preserved in a 30% aqueous solution of sodium chloride (ASSC). In addition, we aimed to determine the ideal time to stop the 1Department of Animal fixation, so that the skin and jejunum present biomechanical characteristics as close as ANATOMICAL TECHNIQUE Morphology & Physiology, São Paulo possible to the control group of fresh animals. Five groups were used: a control group State University (Unesp), (fresh animals without fixation or conservation), and the other 4 groups which differed in School of Agricultural and the time of fixation in EtOH (30, 60, 90 and 120 days). Except for the controls, all groups Veterinarian Sciences, were conserved in 30% ASSC for 120 days. Statistical analysis of variance (ANOVA) Jaboticabal, São Paulo, revealed no difference between treatments and times (P > 0.05) relative to the skin, and Brazil showed at least one time significantly different from the others (P < 0.01) in relation to the 2 Department of jejunum. The non-linear modeling test showed differences in the group fixed in EtOH for Biomechanics, Medicine and Rehabilitation of 30 days, suggesting that this was the best time period for fixing dog cadavers for use in
Locomotor System, surgical training.
College of Medicine, University of São Paulo (Usp), Ribeirão Preto, Brazil 3Department of Exact Sciences, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil KEY WORDS: preservation, biomechanics, jejunum, dog
Correspondence to: Fabrício S Oliveira, Department of Animal Morphology and Physiology, São Paulo State University (UNESP), Via de Acesso Paulo Donato Castelane, Jaboticabal, SP, 14884-900, Brazil. Telephone: 551632097333, e-mail: [email protected] Introduction
Good conservation of anatomical specimens prevents Research on Cancer (IARC) classified formaldehyde as deterioration, and also prevents the proliferation of carcinogenic and teratogenic (IARC, 2006). Glycerin is a pathogens that can spread diseases to laboratory preservative with dehydrating and antiseptic properties, personnel (Corrêa, 2003). Formaldehyde is widely used preventing fungi and bacterial growth (Alvarenga, 1992). as an anatomical fixative and preservative, because it is It does not have harmful fumes such as those released by inexpensive and rapidly penetrates the tissues formaldehyde (Cury et al., 2013), but it is up to ten times (Rodrigues, 2010). However, it is hazardous to health, more expensive than formaldehyde (Krug et al., 2011). and can contaminate the environment through improper handling and through disposal of carcasses and effluent (WHO, 1991). In 2006, the International Agency for
Biomechanical Analysis of The Skin And Jejunum Of Dog Cadavers- 17
There are a number of other examples of fixative solutions The objective of this study was to determine the viability that are capable of preserving cadavers for surgical of a new anatomical technique using EtOH for fixation, training, among them Thiel solution (composed of boric and 30% ASSC for conservation; and to establish the best acid, ethylene glycol, potassium nitrate, time to stop fixation in EtOH for the optimal tissue (chloromethyl)phenol, sodium sulfate, and formalin) resistance in comparison to fresh cadavers without (Groscurth et al., 2001), Klotz's solution (sodium chloride, fixatives/preservatives. sodium bicarbonate, chloral hydrate, formalin and water) and Jores’ solution (distilled water, formaldehyde, sodium Materials and Methods: sulfate, potassium sulfate, sodium chloride, sodium The animals bicarbonate, glycerin, and sodium or potassium acetate) (Rodrigues, 2010), all of which contain formalin. Modified Forty (40) frozen, adult, dog cadavers, 14 male and 26 Larssen solution contains 100 ml of 10% formaldehyde, female were used. The dogs all died due to causes that 400 ml of glycerol, 200 g of chloral hydrate, 200 g of did not involve evident morphological alterations, such as sodium sulfate, 200 g of sodium bicarbonate, 180 g of large tumors, extensive lacerating traumas or bone sodium chloride, and 2 liters of distilled water (Silva et al. fractures. All were from the Zoonoses Control Center of al., 2004). The original Larssen solution did not contain Ribeirão Preto, São Paulo, Brazil, after approval from the liquid glycerin (Menezes, 2012). Laskowski solution Municipal Law Department (process 02.2014/ 000027-1). contains 800 ml of glycerine, 200 ml of ethanol, 50 g of The ages of the animals were determined from their death phenolic acid and 50 g of boric acid, and requires the certificates. preservation of cadavers at 0° C until they are used in surgery classes. Between classes, they must remain The selected cadavers had a mean body weight of frozen, and have to be thawed in water for 24 hours prior 7.6±2.7 kg, mean age 5.6±4.1 years, and body score of 4 to each class. With this solution, the tissues become to 5, which is considered ideal on a scale of 1 to 9 excessively dark (Silva et al., 2007). Of these, Larssen (Laflamme, 1997). They were thawed in a horizontal solution is considered the best solution for maintaining the refrigerator at 8° C, weighed, and then randomly original consistency, color and characteristics of the distributed into groups (Table 1). biological material, and can also remove blood clots (Sampaio, 1989). Table 1. Division of dog cadaver groups in relation to fixation time in ethyl alcohol (ETOH) and storage in a 30% sodium chloride solution (ASSC 30%). Control group Ethyl alcohol (96%) has been found to be efficient for consisting of cadavers of dogs not subjected to fixation or fixing and preserving the pectoral muscles of hens, conservation (fresh). although they became almost five times more rigid during the first six months, and three times more rigid after one Group Fixation Conservation year of immersion in the preservative agent (Nunes et al., (ETOH) (ASSC 30%) 2011). The use of a 30% aqueous solution of sodium Control (fresh cadavers) - - chloride (ASSC) in the preservation of anatomical 1 30 days 120 days specimens previously fixed by formaldehyde was 2 60 days 120 days successfully evaluated for 5 years, with no visual 3 90 days 120 days contamination, presence of putrefaction odors, or alteration of tissue color and softness (Oliveira, 2014). 4 120 days 120 days
There is a need to find a preservation technique that For fixation, 96% EtOH containing 5% glycerin was preserves the body realistically for a long time, serving not infused with a 60 ml plastic syringe, via the common only anatomy classes, but also surgical and clinical carotid artery, at a rate of 120 ml/kg. Each group studies, testing of new radiographic equipment, minimally consisted of eight animals that were fixed for different invasive surgery, and encouraging more scientific times (except for the control group). research to develop protocols that will assist anatomists in the preparation of preserved cadavers, in order to meet After the fixative was injected, running water was used to these demands for high quality parts (Balta et al., 2015). flush out surplus liquid accumulated in the cavities via two incisions, one in the thorax (between the fourth and fifth right intercostal space), and a median abdominal incision.
18 – Rocha, et al
The cadavers were then placed in plastic tanks (1 tank duodenojejunal flexure, the steel template was positioned per group) with a threaded cap (total capacity 310 liters – to delimit the specimen, which was then sectioned Fig. 1), containing 180 litres of EtOH. The abdomen and longitudinally with Metzenbaum scissors. Subsequently, thorax were washed with water for 5 consecutive days. section of the mesenteric edge was performed, exposing The plastic boxes were kept in an area and with abundant the lumen, under which the incision template was ventilation, away from sources of ignition, to avoid any risk positioned with a scalpel blade (Figure 3). of accident. Storage in 30% ASSC for 120 days followed the fixation period. Sodium chloride solution was placed in plastic tubs with a capacity of 310 liters, and the same volume (180 liters) was used for each fixation group.
Figure 2. Skin samples collected transversally to the dog's
Figure 1. Plastic box of 310 liters filled with 180 litres of skin tension line, on the lateral side of the thorax and using EtOH (A). Dogs cadavers kept during ethylic fixation (B). a steel mold, parallel to and 5 cm from the median plane after trichotomy.
Collection of Material
A 1 x 7cm stainless steel template was made for the collection of the skin and jejunum samples (chosen due to the higher tissue sample availability and frequent use in teaching of surgical techniques) during both the fixation in EtOH and 30% ASSC conservation solution. A total of 1,152 samples were taken.
The cadaver was initially placed in right lateral decubitus (left antimere, facing upwards). Using a scalpel (blade Figure 3. Jejunum sample collected with the cadaver number 23) and the template, three sequential, equally- positioned in the right lateral decubitus, after manual traction and using a steel mold. sized samples were collected transverse to the dog's skin tension line (Kirpensteijn and Haar, 2013), on the lateral side of the thorax, parallel to, and 5 cm from, the median Tissue Strength Analysis plane (Figure 2). Shaving had been performed throughout the thorax. Samples from the control group were also To evaluate tissue resistance, a Universal Testing collected in the left antimere and immediately submitted Machine (Instron®- EMIC® - DL2000) was used, with a to biomechanical testing. During the fixation phase in 500 N load cell and electromechanical drive support, with EtOH, samples were collected from the 4 groups in the a speed of 100 mm/min. Traction claws were also used left antimere and, during the conservation phase in 30% by manual compression, in the Laboratory of Surgical ASSC, from the right antimere. Anatomy of the Department of Morphology and Animal Physiology of FCAV - UNESP, Jaboticabal Campus, SP, For the collection of the jejunum samples, the animals Brazil (Figure 4). Tensile testing was conducted up to the were positioned in the right lateral decubitus, exteriorizing point of rupture of the skin and jejunum samples, the jejunum by manual traction. After identification of the obtaining the values of the maximum force applied, in N.
