Efficiency of different decalcification protocols for nasal osseous structures in a rat experimental model of allergic rhinitis,and their effects on epithelial histology: An attempt at standardization

IntroductionDecalcification of osseous specimens is required for histological analysis; this however may cause tissue damage. In rodent models of allergic rhinitis (AR), epithelial histologic assessment necessitates prior decalcification of the nasal osseous structures. However, respiratory epithelium is highly susceptible to damage, and rat nasal architecture is elaborate and its sectioning is challenging. Nevertheless, decalcification is not standardized in experimental AR. We therefore undertook this task, in order to reduce experimental bias.MethodsSix-to-eight week-old Wistar rats underwent an AR protocol. Subsequently, nasal structures were decalcified in the following mediums: (i) formic acid 10% for 5 and 20 days; (ii) formic acid 15% for 5 and 15 days; (iii) Morse Solution for 5 and 20 days and (iv) EDTA for 20 and 40 days. Decalcification efficiency/speed was evaluated via radiographic analysis. Furthermore, specimens were stained with hematoxylin and eosin and assessed for preservation of epithelial features.ResultsSpecimens were appropriately decalcified in 5 days in the formic acid-based mediums and in 20 days in EDTA with minimal epithelial damage. EDTA for 40 days had no unacceptable adverse effects; conversely, 15 and/or 20 days in acid-based agents provided no extra benefit for decalcification and were detrimental to the epithelium.ConclusionsEDTA treatment for 20 days is appropriate for decalcification of nasal structures in rat models of allergic rhinitis; further incubation preserves epithelial integrity but is not required. When urgency is a factor, formic-acid-based decalcification for 5 days yields acceptable results.     

Effect of decalcification agents on immunoreactivity of cellular antigens.

The effects of several strong acids, weak acids, a proprietary decalcifier, and edetic acid on the immunohistochemical staining of cryostat and fixed paraffin embedded sections of tissue from a variety of normal and pathological calcified and uncalcified specimens were studied. Even decalcification in strong acids (HCl, HN03, 5% trichloracetic acid, HCl-edetic acid did not diminish the reactivity of many useful antigens (including leucocyte common antigen, intermediate filaments, S100 protein and epithelial membrane antigen). Weaker acids (formic acid, acetic acid) and edetic acid decalcified more slowly and generally showed greater preservation of antigenic reactivity with better morphology and staining quality. Trichloracetic acid was also useful as a quick one step fixation and decalcifying agent for both cryostat and routinely processed sections. Knowledge of the preservation of antigenic reactivity in decalcified tissue will be useful in the diagnosis of tumours of uncertain histogenesis and origin which affect calcified tissues.     

An evaluation of the immunohistochemistry benefits of boric acid antigen retrieval on rat decalcified joint tissues

This paper describes the evaluation and optimisation of boric acid antigen retrieval (AR) in rat joint tissue immunohistochemistry (IHC), with reference to two sample IHC targets, CD31 (PECAM-1) and Proliferating Cell Nuclear Antigen (PCNA). Sections of buffered formalin-fixed arthritic tibial/talus joints, decalcified with EDTA, EDTA/formalin or Surgipath(R) Decalcifier I(R), were subjected to one of a number of pre-treatments (none, 0.1% trypsin, 0.2 M acetic acid pH 7.0 or 0.2 M boric acid pH 7.0) and then immunostained for CD31 or PCNA. Of the pre-treatment AR regimens, boric acid gave the most consistent and specific immunostaining of both antigens in joints from the three different decalcification protocols. Satisfactory CD31 and PCNA staining was also achieved in EDTA decalcified joints with no pre-treatment. Likewise, PCNA could be demonstrated in Surgipath(R) decalcified tissue without pre-treatment, albeit at slightly lower staining intensity than achieved following boric acid. The remaining decalcification/pre-treatment conditions were unsatisfactory for IHC of the two antigens investigated because of lack of staining, non-specific staining or consistent loss of sections from the slides. Boric acid pre-treatment provides a valuable alternative low temperature AR method where conventional heat-mediated AR methods are normally required but cannot be used due to tissue type.     

