Effect of 10% formalin on radiographic optical ... - BIR Publications

Dentomaxillofacial Radiology (2008) 37, 137–141 ’ 2008 The British Institute of Radiology http://dmfr.birjournals.org

RESEARCH

Effect of 10% formalin on radiographic optical density of bone specimens AA Fonseca1, K Cherubini*,1, EB Veeck2, RS Ladeira3 and LP Carapeto4 1

Stomatology Department, Pontifical Catholic University of Rio Grande do Sul, Hospital Saõ Lucas, Porto Alegre, Brazil; Radiology Department, Faculty of Odontology, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil; 3 Department of Environmental Science, Porto Alegre, Rio Grande do Sul, Brazil; 4Division of Image Diagnosis of Veterinary Hospital, Federal University of Pelotas, Rio Grande do Sul, Brazil 2

Objectives: The aim of this work was to determine whether the fixation process with 10% formalin modifies the radiographic optical density of bone. Methods: Eight rabbit tibiae were placed in separate containers with one of the following fixative solutions: 10% formalin (n 5 3), 10% phosphate-buffered formalin (n 5 3) or 10% calcium carbonate-buffered formalin (n 5 2). Radiographs were obtained at 5 different times: before fixation (time zero), then 1 day, 15 days, 30 days and 90 days following immersion in the fixative solution. Radiographs were scanned and optical density was determined using ImageTool software. Results: There was no significant difference in radiographic optical density among the specimens fixed in 10% formalin (146.60¡32.44), 10% phosphate-buffered formalin (149.84¡32.43) and 10% calcium carbonate-buffered formalin (146.61¡35.92). Regardless of buffering, optical density at time zero was significantly higher than that at 15 days, 30 days and 90 days. However, while optical density at 1 day was significantly higher than that at 30 days and 90 days, it did not differ from that at 15 days. There was also no significant difference in density between 30 days and 90 days (ANOVA, Tukey, at 5% level of significance). Conclusion: The radiographic optical density of specimens stored in 10% formalin diminishes with time, irrespective of buffering, which suggests the occurrence of bone demineralization. Dentomaxillofacial Radiology (2008) 37, 137–141. doi: 10.1259/dmfr/18109064 Keywords: demineralization; bone tissue; formaldehyde; radiograph

Introduction The optical density of bone tissue can be studied using images of dried bones, a method that has the advantage of easy handling. However, there is interference by soft tissues located between the X-ray beam and the radiographic film. Some studies of dried bones have therefore used simulators of soft tissues.1–4 Another possibility in such investigations is to use cadaver specimens preserved in formalin, in which naturally occurring soft tissues are retained. In the study by Barros,2 the optical density differed significantly *Correspondence to: Dr Karen Cherubini, Assistant Professor of Stomatology, Stomatology Department, Pontifical Catholic University of Rio Grande do Sul, Av. Ipiranga, 6690 Sala 231, Hospital Saõ Lucas PUCRS, Porto Alegre, RS 90610000, Brazil; E-mail: [email protected] Received 27 March 2007; revised 11 May 2007; accepted 15 May 2007

between radiographs of cadaver mandibles with intact soft tissues and those of dried mandibles with soft tissue simulators such as water, wax and bovine muscle. We have suggested that it should be taken into consideration that the cadaver tissues were preserved in formalin, which could have interfered with the determination of optical density. Dead tissues, in their natural state, are opaque, soft, fragile and subject to putrefaction. A way of making them more resistant to decomposition and reducing post mortem changes consists of immersing the tissues in chemical solutions called fixatives. These fixatives maintain the integrity of the tissues after death, with minimal alteration in cell structure,5 and their main action is to make tissue proteins insoluble.6 Formaldehyde is one of the main fixatives employed

