| Abstract|| |
Aim: Utility of modified Hammersmith protocol in the deacalcification and/or softening of tissues and samples in a histopathology laboratory were studied. The object of the study was to prepare a novel method for softening/decalcifying tissue for histopathology. Materials and Methods: All the hard tissues received in the histopathology section were received in 10% neutral buffered formalin and then placed in freshly prepared combination of 10 mL of concentrated formaldehyde and 5 mL of 10% formic acid in 85 mL distilled water was used for decalcification. The tissue was checked for evidence of adequate decalcification/softening every 6 hours. Those which were decalcified/softened were sent for routine tissue processing and staining, while those which were not, were again placed in formalin. The process was repeated until the tissue was ready for further processing. The routine sections of these slides were reviewed for morphology and stain quality along with special stains and immunohistochemistry performed. The time taken for decalcification, the variables most likely to affect decalcification, the morphology and staining characteristics were documented. Statistical analysis was done to determine the effect of softening/decalcification process on each variable. Results: A total of 201 blocks in 119 specimens from humans including 61 males and 58 females were studied. Time taken was found to have a significant correlation only with the nature of the tissue (bone vs nonbone) and not with any other parameter viz. age, gender, specimen size, type of bone, and nature of pathology. Conclusion: This novel and modified method has circumvented the common problems of overdecalcification, preserved morphology, and produced consistent results without interfering with special stains and immunohistochemistry.
Keywords: Bone, bone pathology, bone tumors, fixation, immunohistochemistry
|How to cite this article:|
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
|How to cite this URL:|
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 [serial online] 2019 [cited 2020 Dec 4];62:423-9. Available from: https://www.ijpmonline.org/text.asp?2019/62/3/423/263505
| Introduction|| |
In most modern histopathology laboratories, there are a host of conditions warranting appropriate processing of hard tissues. Primary bone pathology, invasive infections or malignancies or nonbone tissues like calcified valves and vessels, osseous metaplasia or hyperkeratotic skin, all need a controlled and optimal softening method to remove the inorganic calcium, to make the tissue soft and to appropriately process the tissue. This is particularly important in the context of the small biopsies done under image guidance for diagnostic purpose. Routine and special staining as well as immunohistochemical characteristics needs to be maintained and the morphology must be well preserved in order to analyze the nature and characteristics of the lesion.,,,, There is a need to have a safe, consistent, reproducible as well as flexible format for fixation, decalcification, processing, sectioning, and staining of the tissue to adapt to most work schedules. Though the additional procedures required can be done in various ways, they often require further investment in terms of equipment, reagents, time, and skill.
The Hammersmith protocol designed at Hammersmith Hospital, London, UK has been in use since 1997 for optimal fixation and decalcification of bone marrow trephine (BMT) biopsies. It incorporates the use of acetic acid–zinc–formalin fixative for the decalcification of BMT specimens followed by 6 hours treatment with a combination of 10% formic acid–5% formaldehyde as decalcification agent. They have used the protocol in over 10000 BMT specimens and found that postdecalcification these specimens can be subjected to processing similar to other biopsy specimens. This has shown to be a safe method producing excellent morphology with hematoxylin and eosin, consistent results with other stains in histochemical analysis, immunohistochemistry, and molecular studies. Following the format of the “Hammersmith protocol” which was designed for processing of bone marrow biopsies, we have developed a modified protocol that was designed and standardized for handling bone and nonbone hard tissues other than the BMTs in the histopathology section in our institute. This aimed at avoiding the conventional hurdles of over- or underdecalcification, and, achieving optimal staining and immunohistochemical characteristics.
| Materials and Methods|| |
All the bone and hard tissues received in the histopathology section over a period of 1 year in our Institute were treated according to the decalcification protocol, as per the details given below:
In the Hammersmith protocol, the fixative used is a combination of 7.5 mL of acetic acid, 12.5 g of z inc chloride, and 150 mL of concentrated formaldehyde (AZF) in 1000 mL of distilled water. The modified protocol differed primarily in the type of fixative used. The former has a corrosive hazard and a likely requirement of added expenses, of which large quantities are required in surgical pathology tissues as compared for BMT biopsy. So, AZF fixative was replaced by the 10% neutral buffered formalin (10% NBF) which has been used as the universal fixative in the histopathology laboratory.
