Decalcification Procedures Research Articles

Liver Status Assessment After Coronary Artery Bypass Grafting.

Background Coronary artery bypass grafting (CABG) is a common surgical intervention used to treat severe coronary artery disease. The Model for End-Stage Liver Disease (MELD) score has become a widely used prognostic index for assessing the severity of liver disease and prioritizing liver transplantation. However, its utility in predicting outcomes in cardiac surgery procedures has not been extensively evaluated. Methods This retrospective study gathered data on patients who underwent CABG or CABG combined with other concomitant surgical interventions, such as carotid common or external carotid artery endarterectomy, thoracic aortic aneurysm repair, and aortic or mitral valve replacement or decalcification procedures, at a single tertiary care facility from January 2011 to December 2020. Researchers collected demographic, clinical, and laboratory information, including MELD score and European System for Cardiac Operative Risk Evaluation (EuroSCORE) data. The patients were divided into two groups: the first group included only those who underwent CABG, while the second group comprised patients who underwent CABG along with other concomitant cardiac interventions. Results The MELD score at discharge was significantly higher in the CABG and other interventions group compared to the CABG-only group (median = 14.09, IQR = 7.41-18.7 vs. median = 6.41, IQR = 4.61-9.44, p < 0.001). However, the difference in MELD score at admission between the two groups was not statistically significant (p = 0.328). A p-value < 0.05 was considered statistically relevant, indicating that liver function worsened postoperatively in the patients with additional interventions. The EuroSCORE was also significantly higher in the CABG and other interventions group, suggesting a higher surgical risk as expected (median = 5.74, IQR = 3.54-11.47 vs. median = 3.34, IQR = 1.97-5.66, p < 0.001). Additionally, differences in laboratory parameters, especially coagulation and hemostasis indicators throughout the postoperative period, including the ICU stay (divided into four equal periods based on each patient's total ICU length of stay) and at discharge, indicate a more complex biological state in patients with additional interventions. These findings may have implications for perioperative management and long-term outcomes. Conclusions The elevated MELD score in patients undergoing CABG with additional interventions emphasizes the need for close monitoring of liver function and coagulation status. Evaluating hepatic status preoperatively would be beneficial, and incorporating liver-protective strategies could help mitigate postoperative repercussions. It may also be useful to include liver function parameters in existing cardiovascular risk scores to improve risk assessment.

Some Useful and Unusual Eponymous Cardiac Surgical Techniques Adopted in Chittagong Medical College Hospital

Background: Innovation is an integral part of cardiac surgery. A number of natural barriers had to be overcome by the cardiac surgery pioneers to establish open heart surgeries in the 1950s. Since then, surgeons are continuously developing and adopting various innovative techniques to ensure safety and efficacy of cardiac surgery. The Department of Cardiac Surgery of Chittagong Medical College Hospital (CMCH) has some unique characteristics as it was developed isolated from the centers of Dhaka. A few interesting methods are in practice here, which worth mentioning for the outside readers. Some of these procedures have been colloquially named in our department often after the visiting surgeon or the team who introduced those here. Methods: The unique and unusual techniques applied in CMCH were sought and discussed. The hospital records, OT operation notes and ICU log books were thoroughly searched for any clue. The local and visiting team members were interviewed for any information. All available data were compiled and analyzed. Results: Six interesting and useful named surgical techniques adopted in CMCH Cardiac Surgery Department were identified. These are: 1. Marine city ASD Closure Technique, 2. Tendolkar’s Maneuver during CPB, 3. Turkish Sling in CABG, 4. Petlar Stitch in VSD closure, 5. Pyongyang Method of LA Closure and 6. NAK-NAKA De-calcification Procedure for valve surgery. The first two were introduced with the help of visiting Indian Professor Anil Tendolkar, whereas the 3rd was introduced by a visiting Turkish team. The 4th technique was adopted from a pediatric cardiac surgeon from Dhaka. The 5th and 6th techniques have long been in practice at NICVD, which were adopted with some modification in our department. Conclusion: The above mentioned six reproducible techniques are unique and useful. Sharing and discussion of these techniques will enable other Bangladeshi surgeons to adopt these useful tips. Cardiovasc j 2024; 16(2): 86-91