Formalin and bone mineral loss AA Fonseca et al

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in the preservation of corpses, whole bodies or individual body parts. Commercial solutions of formalin consist of different dilutions of 40% formaldehyde. Formalin’s acidic pH value, between pH 3 and pH 4.6, can induce demineralization of hard tissues and hardness of soft tissues. For this reason, this fixative is used as a 10% aqueous solution.7 This solution of 10% formalin (4% formaldehyde) is widely used for fixing and preserving tissues8 and in the buffered form it is routinely employed in light microscopy.6 Formalin can be buffered with various reagents, the most common of which are calcium carbonate,9 sodium cacodylate (0.2 M) and sodium phosphate.8 Because of its easy preparation and affordable cost, formalin solutions are often used to fix cadavers.10 A preserved body part can last more than 10 years without substantial modifications in its structure,5 which allows the maintenance of viable cadavers for studies for a long period without deterioration.11 Formalin acts slowly with the rate of fixation being 1 mm of tissue thickness every 8 h, and it decomposes easily into formic acid and water, if exposed to light. Since the demineralization process is induced by an acid medium, tissues submitted to fixation can undergo a loss of minerals if the medium in which they are fixed becomes acid.7 It is therefore suspected that fixation in formalin can induce demineralization and consequently alter the optical density of bone. The aim of the present study was to determine whether the pattern of radiographic optical density of bone is altered by the fixation process in 10% formalin, buffered and non-buffered.

immersion in the fixatives, the specimens were radiographed again with the same positioning and procedures as in the first radiograph, at intervals of 1 day, 15 days, 30 days and 90 days. The radiographs were developed in an automatic processor (Revell, Saõ Paulo, Brazil) and converted to digitized images with an HP Scanjet 6100C scanner (Hewlett Packard, Loveland, CO) at 300 dpi (dots per inch) and 8-bits (256 grey levels), maintaining the original size and without adjusting the brightness or contrast. The images were stored as BMP files and analysed by a single blind observer on a standard personal computer, employing the program ImageTool version 3.0 (The University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX). Optical density was measured in three regions of each radiograph: (1) the central area of 868 pixels in the proximal epiphysis of the tibia; (2) an area of 868 pixels in the middle portion of the diaphysis of the tibia; and (3) an area of 868 pixels in the centre of the distal epiphysis of the tibia (Figure 1). The optical density values recorded by the software were corrected by the optical density of the penetrometer by calculating the proportion. The results obtained were then evaluated by repeated-measures ANOVA, complemented by Tukey’s multiple comparisons test with a 5% level of significance. The protocol in the present study was evaluated and approved by the Committee for Ethics in Research of PUCRS. After the experiments, the animal specimens were donated to the Section of Anatomy of Domestic Animals of the Faculty of Veterinary Medicine of UFPel.

Materials and methods Results The sample was from four adult rabbits (Oryctolagus cuniculus), aged 6 months and weighing a mean of 3825 g, which were obtained from the central animal facility of the Universidade Federal de Pelotas (UFPel). The animals were anaesthetized with Zoletyl 50H at a dose of 20 mg kg21, and submitted to euthanasia by cervical dislocation. Afterwards, the right and left tibiae along with the intact soft tissue were radiographed with an X-ray unit (Phillips, Best, The Netherlands), at 40 kVp and 5 mA. T-MAT G/RA film (24630 cm) (Eastman Kodak, Rochester, NY) was used with a focus–film distance of 100 cm and exposure time of 0.025 s. When radiographs were taken, the specimens were positioned in a standardized fashion and an aluminium penetrometer with five steps was used, where the first two steps were used for calibration of optical density.12–14 After being radiographed, the eight tibiae were each placed in a separate container with the following solutions: (1) 10% formalin (pH 4.0, n 5 3); (2) 10% formalin buffered with monobasic sodium phosphate monohydrate and anhydrous dibasic sodium phosphate (pH 7.0, n 5 3); (3) 10% formalin buffered with calcium carbonate (pH 7.0, n 5 2). The volume of the fixative was ten times that of the specimen. After Dentomaxillofacial Radiology

There was no significant difference in radiographic optical density among the specimens fixed in 10% formalin (146.60¡32.44), 10% phosphate-buffered formalin (149.84¡32.43) and 10% calcium carbonatebuffered formalin (146.61¡35.92) (ANOVA, Tukey, P.0.05). Regardless of buffering, at time zero the density was significantly greater than after 15 days, 30 days and 90 days. After 1 day, the density was significantly greater

Figure 1 analysed

Radiography of the tibiae showing the three regions


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Table 1 Radiographic optical density after different periods of fixation in 10% formalin, non-buffered or buffered with phosphate or calcium carbonate Radiographic optical density 10% Formalin

