JICDRO is a UGC approved journal (Journal no. 63927)

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ORIGINAL RESEARCH - BASIC AND CLINICAL
Year : 2022  |  Volume : 14  |  Issue : 1  |  Page : 52-59

A novel method of decalcification of bone by heat-accelerated method with tissue floatation bath using nitric acid and formic Acid: A comparative study


1 Department of Oral Pathology and Microbiology, Rural Dental College, Pravara Institute of Medical Sciences, Loni, Ahmednagar, Maharashtra, India
2 Department of Oral Pathology and Microbiology, MGV's KBH Dental College, Nashik, Maharashtra, India

Date of Submission15-Nov-2021
Date of Decision16-Mar-2022
Date of Acceptance01-Apr-2022
Date of Web Publication4-Jul-2022

Correspondence Address:
Dr. Amruta Hiraman Galphade
PIMS, PMT Campus, Gate No. 1, Loni, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jicdro.jicdro_80_21

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   Abstract 


Context: Decalcification of bone is a challenging aspect since penetration of decalcifying agents through dense alveolar bone is a slow process. Striking the right balance between speed and quality of staining is essential for accurate and timely diagnosis of bone pathologies. Aims: This study aimed to compare the rate of decalcification of bone by employing conventional method and tissue floatation bath (TFB) using 10% nitric acid and 10% formic acid. It also compared the cellular and staining characteristics of decalcified specimens of bone by the abovementioned methods. Materials and Method: Forty bone slices of hemimandibulectomy specimens were decalcified by employing conventional method and TFB at elevated temperature using 10% nitric acid and 10% formic acid. Following evaluation by chemical endpoint test, they were then subjected to tissue processing and staining with hematoxylin and eosin stain and assessed for various parameters. Statistical Analysis Used: Statistical analysis was done using statistical software SPSS version 18.0. One-way ANOVA was applied to evaluate the significant differences among the mean values in different groups. Results: The heat-accelerated TFB method significantly reduced the time of decalcification of bone without compromising the cellular and nuclear details. The cellular and staining characteristics of TFB method were better than those decalcified by conventional method. Conclusions: TFB method using 10% formic acid at elevated temperature provided the best cellular and nuclear details with reduced decalcification time as compared to conventional methods.

Keywords: Bone, decalcification, formic acid, nitric acid, tissue floatation bath


How to cite this article:
Galphade AH, Mahajan A, Prakash N, Lakshaman P. A novel method of decalcification of bone by heat-accelerated method with tissue floatation bath using nitric acid and formic Acid: A comparative study. J Int Clin Dent Res Organ 2022;14:52-9

How to cite this URL:
Galphade AH, Mahajan A, Prakash N, Lakshaman P. A novel method of decalcification of bone by heat-accelerated method with tissue floatation bath using nitric acid and formic Acid: A comparative study. J Int Clin Dent Res Organ [serial online] 2022 [cited 2022 Aug 7];14:52-9. Available from: https://www.jicdro.org/text.asp?2022/14/1/52/349759




   Introduction Top


The preservation of hard tissues close to the living state is essential for understanding of cellular and subcellular structures. The cutting of thin sections by ordinary methods is impossible in tissues of bone, teeth, and other pathologic lesions which show calcified deposits. Such tissues must be treated to remove the calcium and phosphate salts which are deposited in them by a process known as decalcification.[1] To allow cutting with the microtome knife and to avoid damage to the tissue, it is necessary that the specimen is soft enough.[2] Decalcifying bone is an ongoing challenging aspect of clinical, pathology, and histology research laboratories.[3]

For histopathological examination of bone and other hard tissues, decalcification is the most commonly employed technique yet one of the most technique-sensitive procedures in the histopathology laboratory.[4] It is of utmost importance in oral pathology for the diagnosis of primary bone tumors, bone involvement in malignancies like oral squamous cell carcinoma, and other tumors wherein decalcification is imperative to assess the extent of such tumors.[2]

Chemical agents are generally used for routine decalcification of bone and teeth, though they cause some adverse effects on hard and soft tissues.[5] Decalcification takes a longer period of time by the conventional method (at room temperature), as bone tissue slices have to be kept in the decalcifying solutions for many days or weeks.[6] The staining characteristics are affected due to tissue shrinkage by this method. In addition, this method may cause delay in the diagnosis and subsequent management of patients.