Comparative histologic evaluation of teeth decalcified by conventional method versus microwave induced decalcification

Abstract Introduction: Complete demineralization of hard tissues with preservation of organic components to retain their tissue architecture and to obtain thin section for histological examination is done by a process known as “decalcification”. Tooth decalcification may be achieved by subjecting the tissue to a decalcifying chemical agent and employing heat, vacuum, or electric current to enhance the procedure. Completion of decalcification is adjudged by determination of end-point. Histological examination of hard tooth tissues and pulp is essential for diagnosis of developmental pathologies, pulp pathologies, and forensic odontology analysis as well as for research purpose. Aim: In our study, we studied the time consumed for decalcification procedure by conventional decalcification method and with the microwave-assisted decalcification method for teeth, using 5% nitric acid, 5% trichloracetic acid, and 14% EDTA (Ethylenediaminetetraacetic acid), respectively, along with morphological preservation of dentin and pulp tissue by analyzing preservation of their issue structure (pulp shrinkage, odontoblastic layer damage) and staining efficacy (patchy staining) for the three types of decalcifying agents used. Materials and Method: The sample for this study included 120 freshly extracted teeth consisting of teeth of mandibular dentition. Samples were divided into two study groups: Group I: Decalcified by Conventional method Group II: Decalcified by Microwave method. Each group consisted of, three sub-groups employing a different decalcifying agent from among the three decalcifying agents used, that is, 5% nitric acids, 5% trichloroacetic acid, and 14%EDTA respectively. Result: The difference in speed of decalcification for both groups showed significant result. In either group, decalcification was fastest using 5% nitric acid. The difference in number of decalcified teeth sections showing shrinkage of pulp was not statistically significant for both groups. Damaged odontoblastic layer was evident only in the teeth treated with 5% nitric acid by microwave method. Patchy staining was observed in a few samples of all sample groups except those decalcified with EDTA by conventional method but the results were not statistically different on intercomparison. Discussion and Conclusion: Our results are similar to those of previous studies and we found that microwave method is an option that can reduce the time of decalcification. Tooth tissue morphology also was noted to be well preserved with 5% trichloracetic acid and 14% EDTA in comparison to 5% Nitric acid.

Cytology-histology correlation, time to diagnosis and time to therapy when cytology is performed in addition to biopsies in malignant spinal lesions of previously unknown malignant disease.

e14007 Background: Metastatic spinal lesions are the first manifestation of malignant disease in up to 30% of patients (pts). Spinal care, diagnostic evaluation and initiation of systemic therapy are crucial for outcome in pts with advanced cancer. Histopathology (HP) from bone specimens is time consuming and decalcification procedure can hamper diagnostic information. The additional evaluation of spinal lesions by cytopathology (CP) has the potential to reduce time to diagnosis (TTD) and time to therapy (TTT). Therefore, we performed simultaneous analysis of CP and HP specimens from suspected malignant spinal lesions to evaluate the diagnostic value of additional CP. This work was supported by the German Society for Spine Surgery (DWG). Methods: We obtained CP specimens by transpedicular, intralesional aspiration through a Yamshidi needle or touch preparation from solid tissue samples. CP and HP specimens from spinal lesions were evaluated in parallel in 54 pts (CP/HP group). We analyzed sensitivity and specificity of CP in reference to HP. Furthermore, we compared the TTD and TTT of CP/HP group with a retrospective cohort of 103 patients in whom only HP was performed (HP group). Results: The collection of CP samples was feasible without elongation of surgery time or any complications for the pts. In 52/54 (96%) pts a CP-specimen was successfully obtained. Of the 52 pts analyzed, multiple myeloma was diagnosed in 10 pts, solid malignancy in 34, 1 pt had lymphoma and 7 did not show malignancy. The most common solid malignancies were prostate cancer (n=9), breast cancer (n=5) and adenocarcinoma of the lung (n=5). The sensitivity and specificity of CP in reference to HP is 98% and 71%, respectively. The concordance of CP and HP in differentiating between hematologic or solid neoplastic lesions is 94.2%. TTD in the CP/HP group was significantly shorter compared to HP group (1.7±1.8 and 8.6±3.6 d, respectively; p&lt;0.001). In addition, TTT in the CP/HP group was significantly shorter than in the HP group (19.3±14.4 d and 31.4±24.7 d; p=0.003). In 24.1% of pts a specific tumor therapy was initiated based on CP diagnosis before obtaining the results of HP. Conclusions: The evaluation of CP from malignant spinal lesions shows high sensitivity and concordance with HP and reduced TTD and TTT. Therefore, CP in addition to HP from spinal lesions of suspected malignancy has the potential to improve quality of life and prognosis in pts with advanced cancer. We therefore recommend the implementation of additional CP to HP in previously undiagnosed malignant spinal lesions.