Once received in the laboratory, the specimens were examined grossly. The smaller biopsies found to be hard either due to bone or calcification or due to hyperkeratotic skin or nail were left in containers with 10% NBF, 10–15 times the volume of tissue after noting the gross findings. The larger specimens (resections, amputations, and excisions) were fixed in 10% NBF overnight with multiple incisions to facilitate penetration. The next morning (24 hours later) the smaller biopsy specimens were filtered out following decantation of the fixative. The larger specimens were dissected into smaller sections of 3 mm × 3 mm × 2 mm.
Decalcification for bone and other calcified tissues
Following a distilled water wash, the tissues were labeled and placed in beakers containing decalcification solution. An in-house freshly prepared combination of 10 mL of concentrated formaldehyde (formaldehyde solution 37–41% solution extra pure. Manufactured by Finar Limited, Chacharwadi-Vasna, Bavla, Ahmedabad, Gujarat, India; SAP code 10710LL030 CAS No 50-00-0) and 5 mL of 10% formic acid (Formic Acid, 96% Manufactured by Spectrum Reagents and Chemicals Pvt Ltd, Edayar, Cochin, CAS number 64-18-6, catalog F1089-500MLGL) in 85 mL distilled water was used for decalcification. After 6 hours, the tissue was checked for evidence of adequate decalcification/softening by physical test, i.e., the tissue was gently examined to see if it is soft and could be bent easily. Tissues showing adequate decalcification were washed for 30 minutes in running tap water and then routinely. Tissue which was inadequately decalcified/softened was removed from the decalcifying solution and replaced into 10%NBF until the next day. The tissue was not allowed to remain in decalcifying fluid overnight. Cycles of 6 hour exposure to decalcifying agent were repeated, until the tissue was adequately decalcified/softened. The type of the tissue, size, and the time taken for decalcification was noted.
Softening of hard tissues (including nail and hyperkeratotic skin)
Other hard tissues were also treated in the same method.
Tissue processing, sectioning, and staining
Adequately decalcified/softened tissue was processed along with the other routine histopathology tissue. Tissue processor used was that of Thermo Scientific Spin Tissue Processor Microm STP 120 Version 2.30 (Manufactured by Microm International GmbH part of Thermo Fisher Scientific Otto-Hahn-Str. 1A 69190 Walldorf Germany, Catalog number 813160). It includes the following reagents: Formalin (2 hours, 1 change), alcohol (70%, 80%, 96%-1.5 hours each and 3 jars of 100%-1 hour each), xylol (1.5 hours, 1 change), paraffin (2 hours 2 changes; temperature default value -62°C. Paraffin blocks were prepared at Leica Embedding station EG1150H, Version 2.1 (Leica Biosystems, Nussloch, Germany. Operating temperature range: +18°C to +40°C Working temperatures: 55°C to 70°C adjustable in 5 K increments. Relative air humidity: maximum 60%, noncondensing).
Tissue sections were made at 3–5 μm thickness using semiautomated rotary microtome (Part of Thermo Scientific HM 340E with E blade holder, Catalog number 905190) and transferred to albumin coated slides. Hematoxylin and eosin staining was performed using TissueTek DRS-TM 2000 automated slide stainer as per standard operating procedures (2003 Sakura Finetek USA, Inc.). The special stains and immunochemical testing was performed as and when necessary as per the standard operating procedures.
Immunohistochemistry was performed manually. Formalin fixed paraffin embedded tissue sections (5 μm) were deparaffinized. After treating with 3% hydrogen peroxide in methanol, the sections were taken through changes in running water, distilled water, and citrate buffer (pH- 6.4). Antigen retrieval was done by heat induced epitope retrieval method and the slides were placed in changes of TRIS buffer (Dako company, pH – 7.6) followed by skim milk (3%). The slides were incubated for appropriate antigens. The details of the reagent used are provided in [Table 1].