A demineralization procedure for immunohistopathological use. EDTA treatment preserves lymphoid cell surface antigens

An evaluation of the usefulness of EDTA treatment for decalcification of murine bone tissue in order to preserve both morphological details and immunologically intact cell surface antigens has been performed. The ABC immunohistochemical staining technique employing monoclonal antibodies to subsets of T-lymphocytes, B-lymphocytes and to Ia antigens was used on frozen sections. Treatment of mouse hindlegs with EDTA for 14 days resulted in an efficient decalcification and good preservation of morphological details. When lymphoid tissues were handled in the same manner monoclonal antibodies, defining Ly 1, Ly 2, L3T4, MAS 034 and Ia molecules, were shown to retain their reactivity comparable to that of directly frozen tissues. In contrast, formic acid, the commonly used decalcification agent, destroyed most of the antigenic reactivity. We conclude that EDTA treatment of non-fixed, bone-containing tissues provides a suitable demineralization procedure in the immunohistochemical study of, e.g., arthritis and periodontitis.     

Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I and CD34 expression

 

Aims:

To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.  

Methods and results:

Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and α-smooth muscle actin (α-SMA) required no pre-treatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.  

Conclusions:

This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1–1.5 h.     

Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid (DNA and RNA) recovered from bone biopsies

Molecular studies are part of standard care for cancer patients. Bone, a common and sometimes sole site of metastasis, requires decalcification for morphological examination. Many commonly used decalcification agents contain strong acids that degrade nucleic acids. The paradigm shift in oncology, with biomarker targeted therapy and gene expression profiling analysis, requires sufficient nucleic acid recovery from bone biopsy specimens. We systematically studied the effects of a spectrum of decalcification agents on the quantity and quality of RNA and DNA recovered from bone biopsies. Multiple bone biopsies of similar size and cellularity were fixed in 10% neutral-buffered formalin, randomized to various decalcification agents for 2 hours then processed, and embedded. Tissue lysates were obtained from unstained sections and nucleic acid isolated. DNA and RNA were quantified. Assessment of DNA and RNA integrity was accomplished by comparison of the average cycle threshold by polymerase chain reaction of selected housekeeping genes for each agent. Results were then analyzed by 2-sample t test. There was a significant decrease in both DNA and RNA yield and integrity with strong acids (hydrochloric, nitric) vs 14% EDTA and formic acid. DNA yield was (mean nanograms) 6.15 vs 68.68 (P < .001) and RNA was (mean nanograms) 121.53 vs 288.89 (P = .003), respectively. DNA integrity (mean cycle threshold) was 35.79 vs 30.16 (P < .001), and RNA was 33.03 vs 26.5 (P < .001), respectively. Decalcification of bone biopsies with EDTA or formic acid agents was associated with a significant improvement in recovered nucleic acid quantity and quality.     

Influence of different decalcifying agents on EGF and EGFR immunostaining

This study was designed to verify the influence of three demineralizing agents on EGF and EGFR immunostaining as well as on tissue morphology. We chose submandibular glands that are a source of EGF and its receptor and which could be analyzed using a control in which the decalcification step was not carried out. After sacrifice of adult male Wistar rats by perfusion fixation, the submandibular glands and mandibles were excised and placed together in each of the following solutions: (a) 5% nitric acid in 4% formaldehyde; (b) 4.13% EDTA pH 7.4; (c) 5% trichloroacetic acid. Mandibles served as a parameter for decalcification time in each demineralizing solution. A control group was performed with submandibular glands that were not placed in any demineralizing solution. After mandibles were completely decalcified, glands were processed by embedding in Paraplast^® and immunohistochemical staining was made to detect EGF and EGFR. It was observed that decalcification did not produce noticeable differences in terms of EGF and EGFR immunoreactivity, but had an effect on the quality of the morphology and staining. Our results indicate there is no problem performing immunostaining of EGF and EGFR in tissues that require decalcification. 4.13% EDTA (pH 7.4) is the best choice for decalcification in cases that are not urgent.     

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.     

Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.     