Analysis of Dental Enamel Remineralization: A Systematic Review of Technique Comparisons.

The demineralization process conditions the structure of the enamel and begins with a superficial decalcification procedure that makes the enamel surface porous and gives it a chalky appearance. White spot lesions (WSLs) are the first clinical sign that can be appreciated before caries evolves into cavitated lesions. The years of research have led to the testing of several remineralization techniques. This study's objective is to investigate and assess the various methods for remineralizing enamel. The dental enamel remineralization techniques have been evaluated. A literature search on PubMed, Scopus, and Web of Science was performed. After screening, identification, and eligibility processes 17 papers were selected for the qualitative analysis. This systematic review identified several materials that, whether used singly or in combination, can be effective in the process of remineralizing enamel. All methods have a potential for remineralization when they come into contact with tooth enamel surfaces that have early-stage caries (white spot lesions). From the studies conducted in the test, all of the substances used to which fluoride has been added contribute to remineralization. It is believed that by developing and researching new remineralization techniques, this process might develop even more successfully.

Abstract P6-04-17: High Intra- and Inter-block concordance of HER2 immunohistochemistry (IHC) scores across breast cancer samples and impact of decalcification procedures

Abstract Background &amp; Objective Trastuzumab deruxtecan is effective in HER2-positive (IHC3+ or ISH-positive) and HER2-low (IHC1+; IHC2+/ISH-negative) breast cancer. We assessed consistency of HER2-positive and HER2-low categorization by IHC across and between breast cancer samples with a range of HER2 expression. The impact of decalcification procedures on HER2 IHC staining was also assessed to determine whether HER2 expression is likely to be compromised in decalcified samples from metastatic bone disease. Methods Three non-consecutive tumor sections (approximately 100 μm apart) from 50 commercially obtained breast cancer resections (2 blocks/resection) and 20 biopsies (sections ~40 μm apart) representing a range of HER2 IHC expression, and with known ISH status, were stained using the VENTANA anti-HER2/neu 4B5 IHC assay, scored per ASCO-CAP (2018) guidelines and intra-block/inter-block concordance reported for HER2-positive and HER2-low categorization. To investigate the impact of decalcification, four resection samples were formalin-fixed, divided into four pieces, of which three were pre-treated with either 10% formic acid, Decal STAT or Richard Allan Scientific&amp;lt; sup&amp;gt;TM Decalcifying Solution before processing and staining with the 4B5 IHC assay. Results A single pathologist reviewed all samples. Intra-block agreement for HER2-low (proportion of blocks in which 3 of 3 sections had identical status) was 89.5% for biopsies (17/19) and 80.6% for resection samples (n=79/98). Inter-block agreement (proportion of resections in which two independent blocks had the same majority status for HER2-low) was 91.7%. Intra and inter-block agreement for HER2-positive was &amp;gt;90%. Exposure to Decal STAT or Richard Allan Scientific&amp;lt; sup&amp;gt;TM Decalcifying Solution caused a significant reduction in HER2 IHC1+ and IHC2+ staining in breast cancer samples. 10% formic acid had no apparent impact on HER2 IHC staining in all four samples tested. Conclusion There was high HER2-positive and HER2-low agreement within breast cancer biopsies and resection blocks and highly consistent HER2-positive and HER2-low categorization between different blocks from tumor resections. Our results suggest HER2 staining heterogeneity does not impact determination of HER2-positive and HER2-low status in the majority of cases. Accurate assessment of HER2 IHC1+ and IHC2+ status was observed to be compromised by Decal STAT and Richard Allan decalcification solutions. Decalcification of bone metastasis samples for HER2-positive and HER2-low status determination may be possible using formic acid, but this would require further validation. Citation Format: Asimina Tsirka, Charo Garrido, Marietta Scott, Paul Scorer, Anne-Marie Boothman, Tsuyoshi Matsuo, Craig Barker. High Intra- and Inter-block concordance of HER2 immunohistochemistry (IHC) scores across breast cancer samples and impact of decalcification procedures [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-17.