Secondary antibody [polymer HRP-Rabbit/Mouse- Dako-EnVision(ENV)] and chromogen solution, diaminobenzidine (Company: Dako) were added sequentially. Counterstaining was done with hematoxylin. Slides were dehydrated, cleared, and mounted.
The elapsed decalcification time and the likely variables affecting the decalcification time including age, gender, specimen-size or the type of bone, primary bone pathology or not were documented. The need for repeat processing or tendency to cause artifacts during processing was analyzed including scoring/splitting in sections and ease of ribbon formation. The morphology and staining characteristics were documented based on intensity of hematoxylin and eosin staining of the nuclei and the cytoplasm, respectively. The slides were reviewed by an experienced pathologist. They were evaluated and graded as understained, adequately stained, or overstained. The special stains and immunohistochemistry slides were also reviewed for optimal and suboptimal stain quality. The statistical analysis was done using QuickCalcs (GraphPad software) and Free Statistics Calculator (Version 4.0) taking a 'p' value of 0.05 as cut-off for significance by unpaired 't' test and analysis of variance.
| Results|| |
This protocol was used over a period of 1 year on a total of 201 blocks in 119 specimens [Table 2] from 61 males and 58 females, aged between 5 and 85 years (mean age 38.5 years). The number of blocks per specimen ranged from one to six. Short 6 hourly exposures to the decalcification agent were adapted into the routine laboratory work schedules. None of the tissues required reprocessing or repeat decalcification. These specimens could be integrated into the processing along with the other tissues, without any added investment in terms of manpower or resources. The soft tissue attachment was intact in all the cases with no distortion or shrinkage. Both the soft and hard tissues showed uniform and consistent architectural, morphological as well as staining characteristics, with preserved nuclear and cytoplasmic detail. No evidence of under- or overdecalcification was noted. The overall mean time taken for the decalcification was 13.61 hours (ranging between 6 and 138 hours). The time taken for decalcification in individual cases is depicted in the [Graph 1].
The various probable factors which could possibly affect the time taken for the decalcification time were analyzed namely age, gender, specimen size, type of tissue, type of bone, and the nature of pathology encountered. The age was split into age-ranges in years. The core biopsies and curettage specimens were grouped as 'small' (ranging from 0.3 to 2.8 cm), while the excision, amputation, and resection specimens were grouped under 'big' (ranging in size from 4.2 to 75 cm). The tissues were segregated based on the bone versus nonbone nature. The tissues included as nonbone tissue have been shown in [Table 2] and [Graph 2] [Figure 1].
|Figure 1: (a) Section from a calcified and bony area in a case of mature cystic teratoma showing well-balanced staining characteristics of the myxoid cartilage in distinction from the adjacent soft tissue. (H and E, 400×). (b) Section showing a nail plate with a well-preserved and well-stained thickened and laminated keratin layer (H and E, 200×)|
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The bone tissues were further grouped based on the bone type as long, short, flat, or irregular. Whether or not the final diagnosis showed a primary bone pathology, the nature of the lesion as neoplastic or nonneoplastic (degenerative, infective, inflammatory, metabolic, or hamartomatous) was analyzed. In all the cases where an infiltration was suspected, whether or not there was an infiltration, was also taken into consideration. The results of the mean time taken in the individual subgroups, the standard deviation, and the 'p' value are shown in [Table 3]. The only variable, out of the list reviewed, that was shown to affect decalcification was tissue type, that of bone versus nonbone [Figure 2].
|Table 3: Pathological parameters and the mean time taken for decalcification in various subgroups|
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|Figure 2: Various primary and secondary bone pathologies subject to the decalcifying protocol. (a) Enchondroma showing mature hyaline cartilage in lobules with adjacent bony tissue (b) Low-grade intramedullary osteosarcoma showing osteoid formation. (c) Metastatic adenocarcinoma in an elderly showing malignant clusters within the bony trabeculae. (d) Squamous cell carcinoma of the buccal mucosa with underlying bone showing no evidence of infiltration (H and E, 400×)|
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The specimens were checked for decalcification end point by manual method at 6 hourly intervals. The percentage of specimens decalcified as recorded from various bone specimens in the given time intervals has been shown in [Table 4]. One of the outlier values was found to be a case of synovial chondromatosis [Figure 3].