Microwave decalcification is a novel technique which is rapid compared to the manual conventional method,[4] but it releases toxic fumes. Decalcification of bone using nitric acid in a tissue floatation bath (TFB) has been reported recently.[2] This method decalcified bony tissues much faster than the routine method and is easily available in all histopathology laboratories.

Formic acid produces superior results to nitric acid in terms of cellular and staining characteristics by the conventional method. However, a major drawback is the prolonged duration for decalcification. Till date, decalcification using formic acid in a TFB has not been reported.

We attempted the use of formic acid as a decalcifying agent with TFB since formic acid better preserves cellular and nuclear characteristics and also to offset the long decalcification time normally taken by formic acid. Therefore, the aims and objectives were to compare the rate of decalcification of bone by employing conventional method and TFB using 10% nitric acid and 10% formic acid and also to compare the cellular and staining characteristics of decalcified specimens of bone by the abovementioned methods.


   Materials and Methods Top


Hemimandibulectomy specimens were collected from the Department of Oral and Maxillofacial Pathology in our institute. A total sample size of 40 bone slices from parasymphysis region of approximately equal thickness were divided into four groups. The protocol was reviewed by the institutional review body, and ethical clearance was obtained.

Study groups were divided as:

  1. Group 1 – 10% nitric acid by conventional method
  2. Group 2 – 10% formic acid by conventional method
  3. Group 3 – 10% nitric acid by TFB
  4. Group 4 – 10% formic acid by TFB.


Bone specimens were sliced using a straight handpiece and disk. Specimens were fixed in 10% formalin for 24 h and then washed for 30 min to 1 h before decalcification procedure. The volume of decalcifying solution taken was 20 times greater than that of the specimen size. In conventional method, specimens were placed in decalcifying solution at room temperature and decalcifying solution was changed every 3 days, until decalcification was complete. In the TFB method, the procedure employed by Ojha et al.[2] was employed. Endpoint test was carried out by physical and chemical methods. Routine tissue processing, sectioning, and H and E staining were carried out.

The parameters assessed were yellowishness of tissue, tissue shrinkage, sectioning difficulties and cellular and staining characteristics such as nuclear details, cytoplasmic eosinophilia, nuclear: cytoplasmic contrast, presence of osteocytes in lacunae, and patchy staining.

The statistical analysis was done using statistical software SPSS version 18.0 (IBM Statistical, GNU scientific library, New York). Descriptive statistics were used for demographic data and summarized as mean with standard deviation and as number with percentage for discrete variables. One-way ANOVA was applied to evaluate the significant differences among the mean values in different groups. Post hoc test (Bonferroni test) was applied for pair-wise comparison between all four groups. Chi-square test was used for comparison of individual parameters of two groups. Results with “P < 0.05” was considered to be statistically significant at 95% confidence interval.


   Results Top


The mean decalcification time (in hours) was least with nitric acid in TFB method (19 h) followed by formic acid in TFB method (47.5 h). Among the conventional methods, nitric acid was faster (244.2 h) in comparison with formic acid (726.2 h). These differences found in mean decalcification time were highly significant statistically (P < 0.001) [Graph 1].