In Vitro Model for the Evaluation of Innovative Transcatheter Debridement Device (TDD): Pericardium-Based Scaffold and Stem Cells to Reproduce Calcificated Valves.

Aortic valve stenosis has become the most common valvular disease in elderly patients. Several treatments are available such as surgical aortic valve replacement and transcatheter aortic valve implantation. To date, however, there is a need to discover alternative treatments that can delay the disease progression and, therefore, the implant of a prosthetic valve. In this regard, a decalcification procedure based on the use of ultrasonic waves could represent an innovative solution in transcatheter cardiovascular therapies. In this article, we describe an innovative transcatheter debridement device (TDD) that uses low-intensity ultrasound shock waves for calcium ablation from the native aortic valve and bioprosthetic valve. Mesenchymal stem cells were seeded onto pericardium-based scaffolds and committed into an osteogenic phenotype. After treatment with TDD, cell proliferation was analyzed, as well as lactate dehydrogenase release and cell morphology. The release of calcium and inflammation events were detected. The results confirmed that the TDD was able to induce a safe decalcification without any adverse inflammatory events.

Radiological outcome of PLF surgeries in lumbar spine using DBM

Introduction: Spinal fusion has become one of the most popular methods of treating spinal diseases such as trauma, deformity and degenerative disc disease. Ideally, in order to promote fusion, the bone graft or bone substitute should have both osteoinductive and osteoconductive properties. The demineralized bone matrix (DBM) of demineralized allosteric bone is processed by comprehensive decalcification procedures. These procedures include chemical and radiological steps to reduce the immune response and the risk of infection. The bone inductive activity of demineralized bone matrix (DBM) has been well demonstrated. DBM also appears to support new bone formation through bone conduction mechanisms. Case reports and analyzes of several retrospective non-randomized clinical series indicate that DBM in combination with autologous marrow or bone marrow demonstrate similar performance characteristics to autograft in posterior lumbar spinal fusion. The purpose of this study is to access radiological outcomes of PLF in lumbar spine using demineralized bone matrix (DBM).Purpose: To see Radiological Outcome of PLF (posteriolateral Fusion) Surgeries In Lumbar Spine Using Demineralized Bone Matrix (DBM).Methods: 30 patients were included in the study Patients were followed up postoperatively at one year follow up. The participants were evaluated for Radiological Outcome in the study group was assessed by using Lenke Classification of Posterolateral Fusion Surgeries In Lumbar Spine Using Demineralized Bone Matrix (DBM).Results: All patients followed up for one year. Radiological evaluation of Lumbar fusion rates assessed with Lenke fusion Classification for PLF at the end of one year was 83.33% Grade. A fusion rates which is excellent. Younger patients of either sex had better outcomes compared to the older age group patients.Conclusions: The average improvement across the board was 81.40% which is excellent. The available literature shows similar results compared to an iliac bone graft which is the gold standard for spinal fusion. DBM can be considered a reliable alternative to the autograft when used as a graft expander in combination with the autograft.

Versican (VCAN) Proteolysis Predicts T-Cell Infiltration in Myeloma Bone Marrow Post- Autologous Stem Cell Transplant (ASCT)

