Decalcification of bone specimens using the incubation method: A feasible option for reducing the turnaround time

INTRODUCTION

Bone decalcification is a gradual process of removing calcium phosphate from the bone, making it soft for microtomy. In conventional decalcification, hard tissues are immersed in a decalcifying agent, typically, a low acid concentration at room temperature. The solution is then changed at frequent intervals till the desired level of decalcification is achieved, and then, the bone tissue is processed like the soft tissue bits. This method usually takes days to weeks, depending on the hardness and thickness of the bone.

Strong acid decalcifiers such as 10% nitric acid and 10% hydrochloric acid cause rapid decalcification, but their disadvantage is tissue swelling, severe damage to the tissue, and interference with the subsequent staining. Strong acids cause more damage to tissue antigens for immunohistochemistry (IHC) staining and may cause total loss of enzymes. A strong acid is advised for decalcification (in urgent biopsy specimens) for small bits of bone that need to be processed quickly. Excessive exposure to nitric acid eliminates nuclear staining and colors the tissue yellow.^[1]

Weak acid decalcifiers, such as 5% formic acid, Gooding and Stewart’s fluid (a combination of 10% formic acid and formalin), increase the turnaround time (TAT) for decalcification; however, they will cause less harm to the tissue. They preserve antigenicity for IHC and are recommended for small bone pieces and needle biopsies. The commonly used chelating agent for decalcification is ethylenediaminetetraacetic acid (EDTA) (5.5%), which binds to calcium ions and is progressively eliminated. They are often mild and slow-acting, which makes them suitable fixatives for IHC and electron microscopy investigations.^[1]

Several changes have been made to shorten the duration of the decalcification process. Heating is known to accelerate the decalcification process, no matter what kind of decalcifying agent is typically employed. Decalcification in a microwave oven happens more quickly than regular decalcification. Many studies have emphasized the importance of microwave bone decalcification, which gives comparable morphology on histology and IHC to conventionally decalcified bone.

Pitol et al.,^[2] on rat bone for electron microscopy, concluded that microwave-aided decalcification is more effective than the conventional method in some aspects: A shorter decalcification time, excellent bone tissue morphological preservation, and simple calcium release through microwave.^[2]

Weisberger et al.^[3] proved that the microwave method allows accurate assessment of a complete cross-section of cortical bone concerning the presence or absence of malignant tumor within time limits during resection of the head-and-neck region.^[3] In a study by Sangeetha et al.,^[4] traditional nitric acid decalcification on bone and teeth with microwave decalcification, the staining efficacy and tissue preservation in microwave nitric acid decalcification were good.^[4]

The tissue flotation bath (TFB) is a frequently utilized tool in histopathology laboratories. It can serve as a substitute for the more costly microwave method. A study by Ojha et al.^[5] evaluated the use of TFB for rapid decalcification of mandibulectomy specimens. Here, the time taken for routine decalcification was 7-8 days, while with TFB, the time taken for decalcification is reduced to 18-20 hours. They concluded that TFB is quicker than routine procedures and that it is also more affordable and accessible than microwaves while offering superior staining and comparable preservation.^[5] Galphade et al.^[6] compared the rate of decalcification of bone using the conventional method and TFB using 10% formic acid and 10% nitric acid. They concluded that TFB using 10% formic acid at elevated temperature (45°C) provided the best nuclear and cellular details with reduced TAT as compared to the conventional method.^[6]

On the same lines, we thought decalcification using an incubator, in which hard tissues are immersed in the decalcifying fluid at a higher temperature in an incubator with regular changes of the solution until the endpoint of decalcification is reached, can be done. We chose this method as the TFB in our laboratory is usually busy with the microtomy. The incubator can be checked frequently for the endpoint of decalcification and can be monitored during working hours. In this study, we intend to study the effect of incubation in the decalcification procedure to reduce the TAT without compromising histological findings.

MATERIALS AND METHODS

This was a cross-sectional analytical study done in the histopathology division of the Department of Pathology in a tertiary care center in South India over a year. Anticipating a mean difference of 8 days for completion of decalcification with a standard deviation of 10 days between the conventional method and TFB, with 5% level of significance and 80% power based on comparing two calculating two independent means formula, the sample size was calculated to be 27 in each group. A waiver from the Institute Ethics Committee was obtained (JIP/IEC-OS/2023/011).

The study included 27 bone resections of formalin-fixed tissues received in our department, such as amputation for neoplastic or non-neoplastic pathology, and soft tissues with calcification/ossification. The bone tissues were fixed for 24 h in 10% neutral buffered formalin; four bits were taken from each specimen of size 3 × 3 × 0.5 cm. The bits were washed in running tap water for 30 min before decalcification. Two bits were processed by the conventional method of decalcification using 10% nitric acid and 10% formic acid; the other two bits were processed by the incubation method of decalcification using 10% formic acid and 10% nitric acid. The bone tissues were kept in a pre-heated incubator with a temperature of around 45^°C. Cycles of 8 h of exposure to the decalcifying agent were repeated using a freshly prepared decalcification solution and checked every 2 h until the endpoint was reached by the physical method. The endpoint of decalcification is confirmed by the chemical method using ammonium oxalate and ammonium hydroxide. The tissue was not allowed to remain in the incubator overnight.