CCBF

PBF

Total

Time (days)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Zero 1 15 30 90 Total

167.33 158.63 147.82 133.01 126.22 146.60

38.68 35.83 30.72 19.63 18.85 32.44

170.33 163.33 150.56 139.22 125.76 149.84

38.96 35.66 28.99 20.89 21.89 32.43

171.56 158.51 152.10 129.24 121.63 146.61

39.66 40.53 36.02 19.60 16.21 35.92

169.67A 159.76AB 150.11B 133.15C 124.38C 147.41

37.43 36.04 31.08 19.45 18.00 33.53

CCBF, calcium carbonate-buffered formalin; PBF, phosphate-buffered formalin; SD, standard deviation. Numbers followed by the same letter do not differ significantly (ANOVA complemented by Tukey’s test, at the 5% significance level)

than at 30 days and 90 days, but did not differ from the density at 15 days. Also, there was no significant difference between density at 30 days and at 90 days. The results are presented in Table 1 and Figure 2.

Discussion Buffers maintain the pH of formalin at about 7, while non-buffered formalin is between pH 3 and pH 4.6.7 That is, the fixative medium with non-buffered formalin is acidic, which suggests that it has the capacity of inducing demineralization. Although the present study did not show a significant difference in optical density between the group in which buffered formalin was used and that with non-buffered formalin, there was a tendency for density to diminish with time in both types of fixatives. Such findings corroborate a report by Currey et al15 indicating that exposure to formalin for longer than 24 h is likely to alter the mechanical properties of bone tissue since they are directly related to the degree of mineralization. These authors investigated the effect of formalin on the mechanical properties of bovine bone. In some of their experiments, the test group exhibited mechanical properties that were significantly different from those of the control group. However, in others, no significant

Figure 2 Radiographic optical density after different periods of fixation in 10% formalin, non-buffered and buffered with phosphate or calcium carbonate

difference was observed between the two. The authors pointed out that perhaps the effects were not strong enough to be detected by the evaluation methods employed. Based on the results of the present study, there is no significant difference between non-buffered and buffered formalin with regard to the effect on the optical density of bone tissue. However, it should be noted that density was determined using digitized conventional radiographs. For any change in density to be detected in conventional radiographs, mineral loss needs to be between 30% and 60%. That is, the conventional radiographic technique underestimates variation in bone density.14,16,17 Besides the advantages of higher sensitivity and reproducibility as well as lower exposition time over conventional techniques,18 the digital radiographic technique detects variations in bone density between 1% and 5.3%.14,19 In the present study, the limitation of conventional radiographs in detecting mineral loss was compensated for by the digitization process, which permitted the analysis of images using a software. The sensitivity of the computer program makes it possible to differentiate up to 256 grey levels, which allows the detection of minute variations in optical density. The diagnostic quality of a digitized image depends on the range of grey levels that it displays. Such range is reduced in digitized images compared with conventional radiographs, which have a wide spectrum of grey levels. Nevertheless, the subjective analysis of conventional radiographs limits the distinction of these tones of grey. Digitized images, on the other hand, can be analysed mathematically, which makes interpretation more efficient than for conventional radiographs, which is based on visual acuity.20 Despite being significant, the difference in optical density before fixation and 90 days after fixation is imperceptible when the radiograph is examined with the naked eye, which can differentiate 50 grey levels.21 Also, it is possible that studies applying other methods for measuring the optical density of bone will produce results that differ from those obtained in the present work. The overlapping of anatomical structures is an important limitation of radiographic examinations because it results in the visualization of three-dimensional structures as two-dimensional images. Therefore, foci of demineralization can be negligible in radiographs. Dentomaxillofacial Radiology