Background/ Hypothesis:Versican (VCAN), a large matrix proteoglycan, accumulates in myeloma lesions (Hope et al., Blood 123(21): 3305-3315, 2014). VCAN sustains tumor-promoting inflammation but also generates immunosuppression through IL-6 and IL-10-mediated antigen-presenting cell dysfunction (Tang et al., Cell Rep 13(12): 2851-2864, 2015). VCAN proteolysis generates bioactive fragments (VCAN-derived matrikines, e.g., versikine) that oppose the functions of the parent macromolecule, intact VCAN (Hope et al., Blood 128(5): 680-685, 2016). VCAN-matrikines promote “T-cell inflammation” through co-ordinated effects on antigen-presenting and myeloid regulatory cells. In particular, we have shown that VCAN-matrikines promote Batf3-lineage dendritic cell (DC) differentiation from Flt3L-mobilized bone marrow progenitors and induce IL-12 but not IL-10 production by myeloid cells (Hope et al., J Immunol, in press, 2017). Batf3-DC constitute an intratumorally sparse DC subset with critical roles in tumor antigen cross-presentation and effector T-cell homing. Recently, VCAN-producing macrophages were shown to expand post-ASCT in myeloma and correlate with minimal residual disease (MRD) persistence (Arana, Zabaleta et al., Blood 128(22): 482-482, 2016). However, it is unknown whether VCAN proteolysis predicts T-cell infiltration post-ASCT for myeloma at a timepoint (Day 90-100 post-ASCT) when MRD is routinely quantitated and immunotherapies to control MRD are often instituted.Specific Aims:1.To detect VCAN proteolysis in paraffin-embedded bone marrow tissue obtained post-ASCT for myeloma2.To investigate the relationship between T-cell infiltration and VCAN proteolysis in post-ASCT myeloma bone marrowStudy Design:Bone marrow core biopsies from 40 patients who underwent ASCT for myeloma were analyzed at day 90-100 post-ASCT. ADAMTS protease-mediated VCAN proteolysis was analyzed by immunohistochemistry (IHC) through detection of neo-epitope DPEAAE as previously shown (Hope et al., J Immunol, in press, 2017). DPEAAE staining intensity was scored in the following scale: 0=none, +=patchy, ++= diffuse /weak, +++= diffuse/ strong. T-cell infiltration was scored in the following scale: rare, dispersed and intense. Clusters of T-cells (>5 CD8+ cells clustered together) were independently scored. Granzyme B staining status by IHC was scored using a scale similar to CD8+ status.Results:We found that prior de-calcification procedures did not prevent optimal evaluation and scoring of VCAN proteolysis status by IHC. Intense CD8+ T-cell infiltration and/or CD8+ clustering were only seen in the presence of intense (++ or +++) VCAN proteolysis. 14/14 marrows classified as displaying intense CD8+ infiltration plus CD8+ clusters were also scored as VCAN proteolysis ++/+++. 17/17 marrows with intense CD8+ infiltration were scored as VCAN-proteolysis ++/+++. 18/18 marrows with at least one CD8+ cluster detected in the specimen were VCAN proteolysis ++/+++. 67% of marrows (n=12/18) with +++ VCAN proteolysis had intense CD8+ infiltration. 83% (n=15/18) of marrows with +++ VCAN proteolysis had at least one CD8+ cluster detected. Intense staining for granzyme B was only seen in the presence of ++/+++ VCAN proteolysis (n=10/10).Conclusions/ Future implications:Intense VCAN proteolysis correlated with evidence of adaptive immune activation post-ASCT. T-cell infiltration in myeloma bone marrow post-ASCT was exclusively seen in the presence of intense VCAN proteolysis. Mechanistically, we hypothesize that T-cell infiltration is the result of enhanced Batf3-DC density in VCAN proteolysis-rich myeloma tumor microenvironments. We are currently investigating VCAN proteolysis-detection as an immune biomarker to predict efficacy of anti- myeloma immunotherapies as well as in relationship with MRD status post ASCT. There is a dearth of immune biomarkers that can be analyzed on standard de-calcified, paraffin-embedded bone marrow core specimens. For example, PD-L1 detection is problematic using de-calcified bone marrow specimens. By contrast, detection of VCAN proteolysis was unaffected by the de-calcification process. VCAN-matrikines (e.g., versikine) can also be therapeutically used to boost Batf3-DC whose paucity is often the limiting factor for efficacy of checkpoint inhibition and other immunotherapies. DisclosuresDhakal:Celgene: Honoraria.

Comparison between Conventional Decalcification and a Microwave-Assisted Method in Bone Tissue Affected with Mycetoma.