Analysis of The Skin and Jejunum of Dog Cadavers - 19
An ANOVA test was also performed to verify the differences between the adjusted parameters in the different treatments.
Results
The preservation technique employed proved to be efficient for the fixation and conservation of the animals throughout the experiment. During the storage period in the plastic boxes in ASSC 30%, there was a gradual release of fat from cadavers in groups 3 and 4 (fixed 90 and 120 days in EtOH respectively). Because they remained longer in the alcohol fixative, they were more greasy and viscous at the end of storage in 30% ASSC, making it difficult to manipulate them during sample collection. During fixation in EtOH, stiffening of the skin and jejunum was observed when handling the samples, Figure 4. Universal testing machine (A) for biomechanical and later, there was an increase of the tissue malleability analysis. A dog´s skin sample during a traction test (B). during the period of conservation in 30% ASSC. At the end of the period of alcohol fixation, the alcohol
concentration ranged from 80 to 83%, which is an Statistical Analysis excellent amount of alcohol in the solution, and an appropriate degree of fixation of the cadaver for The data for the maximum force for the rupture of skin and placement in the 30% ASSC for 120 days. jejunal tissues at different times and treatments were analyzed by analysis of variance (ANOVA) with Table 2 - Absolute mean (± SD) of the maximum strength of rupture (N), of skin samples of groups 1, 2, 3 and 4, significance set at 0.05. submitted to different fixation times in ethyl alcohol and preservation in an aqueous solution of sodium chloride. For a more detailed analysis of the maximum jejunum breaking point as a function of time, nonlinear models Time G1 G2 G3 G4 were fitted in exponential form by the ordinary least 131.3 ± 131.3 ± 131.3 ± 131.3 ± squares methodology, according to the following 0 75.6 75.6 75.6 75.6 equations: 152.6 ± 131.1 ± 177.5 ± 110.8 ± 1 −(푋−푋표)/푡1 푌 = 푌표 + 퐴1 × 푒 71.9 53.6 86.6 56.7 139.3 ± 119.1 ± 148.7 ± 118.4 ± 2 푌푒푠푡 − 푌표푏푠 55.0 52.2 83.3 65.9 ∑푁 (| 푖 푖| × 100) 푖=1 푌표푏푠 130.3 ± 106.6 ± 148.7 ± 121.5 ± 푀퐴푃퐸 = 푖 , 3 푁 54.1 51.0 71.9 78.8 148.7 ± 145.6 ± 116.4 ± 110.3 ± 4 63.1 69.6 60.9 64.0 115.1 ± 112.3 ± 112.3 ± 132.4 ± where Y is the maximum force (N), Yo is the mean in the 5 53.5 78.3 60.3 65.5 stabilization of the maximum force (N) at the final times of the treatment, A1 is the weighted amplitude of the values Time: 0 (pre-fixation analysis); 1 (30, 60, 90 and 120 days of Y, Xo is the time of greatest decay rate of the maximum of fixation in ethyl alcohol, for groups 1, 2, 3 and 4, respectively); 2 (30 days 30% ASSC); 3 (60 days 30% force (time), t1 is the mean decay rate (N time-1), and X ASSC); 4 (90 days 30% ASSC) and 5 (120 days 30% ASSC). is the measured time. MAPE is the mean percentage error, Yesti is the estimated value of maximum force at From the general data of the maximum rupture forces of time i, Yobsi is the observed value of maximum force at the skin and jejunum samples submitted to the time i and N is the number of data. biomechanical tensile test, the absolute mean and
20 – Rocha, et al
respective standard deviation of groups 1, 2, 3 and 4 were there were no differences between treatments (G1 to G4) obtained at different times of fixation in EtOH, and (Table 5). conservation in 30% ASSC (Tables 2 and 3). The maximum force required to rupture the skin and jejunum Table 5 - Analysis of variance (ANOVA) between samples (at all times) ranged from 106.7 N to 177.5 N treatments and times for jejunum samples from dog cadavers fixed for different times in ethyl alcohol and (mean 142.1 N) and from 12.9 N to 27.6 N (mean 20.2 N), preserved in a 30% aqueous solution of sodium chloride respectively. for 120 days. There was at least one different time in the treatments (P < 0.01). Table 3 - Absolute mean (± SD) of the maximum force of rupture (N) of jejunum samples of groups 1, 2, 3, and 4 Source of variation SS DF MS F P F critical submitted to different fixation times in ethyl alcohol and preservation in an aqueous solution of sodium chloride at Times 2,392.40 5 478.48 5.45 30% (30% ASSC). Treatments 273.064 3 91.021 1.037 Time G1 G2 G3 G4 0 27.6 ± 27.6 27.6 ± 27.6 ± Interactions 559.24 15 37.28 0.42 1 14.017.5 ± 5.1 14.8±17.5 ± 6.8 18.517.5 ± 9.7 18.317.5 ± 6.1 Total 17,969.80 191 2 15.4 ± 8.9 19.0 ± 20.3 ± 18.0 ± 11.9 11.4 13.0 3 18.9 ± 9.3 20.3 ± 19.6 ± 21.1 ± SS: sum of squares; DF: degrees of freedom; MS: mean 4 20.5 ± 9.3 21.711.7 ± 20.110.6 ± 10 18.417.6 ± square; F: ratio between the model and its error; P: significance level; F critical. 5 12.9 ± 18.312.4 ± 19.7 ± 8.9 24.316.0 ± 16.0 11.6 15.5 Time: 0 (analysis prior to fixation); 1 (30. 60. 90 and 120 As a different time was identified in the statistical analysis days of fixation in ethyl alcohol. for groups 1. 2. 3 and 4. of the jejunum, nonlinear modelling was performed, respectively); 2 (30 days 30% ASSC); 3 (60 days 30% aiming to determine the time. In the modelling, the ASSC); 4 (90 days 30% ASSC) and 5 (120 days 30% ASSC). exponential decay model (equation 1) explained the variation of the maximum force for rupture of jejunum Table 4 - Analysis of variance (ANOVA) between samples as a function of the different times in the treatments and time for skin samples from dog cadavers fixed at different times in ethyl alcohol and preserved in a treatments (Figure 5), since general MAPE was 9.6%. All 30% aqueous solution of sodium chloride for 120 days. values of Yo between the models were statistically similar, There was no difference between treatments (P > 0.05). indicating that the mean maximum strength at the final times of the treatments were equal, thus confirming the Source of SS DF MS F P F variation critical efficiency of the 30% sodium chloride solution as a preservative. All other adjusted parameters showed at Times 5,676.82 5 1,135.36 0.24 0.94 2.27 least one difference between the treatments, indicating that the response of the treatments varied in relation to Treatments 9,460.00 3 3,153.33 0.68 0.57 2.66 the mean amplitude (A1), the time of the highest decay Interactions 3,8855.60 15 2,590.37 0.56 0.90 1.73 rate (Xo), and the decay rate (T1), to the maximum force values. Total 836,112.50 191 In the jejunum samples, a gradual decay of the values *SS: sum of squares; DF: degrees of freedom; MS: mean square; F: ratio between the model and its error; P: was observed for the maximum rupture force until the final significance level; F critical. fixation time in ethyl alcohol and a later stabilization of the maximum force for up to 120 days, thus confirming the For the skin samples, ANOVA showed that there were no preservative effect of the 30% ASSC for this type of differences between treatments and times (Table 4), tissue. Thus, in relation to jejunum, G1 presented A1 and because the P value was always higher than 0.05. The Xo differently from the other groups, as this is the time of ANOVA test also indicated that there were no interactions significance (Table 6). between the treatments and times. For the jejunum samples, ANOVA showed that at least one time was significantly different from the others (p <0.01) and that
Analysis of The Skin and Jejunum of Dog Cadavers - 21
greatest decay rate of maximum force (N); T1: average 28 decay rate (N time-1). 26 G1 Discussion 24 G2 22 Ethyl alcohol was effective as a fixative of dog cadavers, G3 providing good conservation, and avoiding deterioration 20 G4 of the material, per Rodrigues (2010). The method 18 described here is similar to the study involving alcohols G1est 16 used to fix human cadavers for 6 months to 1 year, which G2est left the tissue quality like that of fresh tissue (Goyri-O’Neill 14 G3est et al., 2013).