Yellowishness of tissue was present in all ten specimens decalcified using 10% nitric acid conventionally whereas mild yellowishness of tissue was present in three specimens using 10% formic acid conventionally. The results of these two groups were highly significant statistically (P < 0.001). Yellowishness of tissue was present in all specimens decalcified using 10% nitric acid in TFB [Figure 1] and [Figure 2] method, whereas it was absent in all specimens decalcified using 10% formic acid in TFB [Figure 3] and [Figure 4]. These results were highly significant statistically [Table 1] and [Table 2] (P < 0.001).
Figure 1: yellowishness of tissue before decalcification in 10% nitric acid (tissue floatation bath)

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Figure 2: yellowishness of tissue after decalcification in 10% nitric acid (tissue floatation bath)

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Figure 3: yellowishness of tissue before decalcification in 10% formic acid (tissue floatation bath)

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Figure 4: yellowishness of tissue after decalcification in 10% formic acid (tissue floatation bath)

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Table 1: Comparison of yellowishness of tissue between nitric acid conventional and formic acid conventional (Chi-square test)

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Table 2: Comparison of yellowishness of tissue between nitric acid tissue floatation bath and formic acid tissue floatation bath (Chi-square test)

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Tissue shrinkage when compared between nitric acid and formic acid by both conventional and TFB methods showed highly significant differences in the results [Table 3] and [Table 4] (P < 0.001).
Table 3: Comparison of tissue shrinkage between nitric acid conventional and formic acid conventional (Chi-square test)

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Table 4: Comparison of tissue shrinkage between nitric acid tissue floatation bath and formic acid tissue floatation bath (Chi-square test)

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The sectioning difficulties were less when 10% formic acid was used as compared to that of 10% nitric acid in conventional method. These results found were not significant statistically [Table 5] (P = 0.702). On comparing sectioning difficulties between 10% formic acid and 10% nitric acid in TFB method, we did not find a significant difference [Table 6] (P = 0.891).
Table 5: Comparison of sectioning difficulties between nitric acid conventional and formic acid conventional (Chi-square test)

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Table 6: Comparison of sectioning difficulties between nitric acid tissue floatation bath and formic acid tissue floatation bath (Chi-square test)

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Nuclear details were good in all ten specimens decalcified using 10% formic acid conventionally [Figure 5]. The differences in nuclear staining between 10% nitric acid and 10% formic acid by conventional method were statistically significant [Graph 2] (P < 0.001). There was no significant difference in the nuclear details between formic acid and nitric acid employing TFB [Figure 6] and [Figure 7] [Graph 3] (P = 0.456).
Figure 5: ten percentage formic acid (conventional method) (H and E stain, ×400)

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Figure 6: ten percentage nitric acid (tissue floatation bath) (H and E stain, ×400)

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Figure 7: ten percentage formic acid (tissue floatation bath) (H and E stain, ×400)

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There was significantly increased cytoplasmic eosinophilia in specimens decalcified using 10% formic acid [Figure 5] as compared to 10% nitric acid by conventional method [Figure 8]. The results were found highly significant statistically [Graph 4] (P = 0.019). Specimens decalcified using 10% formic acid [Figure 7] and 10% nitric acid by TFB [Figure 6] showed no statistically significant difference which indicates that both the methods were comparable [Graph 5] (P = 0.757).
Figure 8: ten percentage nitric acid (conventional method) (H and E stain, ×400)

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Nuclear: cytoplasmic contrast was good in all ten specimens decalcified using 10% formic acid conventionally [Figure 5]. However, specimens decalcified using 10% nitric acid by conventional method [Figure 8] showed average contrast in four and poor in six specimens out of ten. The results were found highly significant statistically [Graph 6] (P < 0.001). There was no significant difference in nuclear: cytoplasmic contrast between 10% nitric acid and 10% formic acid utilizing TFB [Figure 6] and [Figure 7] [Graph 7] (P = 0.456).