Mycetoma is a lifelong granulomatous disease of subcutaneous tissues and bones. Histopathology is a substantiated indicative method based on the assumption of a definitive diagnosis of mycetoma. It requires efficient processing of tissues including bone decalcification. The decalcification process must ensure complete removal of calcium and also a proper preservation of tissue and microorganisms' staining ability. Objectives. To compare the conventional method used in decalcification with the microwave method using different decalcification solutions. Different characteristics were tested, including the speed of decalcification and morphological and fungal preservation in bone tissue affected with mycetoma. Materials and Methods. Three decalcification solutions were employed to remove calcium from 50 bone tissue samples affected with mycetoma, including 10% neutral buffered EDTA (pH 7.4), 5% nitric acid, and 5% hydrochloric acid. Conventional and microwave methods were used. Haematoxylin-eosin (HE) stain, Gridley's stain, and Grocott hexamine-silver stain were employed to evaluate the bone and fungi morphologies. Results. The decalcification time of the conventional method compared with the microwave method with 10% EDTA (pH 7.4) took 120 hours and 29 hours, while 5% hydrochloric acid and 5% nitric acid took 8 hours and 3 hours, separately. Also, 10% EDTA is the best decalcifying agent for HE staining and fungal stains. 5% hydrochloric acid and 5% nitric acid can be used for fungal staining. Conclusion. The current study investigated the effects of different decalcifying agents as well as two decalcification procedures on the preservation of the bone structure and fungal staining, which will help to develop suitable protocols for the analyses of the bone tissue affected with mycetoma infection.

Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (<5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

Rapid detection of EGFR mutations in decalcified lung cancer bone metastasis

Detection of molecular alterations in lung cancer bone metastasis (LCBM) is particularly difficult when decalcification procedure is needed. The Idylla™ real-time (RT)-PCR is compared to the routine method used in our laboratory, which combines next generation and Sanger sequencing, for the detection of EGFR mutations in LCBM.LCBM subjected to EDTA or formic acid decalcification were analysed for EGFR mutational status using two methods: first, the Ion Torrent Ampliseq next generation sequencing (NGS) assay +/- Sanger sequencing was used prospectively; then, the fully-automated, RT-PCR based molecular testing system Idylla™ EGFR Mutation Test was applied retrospectively.Out of the 34 LCBM assayed, 14 (41.2%) were unsuitable for NGS analysis and five remained unsuitable after additional Sanger EGFR sequencing (5/34, 14.7%). Using Idylla™, valid results were observed for 33/34 samples (97.1%). The concordance between the NGS +/- Sanger sequencing method and the RT-PCR method was 89.7% (26/29), one false positive EGFR S768I mutation and two false negative results were observed using Idylla™; one of these false negative cases was diagnosed by Sanger sequencing with a rare exon 19 EGFR mutation not covered by the Idylla™ EGFR Mutation Test design.Detection of EGFR mutations in decalcified LCBM is challenging using NGS, more than half of samples showing invalid results. Alternative methods should thus be preferred to spare clinical samples and decrease delay. The Idylla™ EGFR Mutation Test shows a good performance on decalcified bone samples and could be used as a first step. In case of negative results, a sequencing approach is mandatory to check the presence of rare EGFR mutations sensitive to EGFR tyrosine kinase inhibitors.

Comparative Evaluation of Pulpal Response to Tri-antibiotic Paste and Allium Sativum with Formacresol as Pulpotomy Medication in Primary Teeth:An in vivo Study

Background:-To evaluate and compare Pulpal Response to Tri-antibiotic Paste and Allium Sativum with Formacresol as Pulpotomy Medication in Primary Teeth. Materials and Methods:-30 children in age group 7-9 years have bilateral primary teeth indicated for orthodontic serial extraction .children divided into two groups, (group I) included(15) children had bilateral primary teeth, the right side tooth treated with tri-antibiotic past (3 mix) (group IA) and left side tooth treated by formacresol (group IB) and (group II) included (15) children had bilateral primary teeth, the right side tooth treated with allium sativum oil (A. sativum) (group IIA) and left side tooth treated by formacresol (group IIB) . Each tooth was treated with standard pulpotomy procedure. . Patients were recalled after 15 and 30 days’ time interval as a part of orthodontic treatment. Teeth in each group were extracted and were subjected to decalcification procedure for histopathological evaluation. Results:- In group(I A) Nearly normal pulpal architecture was observed. Another finding common to all experimental groups was the presence of inflammatory processes of different levels of severity. Moderate amount of inflammatory cell infiltration was noted about 50-60% in cases after 15 and 30 days. In addition the results of group (IB), (IIB) there was pulpal inflammation was prominently observed in all cases treated with formacresol. After 15 days about 33.3% of cases showed severe inflammation with lymphocytes and foamy macrophages while 66.6% showed moderate inflammatory infiltration. By the end of 14 days cases with severe pulpal inflammation were about 46.6%. Also, in group (IIA) there was pulpal inflammation was prominently observed in all cases treated with formacresol. After 15 days about 33.3% of cases showed severe inflammation with lymphocytes and foamy macrophages while 66.6% showed moderate inflammatory infiltration. By the end of 30 days cases with severe pulpal inflammation were about 46.6%.Conclusion:- 1-Odontoblast remained vital in most cases treated with Both Tri-antibiotic paste and A. sativum . 2-Interal root resorption found in most specimens treated with formacresol .Recommendations: Further clinical studies with long time evaluation periods should be done to investigate the effects of the three medicament on pulp tissues of primary teeth .