Force for the rupture of the jejunum samples (N) 12 0 2 4 6 G4est The 30% ASSC solution was extremely effective in the Moments preservation of the fixed tissues, as described by Oliveira (2014). There was no apparent contamination similar to Figure 5. Nonlinear models of exponential decay relating that described in canine pericardium preserved for a to the maximum force as a function of different treatment minimum period of 90 days in hyper-saturated sodium times and different fixation times in ethyl alcohol and chloride solution (Brun et al., 2002), and that described for preservation for 120 days in a solution of 30% sodium chloride. Note the stabilization of the values during the canine phrenic center preserved in glycerin (Brun et storage in saline solution. al., 2004). The 30% ASSC may be successful because of the difficulty of survival for microorganisms in a medium G1 (30 days in alcohol), G2 (60 days in alcohol), that requires an enormous osmoregulation capacity, G3 (90 days in alcohol), and G4 (120 days in alcohol). M1: final moment of fixation; M2: after 30 days preserved similarly to the oceans (Munro et al., 1989), and the Dead in saline solution; M3: after 60 days preserved in saline Sea (Nissenbaum, 1975). Several concentrations of solution; M4: after 90 days preserved in saline solution; fixatives and preservatives have been evaluated for M5: after 120 days preserved in saline solution. preservation of anatomical specimens, but the use of a sodium chloride solution below 20% has failed to preserve Table 6 - Adjusted values of the parameters of the parts for use in tissue dissection (Friker et al., 2007). exponential non-linear decay model. The general model Statistical modeling analysis evidenced the stability of the indicates the fit for all treatments together. The letters in data in this study, using 30% ASSC as a tissue superscript indicate the ANOVA test verifies at least one difference of the parameters between treatments of preservative for skin and jejunal samples for up to 120 different fixation times in ethyl alcohol and preservation days. for 120 days in a 30% aqueous solution of sodium chloride. G1 (30 days in alcohol), G2 (60 days in alcohol), There was no generation of contaminated effluent, G3 (90 days in alcohol) and G4 (120 days in alcohol). commonly observed when toxic preservatives are used Parameters (WHO, 1991), nor any health-damaging fumes, such as Model MAPE those released by formaldehyde (Cury et al., 2013). In Yo A1 Xo T1 addition, the appropriate waste management of formaldehyde is costly in both financial and environmental Geral 9.6 % 18.748a 1.916a.b 0.122a.b 0.079a.b terms, requiring the search for low-cost and non-risky alternatives (Janczyk et al., 2011). G1 16.7 % 16.393a 1.984a 0.129a 0.074b
G2 10.3 % 18.847a 1.703b 0.098b 0.060a Cadavers prepared for anatomical dissection were described as having better quality when a mid-line G3 2.5 % 19.681a 1.824b 0.111b 0.075b abdominal incision was performed (to enable the preservative to enter the abdominal cavity, to improve a b b b G4 10.5 % 20.078 1.804 0.111 0.077 perfusion of the abdominal tissues) as was done in this MAPE: mean percentage error; Yo: mean in the study, instead of not opening the abdominal cavity stabilization of the maximum force in the final times of the (Janczyk et al., 2011). treatment (N); A1: amplitude of the values of Y; Xo: time of
22 – Rocha, et al
Tissues become stiffer with formaldehyde, which, in 2002: Solução hipersaturada de sal como conservante biomechanical shear tests, caused a considerable de pericárdio canino utilizado na reparação do músculo stiffening in chicken breasts fixed for up to one year (4.4 reto abdominal de ratos Wistar. Ciên Rur 32: 1019- to 5 times higher) (Guastalli et al., 2012). When EtOH was 1025. used as fixative, tissue stiffness increased, making them almost five times more lilely to rupture during the first six Brun MV, Pippi NL, Driemeier D, Contesini EA, Beck months, and three times more likely after one year of CAC, Cunha O, Pinto Filho SL, Roehsig C, Stedile R, immersion (Nunes et al., 2011). However, in this study, Silva TF. 2004: Solução hipersaturada de sal ou de the maximum tensile strength in the skin traction test glicerina a 98% como conservantes de centros frênicos (mean of 142.1 N) and jejunum (mean of 20.2 N) did not caninos utilizados na reparação de defeitos musculares show a great variation in relation to the maximum strength em ratos Wistar. Ciên Rur 34: 147-153. of skin (131.3 ± 75.6 N) and jejunum (27.6 ± 17.5 N) of Corrêa WR. 2003: Isolation and identification of the unfixed cadaver control group. filamentous fungi found in anatomical pieces preserved Conventional procedures for fixing cadavers using in 10% formalin solution. Dissertation (Biological formaldehyde are of limited use for surgical practise, due Sciences). Institute of Research and Development, to the profound alteration in the staining, resistance, and University of the Valley of Paraíba. 59p. fragility of organs and tissues. Artificial anatomical models Cury FS, Censoni JB, Ambrósio CE. 2013: Técnicas are an alternative, and can be used mutiple times anatômicas no ensino da prática de anatomia animal. (Groscurth et al., 2001). However, with the method Pesq Vet Bras 5: 688-696. described here, each cadaver, prepared without the use of formaldehyde, can be used for training in surgical Friker J, Zeiler E, McDaniel BJ. 2007: From formalin to techniques for an entire semester, with life-like softness salt. Development and introduction on a salt-based and tissue malleability. preserving solution for macroscopic anatomic specimens. Tierärztl. Praxis 35: 243–248. ANOVA showed that there were no differences between treatments and times (P > 0.05 in all cases). Thus, among Guastalli BHL, Nunes TC, Gamón THM, Carmo LG, Del the evaluated time periods (30, 60, 90 or 120 days), there Quiqui EM, Oliveira FS. 2012: Análise da textura de is no specific time that is better than others. Therefore, we músculos submetidos à fixação em formaldeído e suggest the shortest time (30 days - G1) for the conservação em benzoato de sódio 0,5% e ácido preparation of the dog cadavers, because, due to the acético 0,5%. Acta SciVet 40: 1041. rapidity of preparation and the lower occupational risk that it confers, it is the best for cutaneous surgery. Goyri-O’Neill J, Pais D, Freire De Andrade F, Ribeiro P, Belo A, O’Neill A, Ramos S, Neves Marques C. 2013: Acknowledgement: FAPESP, process 2015/08259-9. Improvement of the embalming perfusion method: The innovation and the results by light and scanning electron REFERENCES microscopy. Acta Med Port 26: 188-194. Alvarenga J. 1992: Possibilidades e limitações da Groscurth P, Eggli P, Kapfhammer J, Rager GJ, utilização de membranas biológicas preservadas em Hornung P, Fasel JDH. 2001: Gross anatomy in the cirurgia. In: Daleck CR (editor). Tópicos em cirurgia de surgical curriculum in Switzerland: improved cadaver cães e gatos. Jaboticabal: Fundação de Estudos e preservation, anatomical models, and course Pesquisas em Agronomia da Universidade Estadual development. Anat Rec 265: 254–256. Paulista. p 33-39. IARC (International Agency for Research on Cancer). Balta JY, Cronin M, Cryan JF, O´Mahony SM. 2015: 2006: Formaldehyde, 2-butoxyethanol and 1- Human preservation techniques in anatomy: a 21st tertbutoxypropan-2-ol. IARC Monogr Eval Carcinog century medical education perspective. Clin Anat 28: Risks Hum 88: 1–478. 725-734. Janczyk P, Weignera P, Luebke-Beckerb A, Brun MV, Pippi NL, Dreimeier D, Contesini EA, Beck Kaessmeyera S, Plendla J. 2011: Nitrite pickling salt as CAC, Cunha O, Pinto Filho SL, Roehsig C, Stedile R. an alternative to formaldehyde for embalming in
Analysis of The Skin and Jejunum of Dog Cadavers - 23 veterinary anatomy: a study based on histo- and Silva RMG, Matera JM, Ribeiro AACM. 2007: New microbiological analyses. Annals Anat 193: 71–75. alternative methods to teach surgical techniques for veterinary medicine students despite the absence of Kirpensteijn J, Haar G. 2013: Reconstructive Surgery living animals. Is that an academic paradox? Anat Histol and Wound Management of the Dog and Cat. Manson Embryol 36: 220–224. Publishing Ltd. London UK. p.12. WHO 1991: World Health Organization - IPCS Krug L, Pappen F, Zimmermann F, Dezen D, Rauber L, International Programme on Chemical Safety – Semmelmann C, Roman LI, Barreta MH. 2011: Formaldehyde - Health and Safety Guide. n. 57. Conservação de Peças Anatômicas com Glicerina Available at
Laflamme DP. 1997: Development and validation of a body condition score system for dogs. Can Pract 22: 10- 15.