There was a statistically significant difference in the percentage of osteocytes in lacunae by using 10% formic acid as compared to 10% nitric acid by conventional method [Figure 8] and [Table 7] (P = 0.004). There was no significant difference in the percentage of osteocytes in lacunae when 10% formic acid and 10% nitric acid in TFB [Figure 6] and [Figure 7] were compared [Table 8].
Table 7: Comparison of presence of osteocytes in lacunae between nitric acid conventional and formic acid conventional (Chi-square test)

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Table 8: Comparison of presence of osteocytes in lacunae between nitric acid tissue floatation bath and formic acid tissue floatation bath (Chi-square test)

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The patchy staining was less when 10% formic acid [Figure 5] was used as compared to 10% nitric acid by conventional method [Figure 8]. This result found was significant statistically [Graph 8] (P = 0.025). There was no significant difference when 10% formic acid and 10% nitric acid in TFB [Figure 6] and [Figure 7] were compared [Graph 9] (P = 0.210).




   Discussion Top


The diagnosis of many cysts, intraosseous tumors, and fibro-osseous lesions is dependent on assessment of decalcified tissue sections. Information pertaining to the relation of soft tissue to a tooth (in case of dentigerous cyst), involvement of bone by tumor (determining involvement of surgical excision margins), resorption pattern, and remodeling of bone (in lesions like fibrous dysplasia) decide the optimal management of the lesion.[7],[8]

Conventional bone decalcification is a time-consuming process and is not suitable for clinical applications and research projects which are time limited.[9] In histopathology, there is a constant tug of war between the rapidity of decalcification against the quality of the sections that are needed. In an effort to increase the rapidity of the process, strong acids are commonly used, which may compromise the morphology, staining quality, and thereby the diagnostic value.[10]

In order to circumvent these problems, the present study was carried out.

It was found that 10% nitric acid aided in rapid decalcification compared to 10% formic acid and the decalcification time was rapid when TFB at 45°C was used which was similar to the results of Ojha et al.[2]

In our study, there was yellowishness of tissue by both the methods using 10% nitric acid. However, Ojha et al.[2] found that there was reduced yellowishness of tissues in TFB compared to conventional method using 10% nitric acid. In our study, yellowishness of tissue with formic acid was absent in both the methods. The results of Sangeetha et al.[4] were similar to our findings when using 5% nitric acid by both manual and microwave methods for decalcification of specimens, whereas it was absent in formic acid and ethylenediaminetetraacetic acid irrespective of the method used. Tissue shrinkage was determined by measuring tissue size on graph paper. Tissue shrinkage was less in TFB method than routine method. Nitric acid is stronger than formic acid, thereby causing deleterious effects on tissues or specimens such as yellowishness of tissue and tissue shrinkage as the color leaches from the specimen during processing.[4],[5]

A comparison of all the parameters with formic acid in TFB could not be done since there is no literature pertaining to the abovementioned method.

Nuclear details were better with 10% nitric acid in TFB than the conventional method. However, there were no differences in nuclear details between formic acid and nitric acid employing TFB. Therefore, the use of TFB irrespective of acid used aided in preservation of nuclear details.

It was found that formic acid gave the best results for eosin staining by conventional method similar to those documented by Prasad and Donoghue.[11] We found that cytoplasmic eosinophilia was reduced in conventional method as compared to TFB using nitric acid. Our study showed similar results as that of Ojha et al.[2] where nuclear–cytoplasmic contrast was better in TFB method as compared to routine method of decalcification.

A study done by Prasad and Donoghue.[11] found that osteocyte retraction was less in formic acid compared to nitric acid which was similar to the present study. Sangeetha et al.[4] found that osteocytes were better preserved in the microwave oven method as compared to routine method. In our study, osteocytes were markedly better preserved in TFB method than conventional method. This indicates that the agent used, i.e., a weaker acid and the increased temperature as in TFB, better preserved osteocytes in lacunae as there was lesser retraction of osteocytes.

Patchy staining was present in more samples of conventional method compared to TFB method which was similar to the findings of Sangeetha et al.[4] In a study done by Prasad and Donoghue,[11] it was found that nitric acid showed lesser sectioning difficulties than formic acid. These results were contradictory to our study wherein formic acid showed lesser sectioning difficulties than nitric acid irrespective of the method used.