The comparative analyses of decalcification procedures and methyl benzoate pre-treatment on tissue preservation and antigenicity in human acetabular labra.

The histological processing of musculoskeletal tissue might be challenging. The alteration of tissue composition e.g. by calcification of soft tissue in the elderly, after trauma or surgical interventions makes the histological processing of fixed tissue difficult. Additional steps of decalcification are then needed that probably affect the staining quality. In the present work, the effects of different decalcification agents and the intermedium methyl benzoate on histological staining methods and immunohistochemistry have been compared. Acetabular labra were fixed with 4% paraformaldehyde, left untreated or decalcified using 30% ethylenediaminetetraacetic acid (EDTA; Chelaplex®) or 6% trichloroacetic acid (TCA) for 1-4 days to investigate the effects of decalcification duration. Moreover, samples were pretreated with methyl benzoate or conventionally paraffin embedded independent of decalcification procedure and duration. The specimens were evaluated using hemalaun-eosin, Azur II- methylene blue staining or immunohistochemistry against ankyrin B to visualize nerve fibers. Decalcification with Chelaplex® or TCA reduced cutting artifacts without affecting the tissue morphology and proteoglycan staining but decreased antigenicity in immunohistochemistry. Interestingly, methyl benzoate further reduced cutting artifacts without altering tissue morphology and elevated antigenicity for Chelaplex® decalcified tissue samples in immunohistochemistry. The decalcification with Chelaplex® or 6% TCA preserves tissue morphology and proteoglycan staining similar to non- decalcified tissue but facilitates section processing. In immunohistochemistry both decalcification agents decreased antigenicity. Chelaplex® decalcified, methyl benzoate treated samples yielded an improved antigenicity.

Comparison of two decalcification agents using microwave technology: a histochemical assessment of the rat cochlea

ABSTRACTDespite the prevalence of hearing loss, there are no FDA-approved chemical entities available for either otoprotection or treatment of hearing impairment. This lack of drug-based treatments has made the assessment of the middle and inner ear a rare, challenging exercise in the pharmaceutical industry. Recently, advances in the use of various decalcifying agents, as well as heating techniques utilizing microwave technology have proven to be effective at shortening processing times, minimizing tissue damage and potentially lowering the cost of histopathological evaluation and diagnosis. This study tested decalcification agents in order to determine the most efficient decalcification procedure for rat outer, middle, and inner ear structures while preserving tissue morphology and eliminating introduction of artifacts to best facilitate histopathological analysis. Presented here are the results of rat ear histological preparations using two decalcifying agents at increasing microwave times with heat controlled at the physiological temperature of 37 °C. In this analysis, an acid-based combination decalcification-fixative solution was found to significantly speed up the decalcification process when compared with the calcium chelator EDTA.

The effect of prolonged decalcification on the immunohistochemical staining of Leishmania infantum amastigotes in formalin-fixed, paraffin-embedded canine soft tissues (lymph nodes)

The aim of this report was to present the preliminary results of the effect of prolonged decalcification on the immunohistochemical staining of Leishmania infantum in formalin-fixed, paraffin-embedded canine soft tissues. A heavily parasitized by Leishmania infantum amastigotes popliteal lymph node was used in this study. One half of a surgically excised lymphnode was processed according to a decalcification protocol (for 40 days), while the other half was processed without decalcification. Immunohistochemical staining for Leishmania infantum was then applied on both decalcified and non-decalcified lymph node sections. The amastigotes were stained and detected in non-decalcified sections, whereas a significant reduction of immunostainingwas observed in decalcified sections. It is likely that parasite antigens are altered by the decalcification procedure, probably as a result of the long lasting effect of decalcifying acids on both the amastigotes and the host cell cytoplasm. Consequently, it seems that immunohistochemical detection of Leishmania infantum amastigotes is significantly affected following prolonged tissue decalcification.