Menezes CLM. 2012: Preservation of rabbit cadaver with modified Larssen´s solution for training in videolaparoscopic surgery. Federal University of Rio Grande do Sul: Brazil. Dissertation of Veterinary Sciences. 82p.
Munro PM, Gauthier MJ, Breittmayer VA. 1989: Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater. Appl Environ Microbiol 55: 2017-2024.
Nunes TC, Oliveira FS, Gamon THM, Guastalli BHL, Carmo LG, Del Quiqui EM. 2011: Análise da textura de músculos peitorais submetidos á fixação e conservação em álcool. Braz J Vet Res An Sci 48: 464-467.
Nissenbaum A. 1975: The microbiology and biogeochemistry of the Dead Sea. Microbiol Ecology 2: 139-161.
Oliveira FS. 2014: Assessing the effectiveness of 30% sodium chloride aqueous solution for the preservation of fixed anatomical specimens: a 5-year follow-up study. J Anat 225: 118-121.
Rodrigues H. 2010: Técnicas Anatômicas. Vitória-ES, Brazil: GM Gráfica & Editora.
Sampaio FJB. 1989: Study of the human kidney growth during fetal life. Thesis (Doctorate of Morphology). São Paulo State Medicine College, Brazil.
Silva RMG, Matera JM, Ribeiro AACM. 2004: Preservation of cadavers for surgical technique training. Vet Surg 33: 606–608.
The Journal of Plastination 30(1):24 - 26 (2018)
Bleaching of specimens before dehydration in TECHNICAL plastination: a small-scale pilot study using human REPORT intestine
Jie-Ru Chen1 ABSTRACT: Hong-Jin Sui1,2
TECHNICAL Objective: The aim of this study was to explore the factors that influence bleaching of 1Department of specimens prior to plastination. Anatomy, Dalian Medical University, No.9 West Materials and Methods: Four sections of formalin-fixed human intestines were divided Section, Lushun South into two groups, to compare the effects of hydrogen peroxide concentration (5% and 10%) REPORT Road, Dalian 116044, China and temperature (20 °C and 30 °C) on the effectiveness of bleaching.
2Dalian Hoffen Bio- Results: In the first group, a high concentration of bleach appeared to make a better
technique Co. Ltd., No.36, appearance. In the second group, a higher temperature gave a better appearance. Guangyuan Street, Lushunkou Economic Conclusion: A high concentration of bleach and temperature can both lead to a better Department Zone, Dalian appearance of the specimen. 116052, China
KEY WORDS: Bleaching, intestine, plastination
Correspondence to: *[email protected]
Introduction bath. In this study, we investigated the effects of hydrogen peroxide concentration and temperature on samples of Plastination is a technology that can preserve biological human intestines. tissues in a life-like state for long-term preservation without unpleasant odors (Weber et al., 2007); as a result, it has now become more and more popular both for medical/veterinary teaching, and public exhibitions. Materials and Methods Plastination technology is a technique purely for Four sections of formalin-fixed human intestines were preservation, so in order to create a good specimen, it is used in this experiment. The samples were divided into vital to start with a high quality dissection. This is because two groups (Fig. 1); one group (Group 1) was used to plastination cannot make the specimen better if the quality study the effect of concentration of hydrogen peroxide of the dissection is not good enough in the first place, and on the color of the specimen, while the other group a good specimen requires a clear structure and a natural (Group 2) was used to investigate the effect of color (Smodlaka et al., 2005). Color is a very important temperature. factor in a good specimen, because it can make the surface of the specimen more natural, and improve the In Group 1, investigating the effect of hydrogen peroxide appearance of the specimen. It can also give a better concentration, two lengths of intestine were placed into experience for the person who is using the plastinated separate baths of hydrogen peroxide at 24° C for 24 specimen. Bleaching is a key technology, and is very hours. The baths contained 5% and 10% hydrogen important in enhancing the color, as it can make the color peroxide, respectively. brighter (Sui and Henry, 2007). Hydrogen peroxide solution is usually used as the bleaching agent; the In Group 2, the effect of temperature on the bleaching specimen is bleached for 2-5 days at a concentration of process was investigated. Two lengths of intestine were 10%, at a temperature of 24 °C in the hydrogen peroxide
Bleaching of Specimens Before Dehydration - 25 placed into separate baths of 5% hydrogen peroxide at Discussion 20° C and 30° C, respectively, for 24 hours. The color of an anatomical specimen is very important to the people who use it, whether it is used in medical teaching or in popular exhibitions. A good color in a plastinate means that, technically, a better result has been achieved. In plastination, bleaching is an important step before dehydration, in which the color of the specimen can be changed to enhance the appearance of the specimen. From our own experience, we use hydrogen peroxide bleaching solution of sufficient depth to completely cover the specimen (Gao, et al., 2006). The specimen is bleached for 2-5 days in a 10% hydrogen peroxide bath, at a temperature of 24 °C. During the Figure 1. The four intestinal canals were dissected, and divided into two groups prior to bleaching. bleaching process, the specimen should be inspected regularly, until the specimen has turned white or pink. Results There are three factors that can affect the bleaching In Group 1 (Fig. 2), we found that the intestine segment process: the concentration of the bleach, the temperature, which was bleached in 10% hydrogen peroxide had a and, in our experience, sunlight can also be a factor. better appearance, and brighter color, than the other Findings from the small-scale study reported here, specimen, which was bleached in 5% concentration. In confirmed that when the concentration of bleach and the Group 2 (Fig. 3), the intestine segment which was placed ambient temperature are high (20%, and 30 °C, in 5% hydrogen peroxide at 30° C had a better respectively) the bleaching is fast and effective. We have appearance than the specimen that was bleached at also found, through our own practical experience, that 20°C. with exposure to sunlight, the speed of bleaching is faster. However, for those who are new to bleaching, it is advised to use low concentration and low temperature, and a longer period in the bleach solution. This is because the contrast of the tissue may be very low if the bleaching is too fast, and is not stopped in time.
Conclusion
Increasing the concentration or the temperature of the Figure 2. Group 1. The two intestine specimens after hydrogen peroxide during the bleaching process can bleaching at 24° C for 24 hours in 5% hydrogen peroxide enhance the appearance of anatomical specimens. (left) and 10% hydrogen peroxide (right). However, this is not advised for inexperienced plastinators.
References
Gao H, Liu J, Yu S, Sui HJ. 2006: A new polyester technique for sheet plastination. J Int Soc Plastination 21:7-10
Smodlaka H, Latorre R, Reed RB, Gil F, Ramirez R, Figure 3. Group 2. The two intestine specimens after Vaquez-Auton JM, Lopez-Albors O, Ayala MD, Orenes M, bleaching for 24 hours in 5% hydrogen peroxide at 20 °C (left), and at 30 °C (right). Cuellar R, Henry RW. 2005: Surface detail comparison of
26 – Sui, Chen specimens impregnated using six current plastination regimens. J Int Soc Plastination 20:20-30
Sui HJ, Henry RW. 2007: Polyester plastination of biological tissue: Hoffen P45 technique. J Int Soc Plastination 22:78-81
Weber W, Weiglein A, Latorre R, Henry RW. 2007: Polyester plastination of biological tissue: P35 technique. J Int Soc Plastination 22:50-58
The Journal of Plastination 30 (1): 27-36 (2018) TECHNICAL General Issues of Safety in Plastination REPORT
Schill VK ABSTRACT: When people intend to start plastination at their institute, they are sometimes unaware of the scope of equipment, auxiliaries and chemicals they need. They may be even less BIODUR® Products aware of the potential hazards which arise from plastination. Certain chemicals may pose GmbH, Im Bosseldorn 17, acute or chronic health hazards. Acetone, which is mostly used for dehydration and 69126 Heidelberg, defatting, is a flammable liquid and therefore brings about fire and explosion hazards.
Germany In this paper, information about the characteristics of some commonly used chemicals in TECHNICAL plastination is provided. Suitable personal protective equipment must be used to allow for safe working when handling these substances. For chemicals posing an inhalation hazard, technical room ventilation or workplace ventilation is required to keep the
concentration of hazardous vapours below their respective workplace concentration REPORT limits. If ventilation is not sufficient, respiratory protection must be worn. Avoiding the risk of fire and explosion caused by handling of acetone or other flammable
liquids is achieved by a combination of measures: Proper laboratory furnishings (ventilation system, electric installations, etc.) are of importance as well as the design of the equipment used for plastination. Depending on the result of the local risk assessment, some appliances like solvent pumps or fans should be designed to be explosion-proof. Organisational protective measures support the technical measures in order to enhance occupational safety. Here, proper instruction of staff is of particular importance..