Improved methods for immunohistochemical detection of BrdU in hard tissue

Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieval for immunohistochemistry of BrdU in hard tissues including bone, tooth, and periodontal ligament. Mice were subcutaneously injected with BrdU (50 µg/g body weight) twice a day from the postnatal day 11 to day 15 and sacrificed at 2 h after the last injection. Dissected maxillae were fixed (Bouin's solution or 4% paraformaldehyde), demineralized (Morse's solution or EDTA), and embedded in paraffin. Antigen retrieval procedures were performed before incubation with primary antibody. When sections were treated with HCl for DNA denaturation, the staining intensity of BrdU positive cells was not affected by difference of fixatives. Higher sensitivity was obtained by demineralization with Morse than with EDTA. Although heat-induced antigen retrieval techniques in citrate buffer (pH 6.0) showed as well or better sensitivity than acid pretreatment, heating caused tissue damage specifically to tooth dentine and the surrounding tissue. When the LRCs at four weeks after the last injection of BrdU were compared, much more LRCs were observed in specimen demineralized with Morse than with 10% EDTA. Our data suggest that demineralization with Morse with Bouin fixative plus HCl pretreatment gives rise to the optimal results for BrdU immunodetection in hard tissue.     

Decontamination Strategies for Creutzfeldt-Jakob Disease Agent

Abstract

A polymethylmethacrylate technique that permits processing of enucleated eye globes with intact bone and tissue is described. The process includes decalcification in 5% formic acid, 22 hr dehydration in ascending alcohols, and 4hr clearing with xylenes to facilitate sectioning.

The combination of established techniques, decalcification agent, and plastic embedment decreases artifact and damage to ocular tissue and avoids lengthy celloidin procedures. (The J Histotechnol 15:311, 1992)     

Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection

Immunocytochemical detection of p53 protein products in paraffin sections is possible with a number of antisera, monoclonal and polyclonal. Few corroborative results amongst different laboratories have been published due to variations in the antibodies, the fixation protocols, and the immunocytochemical methods applied. Antigen unmasking methods employing microwaves or proteolytic enzymes add to the disparity in the percentage of positive cases reported. In this study, paraffin sections of 55 cases of cervical carcinoma were immunostained with monoclonal antibodies p53-DO7 and p53-1801 (a) without section pretreatment, (b) with pronase digestion, and (c) with microwave antigen retrieval in citric acid buffer. Specimens fixed in buffered formalin required antigen unmasking to show positive staining. Pronase digestion caused false-negative immunostaining. Microwave pretreatment with p53-DO7 yielded 100 per cent positivity for p53 proteins but only 7/55 cases with more than 50 per cent positive cells. Monoclonal antibody p53-DO7 detected more positive cases than p53-1801. Immunostaining with antibodies to p53 proteins must be interpreted cautiously as variations in fixation, antibodies, and section pretreatment will significantly affect results.     

Mucin-Positive Epithelial Mesotheliomas: A Histochemical, Immunohistochemical, and Ultrastructural Comparison with Mucin-Producing Pulmonary Adenocarcinomas

Pathologists routinely use histochemistry, immunohistochemistry, and electron microscopy to differentiate epithelial mesotheliomas from pulmonary adenocarcinomas. Epithelial mesotheliomas are usually mucicarmine-, PAS-diastase, and carcinoembryonic antigen-negative, whereas about 60-75% of pulmonary adenocarcinomas are mucicarmine- and PAS-diastase-positive, and about 90% express polyclonal carcinoembryonic antigen. During a pathologic evaluation of pleural neoplasms between 1975 and 1990, 10 epithelial mesotheliomas were identified that were mucicarmine- and in some instances PAS-diastase-positive (diagnosis of mesothelioma confirmed by ultrastructural examination), with four mesotheliomas focally expressing carcinoembryonic antigen. The mucicarmine, PAS-diastase, and carcinoembryonic antigen staining were usually eradicated or reduced in intensity by pretreatment of the tissue sections with hyaluronidase, suggesting that hyaluronic acid was responsible for the positive mucin reactions. In three cases the epithelial mesotheliomas showed focal regions of mucicarmine, PAS-d-, and Alcian blue-hyaluronidase-resistant staining. In contrast, 10 mucicarmine-, PAS-diastase-, Alcian blue-, and carcinoembryonic antigen-positive pulmonary adenocarcinomas were not affected by hyaluronidase pretreatment of the tissue. Besides the usual ultrastructural features of well- to moderately well-differentiated epithelial mesotheliomas, the mucin-positive epithelial mesotheliomas often showed medium-electron-dense secretory material covering the microvilli, aggregates of medium electron-dense material in association with the microvilli, producing an ultrastructural morphology that has been observed only in epithelial mesotheliomas.     

Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method

We have compared light chain immunohistochemistry in reactive lymphoid tissue and a series of paraffin-embedded B-cell lymphomas using standard trypsin digestion with a heat mediated epitope retrieval method. Fifty-seven B-cell lymphomas (18 high grade, 29 low grade and 10 cases of nodular lymphocyte predominance Hodgkin's disease), two reactive lymph nodes and eight tonsils fixed for known times between 12 h and 2 years were studied. Paraffin-embedded tissue was stained with polyclonal anti-kappa and anti-lambda antibodies. For each antibody staining was performed on two sections, one treated with trypsin digestion and one with microwave heating. Sections were scored from 0 to +++ with 0 representing poor staining and +++ excellent staining. A score of ++ was considered satisfactory. Light chain restriction was recorded if present. Satisfactory staining was obtained in 34/59 cases using trypsin digestion and 56/59 cases using heat retrieval. Light chain restriction was demonstrated in 32/57 (56%) B-cell lymphomas using trypsin digestion and 52/57 (91%) using heat retrieval. Satisfactory staining was obtained in tonsils fixed for up to 48 h using trypsin digestion and up to 2 years using heat retrieval. We have shown that for light chain immunostaining a heat mediated epitope retrieval method produces more consistent and satisfactory results and is effective over a greater range of fixation times than traditional trypsin digestion.     

Demonstration of trivalent iron in decalcified bone marrow specimens. Comparative study of standard Perls stain on sections and the Perls pre-reaction on fragments

In the literature the usefulness of the Perls reaction for Iron (Fe+++) stores estimation on bone marrow is largely accepted. However, due to decalcification, there is a considerable disagreement about the accuracy of this staining on osteomedullary tissue. In fact loss of Fe+++ has been claimed by some Authors, and denied by others. In order to check this controversial point in this paper Perls reaction, as a methodological variant, was directly anticipated on fragments (pre-Perls). In this way numerous osteomedullary sternal specimens from autopsies (non hematological cases) were observed in comparison with normal Perls reaction on sections (standard-Perls). In standard-Perls series (A) decalcification was achieved by HCl (alone or in combination with EDTA) or by formic acid. On pre-Perls treated fragments decalcification was an intrinsic effect of the hydrochloride component during Prussian Blue formulation. Nine different procedures of fixation were tested respectively for standard-Perls and pre-Perls. Pre-Perls was performed on pre-fixed (B series) as well as post-fixed material (C series). For the microscopical evaluation was applied the classical grading system proposed by Lundin for histology. Maximum grade was only observed in B and C, was the same in both these series and varied from 2 to 4 depending on autopsic case. Results strongly indicate the superiority of pre-Perls versus standard-Perls, with a differential grading ranging from 2 to 4 grades, in seven procedures of B (two procedures were equivalent in A and B) and in all the procedures of C. The comparison between B and C series showed inferiority of B in four procedures. For control (D series) standard-Perls was systematically performed on sections from pre-Perls: no differences between pre-Perls and pre-Perls + standard-Perls were observed indicating a complete permeation of fragments by Perls reagents. In general terms morphology in pre-Perls B was better than in pre-Perls C and substantially identical to standard procedures of A. Other data are analytically reported. CONCLUSIONS: Pre-Perls staining (with special reference to five procedures of B) seems to be recommendable for decalcified bone marrow biopsies, in conjunction with standard processing, when an high sensitivity in detection of Fe+++ deposits is required or in absence of aspirate. Pathologists could subsequently decide if this methodological variant is suitable or not for an effective alternative choice in their routine according to morphological and immunohistochemical adequacy.     