Physics and Histologic Evaluation of Rotary, Ultrasonic, and Sonic Instruments.

Rotary instruments (RIs) are the most commonly used to perform osteotomies in many fields of medicine. Owing to a new interest in performing a minimally invasive surgery, over last fifteen years new devices have been used in oral surgery such as ultrasonic instruments (UIs) and, lately, sonic instruments (SIs). Nowadays, bone preservation and regeneration are paramount in many clinical situations and, consequently, it is crucial to rely upon instruments, which cause the least tissue damage during the surgery. Concerning SIs, there is still few information about workload to be applied and related temperature increases; furthermore, there are no comparative in-vivo studies, which analyze the thermal and mechanical effects on bone. Thus, SIs have been compared with UIs and RIs in terms of heat generation, operating time, accuracy, and tissue damage. Decalcification and sectioning procedure resulted in no significant differences between the applied instruments in terms of bone damage. RIs resulted more efficient than UIs (P < 0.001), but demonstrated low accuracy (NRS 4.9), whereas SIs (P = 0.005) required more time to perform the osteotomy. The maximum temperature increase occurred in the ultrasonic group. Even though SI were the slowest, they have proved to be the most accurate (NRS 8.4) in comparison with UI (NRS 7.6) and RI (NRS 4.9). Within the limit of this study, sonic instruments could be considered a safe alternative to ultrasonic instruments.

Root canal morphology of mandibular first molars in a North Indian subpopulation: An in vitro clearing study

Introduction: Limited information is available about the canal morphology of mandibular first molars in North Indian population. The purpose of this study was to evaluate the root canal morphology of North Indian mandibular first molars by clearing and staining technique. Materials and Methods: A total of 863 mandibular first molars collected from various places in North India were subjected to canal staining and decalcification procedures. Access cavities were prepared, and pulp tissue dissolved with sodium hypochlorite. Indian ink was injected into the root canals aided by negative pressure applied at the root tips. The stained teeth were decalcified with 7% hydrochloric acid. Instead of ascending concentrations of alcohol, a nonalcohol-based drying agent (anhydrous calcium chloride) was used to remove traces of acid and water from the specimens. The dried specimens were immersed in methyl salicylate to render them transparent. The following features were observed under operating microscope at ×6 magnification: (i) Number of root canals per tooth, (ii) number of root canals per root, (iii) root canal configuration in each root (Vertucci's classification), and (iv) lateral canals, intercanal communications, and their location. Results: Majority had two roots (85%) with three (61%) and four (30%) canals. Three roots were present in 15% of the specimens. Type IV (49%) and type I (48%) were the most common configurations in mesial and distal roots, respectively. Conclusion: First molars with two roots and three canals are a common feature in North Indian patients. Both roots showed wide variations in canal anatomy with type IV and type I configurations predominating in mesial and distal roots, respectively.

Method for an accelerated reproducible calcite-layering of potable water systems to validate decalcification procedures

This article describes a method for an accelerated, controlled, and reproducible build-up of limescale in potable water systems. The concept of this method is to systematically generate water with a high calcium carbonate precipitation potential (CCPP) by CO 2 -enrichment, hardening with CaCO 3 followed by CO 2 -desorption. Water with a CCPP of about 100 mg/L CaCO 3 led to the intended calcification on inner steel tube surfaces in a simplified test plant. The limescale built-up was performed at ambient temperature. The procedure is an enabler for validation, verification and comparison of both the efficiency of decalcification methods, as well as scale preventing methods, under comparable and defined conditions. An economical and time efficient calcification method has been developed that provides solid calcite deposits of reproducible thickness and characteristics . The method can be used for studies with all water-carrying systems in household, public transportation or industrial applications.

Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer

Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ERα, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen's buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ERα-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen's buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ERα, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen's buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.