KEY WORDS: plastination equipment; inhalation hazard; explosion hazard; explosion protection; occupational safety
Correspondence to: V. Schill, telephone: +49 6221 331165; Fax: +49 6221 331112; Email: [email protected]
Introduction thus, the following information should be considered as a subjective selection, based on several years’ experience The technique of plastination, invented by Gunther von in plastination work and on a number of visits to Hagens in 1977, offers the opportunity for scientists to laboratories in different countries, without any claim to be produce durable preparations in their own labs (von exhaustive. Hagens et al., 1987). While plastinated specimens as final products are non-hazardous, the handling of solvents and Hazard types some other specific chemicals during the production process requires (1) awareness of their hazards and (2) In plastination, we may be confronted with hazards of the some technical, organisational, and personal protective following types: measures to achieve work safety. In the following article, -Health hazards (acute toxicity, sensitisation, the main potential hazards related to plastination work, as carcinogenicity, etc.) well as recommended measures to effectively avoid explosion and health hazards are discussed. -Physical hazards (explosion hazard, mechanical hazards, etc.) Important note: work safety is a wide subject where international, national, and regional regulations must be -Biohazard (when handling fresh, unfixed specimens) observed. This paper naturally can’t cover all regulations,
28 - Schill -Environmental hazard (in case chemicals are accidently Health hazards released) When discussing health hazards, we commonly Biohazard and environmental hazard are related to distinguish three different routes of exposure: skin/eye plastination only in a wider context. Biohazard may occur contact, ingestion, and inhalation. Of these, inhalation is when handling fresh tissue, i.e. before fixation, of major importance when working in plastination. embalming, or transfer into the dehydration bath. Environmental hazards mainly occur where chemicals How can we find out if a chemical poses an inhalation hazardous to the environment are accidently released, hazard? The first approach is having a look at the product and enter the soil or water system. Health and explosion label on the container and/or at the SDS. While the hazard hazards are more closely associated with plastination pictograms alone are often not very clear in their meaning, work, and will therefore be discussed in more detail below. the H-statements are more informative. For example, the H-statements indicating acute toxicity through inhalation Gathering information are very clear. They are, in decreasing severity: H330 “Fatal if inhaled,” H331 “Toxic if inhaled,” H332 “Harmful Information about chemicals presenting health hazards or if inhaled,” and H333 “May be harmful if inhaled”. (Note: explosion hazards can be found H333 is included in the United Nations’ Globally Harmonized System of Classification and Labelling of • on the product container’s label. Chemicals [“GHS”; United Nations, 2017], Annex 3, but • in the Safety Data Sheet (SDS). has not been adopted e.g. by the European CLP o In Europe, all SDS have a standardised regulation.) structure of 16 sections according to the regulation no. 1907/2006 (“REACh”; The Besides the H-statements related to acute toxicity, there European Parliament and the Council of the are numerous others that are, or can be, related to European Union, 2006), and regulation (EU) no. inhalation hazard. Among these are: 2015/830 (The European Commission, 2015), respectively. Information about classification -H334 “May cause allergy or asthma symptoms or and labelling of the product is found in section 2; breathing difficulties if inhaled,” indicating the risk of physical properties are given in section 9. respiratory sensitisation. • in national or international chemicals inventories, e.g. -H335 “May cause respiratory irritation,” indicating a the “Classification and Labelling Inventory of the specific target organ toxicity after single exposure. European Chemicals Agency” which can be found at https://echa.europa.eu/information-on-chemicals/cl- -H372 “Causes damage to [state organ] [state route inventory-database . of exposure],” indicating a specific target organ toxicity after repeated exposure. The route of While information about specialised chemicals is usually exposure, e.g. “through exposure by inhalation,” may (and sometimes exclusively) found in the safety data be given only if other routes of exposure can be sheets provided by the suppliers, chemical inventories are reliably excluded. a good source to review properties of basic substances like solvents. Commonly used solvents for dehydration like acetone or isopropanol either have H335 (“May cause respiratory Minimum information on a chemical product’s hazards irritation”) or H336 (“May cause drowsiness or dizziness”). includes one or more hazard pictograms, hazard BIODUR® gas cure S 6 has H332 (“Harmful if inhaled”). statements (in short “H-statements”, e.g. H225 “Highly H372 applies to all styrene-containing polyester resins flammable liquid and vapour”), and a signal word that are particularly used for plastination of brain slices, (“Warning” or “Danger”). Precautionary statements (“P- with the wording “Causes damage to hearing organs statements”) indicate measures that should be taken in through prolonged or repeated exposure”. order to prevent exposure to the chemical (e.g. P284 “In case of inadequate ventilation wear respiratory Another approach (besides looking at the H-statements) protection”) or to respond to an exposure (e.g. P312 “Call that can also be of help, is to get an idea about the a POISON CENTER or doctor if you feel unwell”). importance of avoiding inhalation: in the majority of the SDS, very often in section 9, you can find information
General Issues of Safety in Plastination - 29 about the vapour pressure of the chemical. This value is usually given for a temperature of 20° C, therefore you can easily compare different chemicals. Acetone, for instance, has a vapour pressure of nearly 250 hPa while ethanol’s is 58 hPa (Table 1). These values indicate that inhalation hazard has to be taken into consideration. On the other hand, unsurprisingly, for a product like the hardener BIODUR® S3 with a vapour pressure of far less than 1 hPa, inhalation hazard is not of relevance under Figure 1. Examples of personal protective equipment (PPE) to avoid exposure to chemicals: protective gloves normal working conditions. made of butyl rubber (left), disposable apron (middle), full face piece respirator (right). While it is comparably easy to protect one’s skin and eyes by wearing suitable safety goggles, protective gloves, arm sleeves, etc., we have to consider several aspects when Physical Hazards we strive to avoid inhalation of hazardous vapours. Depending on the chemicals we work with and the scale Physical factors leading to a potential hazard can arise, of the plastination unit, we have to decide which technical for example when a safety glass plate as part of a and organisational measures are to be taken, alongside plastination kettle bursts, leading to mechanical impact the use of personal protective equipment. As the caused by small fragments (Fig. 2). Therefore, it is atmosphere holding vapours hazardous to health usually important to inspect such glass plates from time to time is identical to the one that poses an explosion hazard, for damage, especially the edges. Damaged glass plates technical and organisational protective measures will be should be replaced with new ones. discussed below, under the heading “Explosion Hazard”. Working in cold temperatures carries the risk of cryogenic burns caused by low-temperature impact (Fig. 2). Wearing insulated gloves or chemical protective gloves Personal Protective Equipment (PPE) with cotton gloves underneath avoids this risk.
Hand/skin protection: protective gloves should be mandatory whenever one works in the plastination lab. Disposable nitrile or latex gloves provide only minor protection against solvents like acetone, though they are most commonly used in laboratories. They are most suited for protecting the hands in situations where there is a risk of minor splashing. Whenever intensive contact with solvent is expected and reliable protection against Figure 2. Physical hazards: when damaged, a safety glass permeation is required, one should choose a high-quality plate shatters into numerous small pieces (left). Working glove material like butyl rubber (IFA, 2017). When in the cold with unprotected hands bears the risk of handling larger amounts of chemicals, disposable sleeve cryogenic burns (right). protectors and aprons made of polyethylene are a comparatively inexpensive and effective solution (Fig. 1). Explosion Hazard Respiratory protection: if technical room ventilation is not available, or is not powerful enough to keep the The most severe hazard in plastination is explosion, concentration of hazardous vapours below the locally caused by the presence of vapour of a flammable liquid in prescribed limits, protective masks should be worn (Fig. the ambient air at a concentration within its lower and 1). Manufacturers of respirators offer full face masks as upper explosion limits (Fig. 3). If the temperature is above, well as half-masks. Combination filters, which absorb or near the flash point (see appendix) of this liquid, any different kinds of vapours, render the purchase of several source of ignition able to set free enough energy (inside filter cartridges superfluous. or in proximity to the air-vapour-mixture) will ignite this hazardous atmosphere, thus causing an explosion. The
occurrence of such situations during work procedures
30 - Schill must be identified and considered in a risk assessment. formation of a hazardous atmosphere, and the spatial Areas where an explosion hazard may occur should be expansion of this atmosphere, must be considered when defined. They are often named “hazard zones”. determining the explosion hazard in the plastination lab.
Classification of flammable liquids into categories
The hazardous potential of different flammable liquids varies markedly. In the UN “Globally Harmonized System of Classification and Labelling of Chemicals” (GHS; United Nations, 2017), flammable liquids are classified into category 1, 2, or 3, according to their respective flash points and boiling points (Table 2).