Conventional method of decalcification usually takes a longer duration for decalcification which in case of urgency will delay diagnosis. Therefore, rapid decalcification of hard tissues is necessary in such conditions to reduce the time of decalcification. Other methods mentioned in literature which are used to reduce the time of decalcification such as a microwave require a specific instrument and also additional space and have cost constraints. We used TFB for decalcification procedure which is readily available in almost all histopathological laboratories. It minimizes the requirement of any special instrument and is a cost-effective procedure. This instrument also has an adjustable temperature control which is necessary for proper and even decalcification. This decalcification method is comparable with other methods with respect to duration of decalcification and staining quality.

Limitations of the study

  • A larger sample size would have given more conclusive results
  • Specimens of approximately similar sizes and thickness were used, so the decalcification time was likely to be different as the weights of the specimens might be dissimilar.


Future prospects

  • It would be of value to determine the effect of TFB method on the tissue morphology utilizing special stains on bone sections
  • Furthermore, feasibility of immunohistochemical studies for specimens decalcified using TFB method should be evaluated.



   Conclusions Top


Bone decalcification is a time-consuming process for conducting routine histological analysis of specimens in research and pathology. Various decalcifying agents and methods have been described, but none of them have proved to be effective enough to achieve a high standard of hard tissue section preparation. Heat is known to expedite the rate of decalcification, and therefore, the above study was conducted using a TFB.

The TFB method significantly reduced the time of decalcification for bone specimens. The cellular and staining characteristics of bone specimens decalcified using TFB method were better than those decalcified by conventional method. As it requires very less time for decalcification, it can be utilized in case of urgency for diagnosis of biopsy reports. The TFB method is a safe, cost-effective, and easily available technique compared to other methods of decalcification.

This novel method using 10% formic acid in a TFB proved to be the best among all four methods for decalcification of bone. Thus, TFB method can definitely be employed for decalcification of bone and other calcified tissues.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Lynch MJ, Raphel S. Lynch's Medical Laboratory Technology. 3rd ed. London: W.B Saunders; 1976.  Back to cited text no. 1
    
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Ojha S, Haritwal A, Patel R, Meenai F, Chaurasia R, Gupta S. Rapid decalcification technique using tissue floatation bath (TFB) in a tertiary care oncology centre. Indian J Pathol Oncol 2016;3:226-30.  Back to cited text no. 2
    
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Sangeetha R, Uma K, Chandavarkar V. Comparison of routine decalcification methods with microwave decalcification of bone and teeth. J Oral Maxillofac Pathol 2013;17:386-91.  Back to cited text no. 4
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Kapila SN, Natarajan S, Boaz K, Pandya JA, Yinti SR. Driving the mineral out faster: Simple modifications of the decalcification technique. J Clin Diagn Res 2015;9:C93-7.  Back to cited text no. 7
    
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Callis G. Decalcification of bone: Literature review and practical study of various decalcifying agents. Methods and their effects on bone histology. J Histotechnol 1998;21:49-58.  Back to cited text no. 8
    
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Bogoevski K, Woloszyk A, Blackwood K, Woodruff MA, Glatt V. Tissue morphology and antigenicity in mouse and rat tibia: Comparing 12 different decalcification conditions. J Histochem Cytochem 2019;67:545-61.  Back to cited text no. 9
    
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Pradhan P, Rajesh NG, Badhe BA, Ilanchezian K, Manimehalai D, Jyothish A. A novel approach to decalcification in histopathology laboratory: An adaptation from the Hammersmith protocol. Indian J Pathol Microbiol 2019;62:423-9.  Back to cited text no. 10
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Prasad P, Donoghue M. A comparative study of various decalcification techniques. Indian J Dent Res 2013;24:302-8.  Back to cited text no. 11
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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