A simple and rapid decalcification procedure of skeletal tissues for pathology using an ultrasonic cleaner with D-mannitol and formic acid

Decalcification procedures are required in order to prepare histopathological preparations of hard tissues such as bone and teeth. Decalcification is usually performed by immersing the hard tissue in different decalcification fluids with various properties. These decalcification fluids typically include inorganic and organic acids, a neutral fluid containing a chelating agent, or a mixture of solutions. Unfortunately, there is no universal decalcification fluid that satisfies all the requirements of pathologists such as rapid decalcification, easy handling, and minimal tissue damage. Techniques involving use of microwaves (MW) or ultrasonic apparatus (US) have been shown to be useful for shortening the time for decalcification procedures. In the present study, we investigated a unique decalcification procedure that uses a common commercial ultrasonic cleaner and a decalcification fluid (formic acid) containing a free-radical scavenger (D-mannitol). The time required to complete the procedure is approximately half of that required to complete a standard decalcification procedure. In addition, tissue morphology and antigenicity is fairly well preserved after decalcification. The procedure is quick, easy to perform, and achieves decalcification of hard tissue with minimal tissue damage.     

The Cutting Edge: Abstracts from Current Literature

Abstract

A tape-transfer technique for reducing technical problems associated with the sectioning of large paraffin blocks is described. Ten different tapes were attached to the surface of tissue blocks containing decalcified bone, and sections were cut with a microtome. The tapes were evaluated for adhesion to the block face, ease of handling during sectioning, and removal before staining without disruption of the tissue. 3M Company tape numbers 3710–2 and 3750-G were found to be the most satisfactory. This tape-transfer technique yields excellent quality sections with greater ease, less distortion, and fewer artifacts than unsupported sections do. ( The J Histotechnol 13:207, 1990)     

Enzyme histochemistry of undecalcified bone and cartilage embedded in glycol methacrylate.

Undecalcified bone and cartilage tissue blocks were fixed for 3 h in cold formol-calcium, rapidly dehydrated with a graded series of cold ethanol, and embedded in glycol methacrylate. 2 mum sections were produced with a Sorvall JB-4 microtome using glass knives. The quality of the sections were usually excellent except for hard bone from old subjects where the bone sometimes shattered while sectioning. This method is short, relatively uninvolved and eliminates en bloc decalcification. Moreover, the method is gentle enough to allow the histochemical demonstration of alkaline and acid phosphatase by the azo dye methods, and acid phosphatase, 5'-nucleotidase and ATPase by the lead precipitation methods.     

The effect of decalcification solutions on kappa and lambda in situ hybridization

Abstract

Consistently reliable in situ hybridization (ISH) staining and microtomy on decalcified formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies has been a significant challenge. This study evaluates five decalcification solutions’ decalcification cutting and kappa/lambda ISH staining characteristics. Five random benign tonsils and one femoral head were decalcified at intervals from 0·5 to 4 hours in five decalcification solutions. Tonsil sections were stained for kappa and lambda by ISH. Stain characteristics were blindly graded and compared to non-decalcified tonsil sections using hierarchical linear model (HLM) analysis. The femoral head was cored with a standard bone marrow core biopsy needle, and cutting adequacy was evaluated at the same time intervals. Specimens were blinded and judged as ‘acceptable’ (A), ‘cutting not optimal’ (NO), or ‘unacceptable’ (U) by an experienced histotechnologist. Decalcification solutions Formical-2000, Formical-4, and Immunocal demonstrated approximately equivalent quality in staining (average score?=?7·642, 7·952, and 7·805, respectively) through-out 4 hours. Both ethylenediaminetetraacetic acid (EDTA) and Nitrical showed significant drop-off in stain performance. Four solutions had acceptable cutting performance at 1 hour of decalcification, but Immunocal was slower, with adequate cutting obtained at 2 hours. Formical-2000, Formical-4, and Immunocal demonstrated acceptable, similar staining performance, but Immunocal required longer decalcification times. These solutions should be considered as leading candidates for use in histology laboratories.