Category 1 represents the most severe hazard. It is linked to the H-statement H224 “Extremely flammable liquid and
Fig. 3: Potential explosion hazard occurs when the vapour”. If we look at some solvents commonly used in concentration of vapour in the surrounding air ranges dehydration we find that they all fall into category 2 with between the lower explosion limit (LEL) and the upper H225 “Highly flammable liquid and vapour” (Table 1). explosion limit (UEL). Table 1: Some characteristics of solvents used for Measures to improve work safety consist of steps to dehydration (source: “GESTIS” database of the preferentially avoid the formation of a hazardous air- German Statutory Accident Insurance, 2017) vapour-mixture, e.g. by effective and reliable removal of the vapour through ventilation. This approach is referred Characteristic Ethanol Acetone 2-Propanol to as “primary explosion protection”. In cases where Boiling point 78 °C 56 °C 82 °C primary explosion protection is not consistently possible, Flash point 12 °C < -20 °C 12 °C precautions should be taken so that the hazardous Vapour pressure 58 hPa 246 hPa 42.6 hPa atmosphere will not ignite (“secondary explosion at 20 °C protection”). Secondary explosion protection consists of Density at 20 °C 0.79 g/cm³ 0.79 0.78 g/cm³ identifying and eliminating all potential sources of ignition g/cm³ (see below) from the hazard zone. Viscosity 1.2 mPa*s 0.32 2.4 mPa*s When working in plastination, we cannot completely (dynamic) at 20 mPa*s exclude the formation of an air-vapour mixture, especially °C during the dehydration and defatting steps, where Upper explosion 27.7 Vol-% 14.3 Vol-% 13.4 Vol-% acetone or other solvents are used. Some recurring steps limit (UEL) involve working at open containers. If we look at the Lower explosion 3.1 Vol-% 2.5 Vol-% 2.0 Vol-% silicone standard method we find that handling of limit (LEL) flammable liquids happens during the following activities: Partition -0.3 -0.24 0.05 coefficient -Storing flammable liquids (mostly acetone) n-octanol/water -Transporting them within your department -Filling /decanting (log POW) -Immersing specimens into or removing them from Miscibility with miscible miscible miscible the dehydration bath water -Taking acetone measurements with a density areometer (“acetonometer”) Some countries like the US have adopted a fourth -Disposing of acetone category of flammable liquids in addition to the GHS: if the
flash point of a liquid is higher than 60 °C but does not Thus, flammable liquids play a role in numerous steps exceed 93 °C it is classified into category 4 with H227 while working. All of them pose some risk of evaporation. “Combustible liquid”. The likelihood of this evaporation, the subsequent
General Issues of Safety in Plastination - 31 Figure 4 gives an example of explosion hazard zone determination. Zone 0 is usually restricted to the interior Table 2: Criteria for the classification of flammable liquids of conveyor pumps, barrels, and other kinds of as defined by the Globally Harmonized System of receptacles. However, there is no general information on Classification and Labelling of Chemicals” (GHS). the spatial extent of zones 1 and 2. They must be defined by the person who is responsible on-site. Category Criteria 1 Flash point < 23 °C and initial boiling point ≤ 35 °C 2 Flash point < 23 °C and initial boiling point > 35 °C 3 Flash point ≥ 23 °C and ≤ 60 °C
Determination of explosion hazard zones
In many countries it is mandatory to designate special rooms or areas where people handle flammable liquids as “explosion hazard zones”. An explosion hazard zone is an area where an explosive air-vapour mixture may form with a certain probability and frequency of occurrence. Within Fig. 4: Example: determination of explosion hazard zones Europe, directive 1999/92/EC gives the definition of when pumping solvent from a drum into a smaller receptacle. Example drawing given by the German hazard zones 0, 1, and 2 (Table 3) (The European statutory accident insurer VBG (2010) Parliament and the Council of the European Union, 1999). In the United States and Canada, safety officers in charge Table 3: Explosion hazard zones 0, 1, and 2 caused by of explosion hazard assessment follow either the zone gas, vapour or mist, as defined by the European directive classification system or their traditional definition of 1999/92/EC. hazard class and division, given by the US National Electrical Code (NEC 500) and the Canadian Electrical Hazard Explosion Explosion Explosion caused hazard hazard hazard Code (CEC J18), respectively (Stahl AG, 2016; Siemens by zone 0 zone 1 zone 2 AG, 2010). In brief, NEC 500 and CEC J18 define Class I Gases, A place in A place in A place in locations as places in which flammable gases or vapours vapours which an which an which an are or may be present in the air in quantities sufficient to or mists explosive explosive explosive produce explosive or ignitable mixtures. In class I, division atmosphere atmosphere atmosphere consisting of a consisting of a consisting of a 1 areas, ignitable concentrations of flammable gases, mixture with mixture with mixture with vapours or liquids may exist under normal operating air of air of air of conditions and during repair or maintenance works. In flammable flammable flammable division 2 areas, these ignitable concentrations should not substances in substances in substances in exist under normal operating conditions but only in case the form of the form of the form of gas, vapour or gas, vapour or gas, vapour or of a malfunction, e.g. a container leakage. mist is present mist is likely to mist is not continuously or occur in likely to occur Note: Class II refers to explosion hazard caused by for long normal in normal combustible dust in the air. Class III refers to explosion periods or operation operation but, hazard caused by easily ignitable fibres. frequently. occasionally. if it does occur, will persist for Potential sources of ignition a short period only. If reliable avoidance of a hazardous atmosphere is not always possible, it will be necessary to consider potential Note: explosion hazards not caused by the presence of ignition sources. In theory, there are many different types, gas, vapour or mist, but by combustible dust in the air, however, not all of them are likely to occur in a plastination would lead to a classification into hazard zone 20, 21, or laboratory. For instance, the European standard EN 22. This type of hazard is outside the scope of this article.
32 - Schill 1127-1 lists 13 different types (German Institute for stated in the “T-O-P” principle, according to which, Standardisation, 2011). Among these, flames and hot technical measures have priority over organisational gases, static electricity, hot surfaces, and electrical measures which, in turn, have priority over personal equipment are probably of major importance in a typical protective measures. plastination laboratory (nevertheless, all types of ignition sources and the likelihood of their occurrence have to be Effective room ventilation is of major importance when considered!) running a plastination laboratory. At this point, people are often uncertain about the appropriate air-change rate. The Types of ignition sources (examples) information booklet “Working Safely in Laboratories – Basic Principles and Guidelines” (German Statutory Hot surfaces Electromagnetic waves Accident Insurance, 2008) can serve as a reference. Flames and hot gases Optical radiation Therein, a value of 25 m3 per m2 of floor area per hour is Mechanically produced sparks Ultrasound given. In other words: the air changes/hour should be approximately 10 (assuming that the ceiling height of the Electrical equipment Chemical reactions room is approx. 2.5 m). A ventilation system offering two, Transient currents Lightning strikes or several, power levels is recommended, enabling the operator to choose the extraction capacity according to Static electricity the current need.
Flames and hot gases are present for example when If individual ventilation for the laboratory is to be newly using a Bunsen burner. installed, one should consider that the vapours of acetone, ethanol, and all other commonly used solvents Static electricity can occur during friction between certain are heavier than air and therefore will gather near the materials, for example when wearing clothes made of floor. For this reason, it is advantageous to provide for air synthetic fibre. extraction openings close to the floor (Fig. 6).
Hot surfaces are found in heating cabinets, magnetic stirrers, and similar laboratory apparatus.
Electrical appliances, permanent installations or mobile devices, have to be removed from the explosion hazard zone unless they are manufactured with explosion-proof design (Fig. 5).
Fig. 6: Room ventilation with a ventilation opening beside Fig. 5: Electrical devices as potential ignition sources a freezer chest. As solvent vapours are heavier than air, it (examples): Magnetic stirrer with a heating plate, is advantageous to extract them close to ground level. Computer.
In some situations, e.g. when working with polyester resin or when preparing epoxy for injecting vessels of organs, Technical Protective Measures workplace ventilation with a flexible extraction arm (Fig. 7) or working under a fume hood is advisable. Technical protective measures should always have priority over other measures. For instance, installation of A potential equalisation bar should be present in every a workplace ventilation system takes precedence over plastination laboratory, in order to provide an earth for having the staff wear respirators. The order of priority is
General Issues of Safety in Plastination - 33 pieces of equipment (Fig. 8). In this way, electrostatic removal is not possible, as might be the case for some charging can be prevented, and the risk of spark fixed electrical installations, one should consider their generation will be reduced. disconnection from the mains, thus achieving permanent inactivation.