|Table 4: Percentage of cases reaching end point of the decalcification process in 6-hourly time intervals|
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|Figure 3: Case of synovial chondromatosis which was an outlier in the time taken. (H and E (a) low power 100x, (b) high power 400×)|
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Special stains, immunohistochemistry, and molecular techniques
Ziehl–Neelsen (ZN) staining and gomorimethenamine silver (GMS) staining were done in four cases each for highlighting microorganisms including Mycobacteria, while periodic acid-Schiff (PAS) stain was done in seven cases to highlight infective organisms or for demonstration of glycogen. Of the four cases that were stained by ZN technique, there was one positive and three were found to be negative for acid-fast bacilli. The positivity correlated with the subsequent culture. The positivity pattern, the staining quality, and the staining intensity were found to be unaffected by the decalcification process. Out of three cases which were negative for acid-fast bacilli on ZN stain, one case showed a positive culture for Mycobacteria.
The immunohistochemistry was performed in 24 cases the details of the antigen are as shown in [Table 5] and the procedure is accounted in the methodology. The strong consistent staining, for membranous for epithelial membrane antigen, leukocyte common antigen (LCA), CD99 and CD34, cytoplasmic for vimentin, desmin, and pancytokeratin, nuclear staining for estrogen receptor (ER), and FLI-1 and nuclear as well as cytoplasmic staining for neuron specific enolase (NSE), S100 [Figure 4]. However, this decalcifying technique was not studied on the samples for molecular studies. Therefore, their usefulness in such situations is yet uncertain.
|Figure 4: Sections from lytic lesions in femur in an elderly female, a known case of infiltrating ductal carcinoma, showing nests of cells with desmoplastic reaction (a and b, H and E 400×) which are positive for pan-cytokeratin (c, 400×) and estrogen receptor (d, 400×)|
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Biosafety and cost
The reagents could be readily prepared to the required concentration from the available laboratory chemicals at no extra expense. The decalcification agents could be easily disposed as per the standard operating procedures, based on the Bio-Medical Waste Management Rules, 2016 (Ministry Of Environment, Forest And Climate Change, Government Of India), without any serious harm to the laboratory personnel. No hazards or occupational accidents were encountered during handling of these cases.
| Discussion|| |
There has been no “universal” solution, technique, or protocol for decalcification available for the histopathology laboratory.,, A number of studies have aimed at identifying solutions or techniques which remove calcium at a good speed without causing damage to the attached tissues or compromising the staining characteristics.,,, One of the innovations in the present study was that various different hard tissues and not just BMT or teeth were studied. Also, the effect of the nature of the underlying pathology of the bone on the decalcification procedure is assessed.,,,,
In histopathology, there is a constant tug-of-war between the rapidity of decalcification that is required against the quality of the sections that are needed.,, If, in an effort to increase the rapidity of the process, strong acids are used, there is a likelihood off compromising on the morphology, the staining quality, and thereby the diagnostic value. This particularly becomes a problem in smaller biopsies, with limited tissue for diagnostic purpose. As a result, these reagents need very close monitoring to prevent overdecalcification, making it a very stressful exercise. Furthermore, the strong acids pose a greater threat of accidents and injury to the laboratory personnel. In contrast, the weaker acids take longer time for the same. To avoid these issues with acid decalcification, often laboratories used chelating agents like ethylene diamine-tetra-acetic acid (EDTA), which, preserved the histomorphology and does not interfere with molecular testing like comparative genomic hybridization and in-situ hybridization. They often take longer duration to reach an end-point which might not be always feasible for adopting into routine histopathology practice., Use of heat, agitation and vacuum to enhance the speed of decalcification have been recommended. These are shown to produce maceration and severe distortions of the tissue architecture., Microwave method of decalcification is shown to reduce the time taken, but required added investment.,,,
Among all these studies, the authors have studied only the use of various modalities and their impacts and these often required added resources. The pioneering work done by Naresh and co-workers was unique in proposing a 'protocol' based way of dealing with BMTs. It was shown that having a practical and easily adaptable “protocol” could produce consistent and reproducible results. However, this approach was confined to the BMT biopsies. An adaptation of this unique protocol using a weak acid, such as formic acid, in conjunction with a fixative, here formalin, with 6-hour exposures at 24 hour interval was customized in the present study to use in a number of nonmarrow bony and hard tissues. A novel attempt was made to study how different pathological variables affect the decalcification time. In our set-up, the tissue was allowed to fix in 10% neutral buffered formalin. This was documented earlier in literature to provide better results than with the use of formic acid alone.,
This study proved the given solution to be more useful and consistent in the context of bone tissues as compared to nonbone tissues. Most of the bone specimens were decalcified within 24 hours. Interrupting the decalcification process in installments of 6 hours helped in gaining an insight into the time taken by various specimens. As was noticed here, 66% of the specimens were decalcified in the first 6 hours itself. If all these samples were allowed to remain in decalcifying solution overnight, these were likely to show evidence of excessive decalcification. Having this protocol-based approach helped timely processing of these tissues.