The TAT for both decalcification methods (conventional and incubation), both decalcifying fluids, and both types of tissues (hard bone and soft tissue with areas of calcification and ossification) were calculated. A pathologist scored the histo-morphological details, such as staining intensity, nuclear morphology, microtomy artifacts, and tissue integrity, as poor, good, and excellent.

RESULTS

Four bone tissue bits from all 27 samples were processed by conventional and incubation methods using 10% formic acid and 10% nitric acid. The decalcification time taken by the incubation method is a median of 5 h with an interquartile range (IQR) of 2.7-10 h, compared to the decalcification time of a median of 9 h with an IQR of 6-19.6 h by the conventional method. The decalcification time for the four groups, incubation method with formic acid and nitric acid, and the conventional method of decalcification with formic acid and nitric acid showed significant differences [Table 1 and Figure 1a]. As the decalcification time also depends on the type of bone, we separated the tissues into hard bone and soft bone (soft tissue with calcification and ossification). The median duration of decalcification was lowest with 10% nitric acid, with the incubation method, 5.5 h for hard bones and 2 h for soft bones [Table 2 and Figure 1b].

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Figure 1:

(a) Bar diagram showing the median decalcification times by conventional and incubation methods. (b) Bar diagram showing the comparison of decalcification times- Hard bone versus soft bone.

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The histo-morphological details, such as staining intensity, nuclear morphology, microtomy artifacts, and tissue integrity, were scored as poor, good, and excellent by a pathologist and analyzed. The staining intensity was excellent with formic acid by the incubation method, and 74.1% showed excellent staining compared to 59.3% of the conventional method. The nuclear morphology was excellent with the formic acid–incubation method showing 51.9% excellent staining compared to 29.6% of the conventional method. Nitric acid showed good nuclear morphology in 70.4% of cases by the incubation method and 74.1% by the conventional method [Table 3 and Figure 2]. The two methods showed no significant difference in microtomy artifacts and tissue integrity.

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Figure 2:

Microphotographs showing the staining intensity by conventional and incubation methods. (a) Conventional method by 10% formic acid, (b) Incubation method by 10% formic acid, (c) Conventional method by 10% nitric acid; (d) Incubation method by 10% nitric acid X H&E 20×.

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DISCUSSION

Heat is recognized for its ability to accelerate the decalcification process by increasing the chemical reaction and rate of diffusion.^[7] Verdenius and Alma found that when the temperature increases, the time needed for decalcification is decreased.^[8] A study by Kapila et al.^[9] employed a magnetic stirrer and a hot air oven to speed up the decalcification process, significantly reducing the time needed and improving the staining quality.^[9] Our study found that exposing tissues to a constant temperature of 45°C shortened the time required for decalcification. In our study, conventional decalcification took more time than the incubation method to complete decalcification. Similar to Ojha et al.’s^[5] findings, it was discovered that 10% nitric acid facilitated faster decalcification than 10% formic acid and that decalcification times were accelerated when incubation was set at 45°C.^[5] The TAT of various reagents in different studies has been tabulated in Table 4.

Nitric acid causes yellowness of tissue during decalcification due to the production of nitrogen dioxide (NO₂) during its decomposition. NO₂ reacts with tissue and produces the yellow tint. The yellow tint does not directly interfere with the decalcification process or interpretation of tissue, but indicates that the solution may be less effective. However, Ojha et al.^[5] discovered that the tissues’ yellowness was reduced by employing TFB instead of the traditional procedure with 10% nitric acid.^[5] According to Culling, this yellow discoloration could get in the way of further staining.^[7] In our study, yellowing of the tissue was not seen when treated with formic acid. Sangeetha et al.^[5] also stated in their study that when 5% nitric acid was used for specimen decalcification using both manual and microwave procedures, yellowness of tissue was seen; however, regardless of the method, it was absent when employing formic acid and EDTA.

Our study also showed that the histo-morphological outcomes of the incubation method were comparable to those of the conventional method. The staining intensity and nuclear morphology were better in the incubator method using 10% formic acid when compared to all other methods. Similar results were shown by Galphade et al.^[6] using the TFB method, and they proved that 10% formic acid in TFB is better than all other methods.^[6] For parameters such as microtomy artifacts, conventional formic acid and conventional nitric acid had fewer artifacts compared to incubator formic acid and incubator nitric acid. We did not find any literature or study comparing the incubation method of decalcification and the conventional method of decalcification.

Incubators are commonly seen in laboratories and can be used to decrease the time duration of decalcification. In addition, this instrument features additional adjustable temperature control for proper temperature control throughout the process. The incubation methods offer decalcification with good staining quality and shorter TAT.

CONCLUSIONS

The incubation method of decalcification drastically reduced the TAT compared to the conventional method. In terms of TAT, 10% nitric acid performs better. Regarding histomorphology, 10% formic acid by the incubation method is excellent compared to all other methods. We suggest the incubation formic acid method for routine decalcification due to decreased TAT and excellent histomorphology.