For electrical devices that are intended to be operated inside an explosive atmosphere, those with explosion- proof design should be chosen. The suitability of an appliance for use in hazard zone 0, 1, or 2 is shown on the type plate (Table 4) (The European Parliament and the Council of the European Union, 2014):
Table 4: Marking of electrical devices according to directive 2014/34/EU (examples). Equipment group and category for use in
II 1 G zone 0 or zone 1 or zone 2
II 2 G zone 1 or zone 2 Fig. 7: Extraction arm with explosion-proof fan on top, offering workplace ventilation. II 3 G zone 2
The Roman numeral, I or II, indicates the equipment group. For plastination, as well as for general laboratory purposes, only group II comes into consideration. The Arabic numeral, 1, 2, or 3, indicates the equipment category, which determines the hazard zone where it is approved for use.
The capital “G” indicates that the equipment is approved for use in hazardous atmosphere caused by the presence of gas, vapour or mist. See Figure 9 for an example type plate. A capital “D”, on the other hand, would indicate the fitness for use in a hazardous atmosphere cause by the presence of dust.
Fig. 8: Wall-mounted potential equalisation bar with several connection options.
Choosing proper explosion-proof equipment
Primary explosion protection, i.e. avoiding the formation Fig. 9: Example: type plate of an explosion proof fan of an explosive air-vapour-mixture, is always desirable approved for use in explosion hazard zone 1 or 2. and has priority. However, as mentioned before, in some steps of the plastination procedure it is necessary to handle flammable liquids in open containers, and Organisational Protective Measures therefore we need to apply secondary explosion protection, i.e. avoiding ignition of the explosive Technical protective measures usually are accompanied atmosphere. This means that all potential sources of by organisational measures to enhance safety, especially ignition are to be removed from the hazard zone. If when working in a large plastination laboratory.
34 - Schill Organizational measures mainly consist of meaningful protective glasses, are standard and are identical to those labelling, instructions, or restrictions, such as: required in a normal chemical laboratory. Working underneath an air extraction arm or under a fume hood - Access restrictions – only authorised personnel avoids health hazards caused by inhalation, e.g. when are allowed to enter the lab. handling solvent-containing epoxy for vessel injection or - Written and oral working instructions – instruct polyester resin. the staff before they start working in the plastination lab, and then recurrently, e.g. on a The main characteristic of a typical plastination laboratory yearly basis. Mandatory in many countries. is the fact that people store, transport, and handle unusual - -Working alone is not allowed (night time, amounts of solvent, mostly acetone. This requires more weekends) – two or more persons are more alert specific measures in order to prevent (1) health hazards than one, thus avoiding for instance, handling caused by acetone vapour inhalation, and (2) explosion errors. Also enables quick first aid. hazards caused by the presence of a vapour-air-mixture. - -Regular inspection of equipment for safety – Effective room or workplace ventilation is essential to often prescribed at least once-yearly. To be done extract the vapour from the workspace. Despite the by a qualified person, e.g. by an electrician. ventilation system, however, whenever an explosive - -No smoking! Relevant instructions and easily- atmosphere may form, it is essential to ensure that no visible prohibition signs making it clear, even to sources of ignition are present. Potential sources of visitors, that smoking is not allowed in the area ignition include spark-producing electrical installations (Fig. 10). such as light switches, or portable electrical appliances, for instance stirrers with heating plates, or laptop computers. Electrostatic charging of apparatus may also lead to sparking. This charging can be reliably avoided by earthing the equipment via a potential equalisation bar. In some work steps when you need an electrical appliance inside the explosion hazard zone, you have to use explosion-proof equipment. The suitability of a pump, fan, etc. for operation inside the hazard zone is given on the equipment type plate. All technical protective measures should be accompanied by organisational measures, whereby comprehensive and repeated instructions to staff are of particular importance.
When carrying out a risk assessment for a plastination laboratory one always has to consider the overall situation, including the lab dimensions, the types of Fig. 10: Prohibition signs at the entrance to a solvent chemicals used, their quantities and their physical storage. The yellow-black warning sign indicates the potential explosion hazard. properties, the capacity of the ventilation system, the number of people who work in the lab (and their level of
knowledge), the items of equipment, etc. When estimating Summary the hazard caused by flammable liquids, their respective flash points are the most important characteristic. The objective of this article was to review the major types of hazards which may occur when working in plastination, As the conditions differ from department to department, and to create awareness of the precautions that should and from one lab to the next, there is often no general be taken before starting a plastination laboratory. “right” or “wrong” when people ask, for example, about the suitability of existing premises, or of a certain appliance Information about the specific hazards of chemicals used like a freezer, for plastination. It is necessary to know the in plastination can be obtained from the product labels, overall situation on site, or the specifics of a plastination from safety data sheets, or from chemical substance project before a decision can be made. databases. Some protective measures for preventing health hazards, such as wearing safety gloves and
General Issues of Safety in Plastination - 35 It is quite possible to run a plastination lab safely, as Explosion group – a classification system for gases and proven by a number of labs all over the world where vapours, derived from a combination of two substance- people have worked for decades without any accidents. specific physical characteristics, “minimum ignition Everyone who is interested in plastination and is able to current ratio” and “maximum experimental safe gap”. provide a suitable surrounding to set up the equipment Describes the “readiness” of a gas or vapour to ignite and should feel encouraged to start their own lab. In any case, the ability of the explosion to pass a narrow gap of given whether you are a novice or routinely plastinating, it is dimensions. Important for the design of certain explosion- good to be aware of the hazard situation during all steps proof equipment. of the procedure. Groups are, in increasing severity: IIA -> IIB -> IIC. Appendix Examples: acetone: IIA; ethanol: IIB; isopropanol: IIA (Table 5). A brief explanation of the characteristics of gases or vapours related to explosion protection follows. Values Note: The traditional North American system of thereof can sometimes be found in safety data sheets, or classification divides explosive gases, vapours or mists in some online databases or reference books. into gas groups A, B, C or D, with A posing the most severe hazard. Group A and B correlate with the Flash point - the lowest temperature at which a liquid international group IIC, group C correlates with IIB, and generates flammable vapours above its surface which group D correlates with IIA. can be ignited in air by a flame. Examples: acetone: approx. -20° C; ethanol: 12° C; isopropanol: 12° C. (Auto-)Ignition temperature – a substance-specific temperature at which an explosive air-vapour-atmosphere Lower explosion limit (LEL) – a substance-specific will ignite, even in the absence of any flame or spark. concentration of vapour in air. Below the LEL, the quantity Examples: acetone: approx. 530 °C, ethanol: approx. 400 of flammable gas in the air is not sufficient to propagate a °C; isopropanol: 425 °C. flame in the surroundings of the ignition source. Practically, the atmosphere will not burn or explode. Temperature class – according to their ignition temperatures, gases and vapours are classified into six Upper explosion limit (UEL) – a substance-specific temperature classes, T1 -> -> T6, with T6 constituting the concentration of vapour in the air. Above the UEL, the lowest temperature range and therefore posing the most concentration of flammable gas or vapour in the air is so severe hazard (Table 5). high that there is not enough oxygen left to have the reaction of combustion or explosion propagated.
Table 5: Explosion groups (IIA -> IIC) and temperature classes (T1 -> T6) of some gases and vapours. Example: The surface temperature of an electrical appliance labelled “T4” must not exceed 135 °C. Temperature subclasses may apply, especially in North America (e.g. T4A). T1 T2 T3 T4 T5 T6 Ti > 450 °C 450 > Ti > 300 °C 300 > Ti > 200 °C 200 > Ti > 135 °C 135 > Ti > 100 °C 100 > Ti > 85 °C IIA Acetone Iso- Gasoline propanol
IIB Ethanol BIODUR® S 6 Ethyl ether Ethylene
IIC Hydrogen Acetylene Carbon disulphide
Ti = ignition temperature.
36 - Schill References Evaluation, Authorisation and Restriction of Chemicals (REACH). German Institute for Standardisation (2011): DIN EN 1127-1 Explosive atmospheres – Explosion prevention The European Parliament and the Council of the and protection – Part 1: Basic concepts and methodology; European Union (2014): “Directive 2014/34/EU of the German version. Beuth, Berlin European Parliament and of the Council on the harmonisation of the laws of the Member States relating German Statutory Accident Insurance (2008): Working to equipment and protective systems intended for use in Safely in Laboratories – Basic Principles and Guidelines. potentially explosive atmospheres (recast),” Official German version. Jedermann-Verlag, Heidelberg. Journal of the European Union, L 96, 309 – 356.