A novel approach was taken in the present study to correlate the nature of pathology with the decalcification process. The age, gender, specimen-size, or the type of bone did not influence the decalcification time. The time taken was also irrespective off, whether the resection was for a primary bone pathology or not and the nature of pathology affecting the bone. This highlights on the versatility of its use in various settings where bony tissues needed decalcification. The morphology was greatly preserved and there was no distortion in the relationship and architecture of hard or soft tissues. This was quintessentially the primary objective of the study.,,
From the technical standpoint, this protocol was very easy to adopt. The constant fear of over decalcification can be mitigated, using a weak acid. The 6-hourly evaluation of end-point, helped in having a planned, predictable set of timing, and a schedule for managing bone and other hard tissues. The biosafety and adaptability offered ease of use and can be readily modified as per the laboratory timings, at no added cost. Neither the processing nor the section cutting was affected. Similar observations were made by other studies using the same decalcification reagents.,,,
Studies had attempted to see the impact of decalcification procedures with various agents on the stainability of Mycobacteria and gram-positive bacteria in tissue sections. Except for the use of strong acid, none of the other modalities were documented to be associated with reduced detection of Mycobacteria, while the stainable gram-positive bacteria in tissue sections were found to be reduced in number with decalcification with weak acids like formic acid, though the use of neutral formaldehyde mitigated the same to some extent., In our study, there was only one case where the follow-up culture showed Mycobacteria with the tissue section staining negative. But this can be explained by the overall poor sensitivity for detection of Mycobacterium tuberculosis in tissue sections.
In this study, no deleterious impact of the acid treatment on the special stains with PAS and GMS was found. Special stains like PAS and GMS were said to be minimally affected by decalcification excepting when heat is applied along with the acids. Moreover, formic acid had been shown be of superior quality in the study of proteoglycans in cartilage and bone as compared to even EDTA decalcification.,,,,
The immunohistochemistry done in all of our cases gave consistent results and there were no difficulties or interference in the intensity or stain quality. This is a major advantage in view of routine, high volume usage of immunohistochemical markers for diagnosis in surgical pathology. A number of studies have attempted to study the impact of decalcification on antigenic expression. Some studies have concluded that even after decalcification, the cells showing positivity for various immunomarkers are not decreased in number. The weak acids, with their slow decalcification rates, show higher degree of maintained antigenic reaction with better staining quality and preserved morphology.,,, However, the same was not seen with all studies. Some studies have shown progressive antigenic loss with prolonged time spent in decalcifying solutions, with a significant therapeutic impact in almost 9% of the cases.,,,
The suitability of our decalcifying technique to DNA or RNA isolation was not studied. However, the need for molecular diagnostics can be met with better in unfixed tissues than in formalin fixed paraffin embedded tissue.
| Conclusion|| |
This novel and modified method has circumvented the common problems of overdecalcification, preserved morphology, and did not interfere with special stains and immunohistochemistry. Though this method provided a great deal of flexibility in the methods of hard tissue processing, it needs to be customized as per the requirements of the individual laboratory. In our experience, it reduced the time taken and produced reproducible results with limited resources. However, its role in molecular studies is not studied.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Nachiappa Ganesh Rajesh
Department of Pathology, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]