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Besides the conventional radiographic technique, other methods can be used in the determination of bone optical density. Examples of these are quantitative computerized tomography (QCT), microcomputerized tomography (pQCT), quantitative ultrasound (QUS), dual energy X-ray absorptiometry (DXA) and dual energy X-ray absorptiometry for peripheral tissues (pDXA). The techniques vary in radiation dose, accuracy, applicability and information on bone quality or optical density.22 They show greater specificity than the radiographic technique, but require specific sophisticated instrumentation, the cost of which is high and access limited. Lochmu¨ller et al23 investigated the effect of fixation, soft tissues and scan projection on bone mineral measurements with DXA. It was demonstrated that fixation has no significant effect on bone mineral measurements. Kikugawa and Asaka24 investigated the effect of the formalin fixation method on the flexibility and fracture resistance of bovine cortical bone after short- and relatively longterm preservation. The study revealed that formalin affects inorganic as well as organic components of bone from the initial stage of preservation. In the present study, regardless of buffering, optical density at time zero was significantly greater than that at 15 days, 30 days and 90 days, but did not differ from that at 1 day. The latter in turn differed significantly from that at 30 days and 90 days. Such a finding confirms the importance of controlling the time of fixation of specimens. For diagnostic purposes, 24 h fixation is considered ideal25 and any longer is considered damaging, particularly for immunohistochemical assays. However, in the preservation of tissues for anatomical studies, the length of time that bone specimens can be maintained in fixative solutions can be much longer, as long as years. The fact that time zero and 1 day after fixation did not differ significantly in density measurements suggests that the results of investigations of optical density of bone specimens fixed in 10% formalin for 24 h should not be influenced by the fixation process. 1 day after fixation also showed a significantly higher density than 30 days and 90 days after fixation, which is at odds with the results of Tomasi et al,26 who did not detect significant variations in the mineral substance of bone tissue preserved for 10 months in different concentrations of formaldehyde. Perhaps the low level of demineralization observed by Tomasi et al26 resulted from the detection method employed. The authors determined weight loss in investigating demineralization in bone fragments immersed in buffered and nonbuffered formalin solutions. The loss of mineral substance, at intervals of 15 days, 3 months and 10 months, oscillated between 0.3% and 1.3% weight loss and was considered minimal. The determination of optical density in digitized radiographs can be influenced by variables such as the oscillation of electrical current at the moment of X-ray emission and during digitization, as well as by variations Dentomaxillofacial Radiology

in ambient temperature and humidity during the chemical processing of the film.27 Bias was controlled in the present study with the use of a penetrometer.12–14 With time, there is a decrease in the radiographic optical density of bone tissue fixed in 10% formalin, whether buffered or not. In the evaluations performed up to 90 days following fixations, there was no significant difference in results between the buffered and non-buffered solutions. On the other hand, density measurements at 15 days and 30 days showed a significant difference in density in relation to time zero, but comparison of density with 30 days and 90 days after fixation did not show a significant difference. Considering that the interval between these two times is double that between time zero and 30 days after fixation, there appears to be stabilization of the demineralization process by 30 days of preservation in formalin. This is probably due to the saturation of the fixative solution. It is also possible that the determination of calcium concentration in the fixative solution, before and after the fixation period, could provide more reliable data on the decalcification of the tissue during the process. Another point to elucidate in further research is the alteration of soft tissue during the fixation process. As formalin is considered to harden collagen fibres in a relatively short period of time,24 it is plausible to accept that it also modifies the soft tissue density. Considering the extrapolation of these results to human bone, some comments about the animal model employed are necessary. It is known that there are gross differences in the bone anatomy of rabbits and humans both in the size and in the shape of the bones.28 Also, histologically, rabbits’ long bones have very different microstructure from those of humans. On the other hand, while there is minimal literature on the difference between human and rabbit bone composition and density, some similarities are reported in bone mineral density.29 The rabbit is one of the most commonly used animals for medical research, being used in approximately 35% of musculoskeletal research studies.30 This is in part due to ease of handling and size. The rabbit is also convenient in that it reaches skeletal maturity shortly after sexual maturity, at around 6 months of age.31 In conclusion, there is no significant difference in the radiographic optical density of bone specimens preserved up to 90 days in 10% formalin, 10% phosphatebuffered formalin or 10% calcium carbonate-buffered formalin. Regardless of the buffering of the fixative, the radiographic optical density of the specimen preserved in 10% formalin diminished progressively, which indicates a demineralization process. Further investigation is needed to examine fixation periods longer than those employed in the present study, possibly using other techniques as well to evaluate optical density, with the aim of obtaining a better understanding of the effect of fixation with 10% formalin on the optical density of bone tissue.


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