German Statutory Accident Insurance (2017): “GESTIS” The European Parliament and the Council of the Hazardous Substance Information System European Union (2006): Regulation (EC) No 1907/2006 http://www.dguv.de/ifa/gestis/gestis- of 18 December 2006 concerning the Registration, stoffdatenbank/index.jsp (in German). Viscosity data Evaluation, Authorisation and Restriction of Chemicals taken from various safety data sheets from solvent (REACH), establishing a European Chemicals Agency, suppliers. amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation IFA - Institute For Occupational Safety And Health Of The (EC) No 1488/94 as well as Council Directive 76/769/EEC German Social Accident Insurance (as of 09/29/2017): and Commission Directives 91/155/EEC, 93/67/EEC, “Practical solutions for occupational health and safety at 93/105/EC and 2000/21/EC. company level” http://www.dguv.de/ifa/praxishilfen/index.jsp, German The European Parliament and the Council of the version. European Union (1999): “Directive 1999/92/EC of the European Parliament and of the Council on minimum Stahl AG (2016): Grundlagen Explosionsschutz requirements for improving the safety and health (“explosion protection basics,” brochure in German) protection of workers potentially at risk from explosive https://r- atmospheres.” stahl.com/fileadmin/user_upload/mitarbeiter/PDF/ex- grundlagen-explosionsschutz-rstahl-b-de.pdf (as of United Nations (2017) “Globally Harmonized System of 10/13/2017) Classification and Labelling of Chemicals (GHS)” Seventh revised edition Siemens AG (2010): Explosion Protection – Answers for https://www.unece.org/fileadmin/DAM/trans/danger/publi Industry (brochure in German) /ghs/ghs_rev07/English/ST_SG_AC10_30_Rev7e.pdf https://www.automation.siemens.com/salesmaterial- (as of 10/13/2017) as/brochure/de/brochure_explosion_protection_de.pdf (as of 10/13/2017) VBG - Verwaltungs-Berufsgenossenschaft (2010): Broschüre Explosionsschutz – Praxishilfe (brochure in The European Commission (2015): Commission German). VBG, Hamburg. Regulation (EU) 2015/830 of 28 May 2015 amending Regulation (EC) No 1907/2006 of the European Von Hagens G, Tiedemann K, Kriz W (1987): The current Parliament and of the Council on the Registration, potential of plastination. Anat Embryol 175:411-421.
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Journal of Plastination Copyright Transfer Form may be downloaded from Instructions for Authors http://www.journal.plastination.org/downloads/copyright.pdf. (Revised July 2017) After the form is completed and signed by all the authors, it should be submitted to the Editorial Office JOURNAL OF PLASTINATION is owned and controlled by the ([email protected]) as a pdf or jpeg file via an e-mail International Society for Plastination (ISP). attachment.
Goals - The Journal of Plastination (ISSN 1090-2171) aims to Manuscript preparation provide a medium for the publication of scientific papers dealing Cover Letter with all aspects of plastination and preservation of biological The cover letter should include a statement of authorship, specimens. notification of conflicts of interest, ethical adherence, and any Submission Guidelines financial disclosures. All manuscripts must be submitted to the Editorial Office via Cover letters may be addressed to the Editor-in-Chief, Journal of the e-mail: [email protected]. If you experience Plastination. any problems or need further information, please contact Manuscript Philip J. Adds, [email protected]. The manuscript should consist of subdivisions in the following Authors must have an e-mail address at which they may be sequence: reached. Title Page Abstract with keywords Necessary Files for Submission Include: Text • Cover letter Introduction • Manuscript (including references and figure legends) Materials and methods • Table(s) (when appropriate) Results • Figure(s) (when appropriate) Discussion • Copyright Release Form (after acceptance) References Figure Legends Note: The above items should be prepared as separate files. Each file must contain a file extension (.doc, tif, jpg, eps). Title Page • File formats appropriate for text and table submissions: The first page of the manuscript should include: Microsoft Word • Title of paper • File formats appropriate for figure submissions: TIFF, JPEG • Each author’s name (JPG) and EPS • Institution from which paper emanated, with city, state, and postal code. Each affiliation should be listed as a separate Categories of submissions: entity, with a superscript number that links it to the Articles published in Journal of Plastination are grouped into individual author. general article types (listed below). Final designation of a For example: manuscript’s article type is determined by the EDITOR. S. D. HOLLADAY1*, B. L. BLAYLOCK2 and B. J. SMITH1 1 • Original Research – Plastination Department of Biomedical Sciences and Pathobiology, • Original Research – preservation Virginia Maryland Regional College of Veterinary • Education Medicine, Virginia Polytechnic Institute and State • Case reports University, Blacksburg, VA 24061-0442, USA. 2 • Technical brief notes College of Pharmacy and Health Sciences, University of Louisiana at Monroe, Monroe, LA 71209, USA. • Review - by invitation only • Corresponding Author’s name, address, telephone and • Legacy – institutions and people telefax numbers, and e-mail address. • Correspondence For example: • Editorial *Correspondence to: Dr Shane D. Holladay, Department of Biomedical Sciences and Pathobiology, Virginia Acceptance of a submission implies the transfer of copyright Maryland Regional College of Veterinary Medicine, from the authors to the publisher. It is the author's responsibility Virginia Polytechnic Institute and State University, to obtain permission to reproduce illustrations, tables and Blacksburg, VA 24061-0442, USA. Tel.: +001 404 739 figures from other publications. 6403; Fax: +001 404 739 6492; E-mail: [email protected]
The Journal of Plastination 30 (1):38 (2018)
It is the corresponding author’s responsibility to notify the Von Hagens G. 1985: Heidelberg plastination folder: Editorial Office of changes of address. Only the corresponding Collection of technical leaflets for plastination. Heidelberg: author should communicate with the Editorial office for matters Anatomiches Institut 1, Universität Heidelberg, p 16-33. regarding each manuscript. • For other publications: • Internet references: Author last name, initial(s). Year: Abstract & Key Words Title of article. URL: Internet address [accessed month, The abstract should be no longer than 250 words. It should year]. contain a description of the objectives, materials and methods, results, and conclusions. The abstract should include a section Figure legends on technique/technical development if the paper is significantly • Legends for all figures should be brief, specific and not be a technical in nature. The abstract must be written in complete substitute listing for the result section, and appear on a sentences and be intelligible without reference to the rest of the separate page at the end of the manuscript, following the paper. No references should be used in the abstract. list of references. • Legends must be numbered consecutively as they first On the same page, list, in alphabetical order, five Key Words that appear in the text. All symbols or abbreviations appearing reflect the content of the manuscript. Consult the Medical in any figure must be defined in the legend. Subject Headings for appropriate key words. Key words should be set in lower case (except for essential capitals), separated by Tables a semicolon and bolded. • All tables must be cited in the text and have titles. Table titles should be complete but brief. Information other than Text that defining the data should be presented as footnotes. The body of the text should be written using American English • Create tables using the table creating and editing feature of spelling. Microsoft Word. Do not use Excel or comparable Where quantities are specified, S.I. units should be used. spreadsheet programs. Equivalent Imperial or U.S. units, if desired, should follow in • Each table should be simple and uncomplicated, with NO parentheses e.g. 1 Kg (2.2 pounds). vertical and as few horizontal lines as possible. • Each table is to appear on a separate page and must include References the table title and appropriate column heads. • References to published works, abstracts and books must • Save each table in a separate word document file and include all that are relevant and necessary to the upload individually, like figures. manuscript. • Do not embed tables within the body of the manuscript. • Citations in the text should be in parentheses and listed chronologically; e.g. (Bickley et al., 1981; von Hagens, 1985; Figures Henry and Haynes, 1989) except when the authors name is • All figures must be cited in the text and must have legends. part of a sentence; e.g. "…von Hagens (1985) reported • Each figure should be attached as a separate file and labeled that…" When references are made to more than one paper with the appropriate number. by the same author published in the same year, designate • Figures should be created, saved and submitted as either a each citation as 1999 a, b, c, etc. TIFF, JPEG (JPG) or an EPS file. • Literature cited may only include the publications, which • Line drawings must have a resolution of at least 1200 dpi, are cited in the text. References are to be listed and electronic photographs, scanned images, radiographs, alphabetically using abbreviated journal names according to CT and MRI scans must have a resolution of at least 300 dpi. Index Medicus. Page numbers of the citation must be • The size of each figure should be at least 8.25 cm / 3.25 included. inches (one-column width) or 16 cm / 6 inches (two-column • Examples of the reference style are as follows: width). • For a journal article: • Magnification must be recorded and have a “scale bar” in Bickley HC, von Hagens G, Townsend FM. 1981: An the photo. Since reproduction of illustrations is costly, improved method for preserving of teaching specimens. authors should limit the number of figures to those which Arch Pathol Lab Med 105:674-676. adequately present the findings, and add to the • For a book section: understanding of the manuscript. Henry R, Haynes C. 1989: The urinary system. In: Henry R, • Figures that are submitted in color must be published in editor. An atlas and guide to the dissection of the pony, 4th color. ed. Edina, MN: Alpha Editions, p 8-17.
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