One-Step Preservation and Decalcification of Bony Tissue for Molecular Profiling

Mueller, Claudius; Harpole, Michael G.; Espina, Virginia

doi:10.1007/978-1-4939-6990-6_6

Cited by 4 publications

(3 citation statements)

References 27 publications

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“…Decalcification is most often accomplished using strong (e.g., hydrochloric acid or nitric acid) or weak (e.g., acetic acid or formic acid) inorganic acidic solutions to aid in the demineralization of bony specimens [ Table 1]. Although generally adequate for preserving histomorphology, decalcification with acidic solutions may lead to the degradation of genomic material and cause interference when molecular testing is performed [29,30] . Acid decalcification with formic acid (a weak acid) has been reported to preserve genomic material for sequencing analysis although it can cause severe damage to DNA and RNA when longer decalcification times are used [31] .…”

Section: Specimen Handlingmentioning

confidence: 99%

“…Alternative methods or modifications to standard decalcification that exist include the use of ethylenediaminetetraacetic acid (EDTA), microwave, ultrasonography, and other types of acid. Acids or EDTA may be used in bone decalcification and combined with microwave or ultrasonography to reduce the time needed to decalcify, and EDTA decalcification alone appears to provide some measure of genomic material preservation compared to stronger acid solutions [33][34][35][36][37] . Decalcification times vary for both acidic solutions and EDTA and can be modified with additional factors such as temperature or mechanical agitation [38] .…”

Section: Specimen Handlingmentioning

confidence: 99%

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Genetic characteristics and molecular diagnostics of bone tumors

Suster¹,

Suster²

2021

JCMT

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The diagnosis of primary tumors of bone relies heavily on clinicopathological and radiological correlation and is often best performed in a multidisciplinary setting. Bone tumors comprise a heterogenous category of human lesions ranging from benign to malignant neoplasms. These tumors affect a wide age range and can become problematic for diagnosis when less common entities are encountered. Traditionally the pathological diagnosis of many bone tumors has been based primarily on the evaluation of hematoxylin and eosin-stained glass slides, sometimes combined with ancillary diagnostic techniques such as immunohistochemistry, conventional cytogenetics, fluorescence in situ hybridization, and polymerase chain reaction-based assays. More recently, the advent of massively parallel sequencing-based techniques has opened new avenues for diagnostic testing in bone tumors; however, these new testing modalities are sensitive to traditional decalcification procedures that are commonly used in the routine processing of bony specimens. Herein we provide a focused review concentrating on the molecular genetic features of bone tumors with specific, recurrent genetic alterations that make them appealing targets for directed ancillary testing by conventional or molecular techniques. In addition, specimen handling with regards to decalcification procedures are discussed and the different types of testing modalities available are reviewed.

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Section: Specimen Handlingmentioning

confidence: 99%

Section: Specimen Handlingmentioning

confidence: 99%

Genetic characteristics and molecular diagnostics of bone tumors

Suster¹,

Suster²

2021

JCMT

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“…The overall level of progress, the tasks faced and the opportunities available within dentistry have increased and grown to become more complicated in recent decades. The wide scope and a better level of the dental care, coupled with further development and introduction of advanced diagnostic methods for dental patients, have set new tasks on conducting in-depth studies and applying modern morphological methods [7][8][9][10][11][12][13][14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Morphological Features of Dental Tissues in Streptozotocin-Induced Diabetes Mellitus Model

Domenyuk,

Ostrovskaya,

Eremin

et al. 2023

ArveMED

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The global issue of diabetes mellitus (DM), which is a serious burden faced by the healthcare systems and economies in many countries, has been increasing steadily in the recent decades. The fast pace of DM growth and prevalence, as well as associated complications (macro- and microangiopathy, neuropathy, nephropathy, impaired vision), high economic costs and social damage, high disability and mortality rates entailed by this disease take constant improvement of the respective diagnostics, prevention and treatment approaches. Dental manifestations of DM are diagnosed in most patients and reveal themselves through demineralized hard dental tissues, high intensity of dental caries and its prevalence, as well as the prevalence of periodontal diseases and issues affecting the oral mucosa. Our research included 60 male Wistar stock rats, all divided into two groups: the comparison group with no DM (n=10) and the main group with induced DM (n=50). DM modeling was done by a single intraperitoneal injection of streptozotocin (dosage – 65 mg/kg). The main group lab animals were removed from the experiment on Days 8, 16, 24, 32 and 60 under anesthesia by decapitation. All the histological and morphometric changes observed in the dental tissues were recorded in the ImadgeJ software package using a Leica DM2500 hardware and software set. The studied sections were used to evaluate the thickness of enamel, dentin, predentin, cement, pulp, the dentin tubule diameter, as well as the density of odontoblasts and ameloblasts. On Day 60 into the experiment, the following changes were identified in relation to the hard dental tissues: regarding the enamel − demineralization, disintegration of the interprism substance; deformed enamel prisms; erosion-type defects developing; regarding the dentin − expanded dentin tubules, vacuole dystrophy of odontoblasts, accumulating edematous fluid under the odontoblast layer, deformation and atrophy of odontoblasts; regarding the cement – layer thinning, cementolysis, cavities developing. Quantitative evaluation of rat dental tissues on Day 60, if compared with intact animals, revealed a multidirectional dynamics in the morphometric values. There was a statistically significant (p≤0.05) decrease in the enamel thickness identified (from 31.06±2.17 microns to 18.19±1.36 microns), in predentin (from 25.19±1.48 microns to 20.93±1.16 microns), in cement (from 37.28±2.04 microns to 31.04±1.46 microns), in the dentin tubule diameter (from 1.82±0.14 microns to 1.56±0.13 microns), as well as in the density of odontoblasts (from 7683.24±319.76 units/mm2 to 7312.61±256.19 units/mm2) along with a statistically reliable (p ≤0.05) increase in the thickness of dentin (from 96.54±3.39 microns to 105.11± 3.16 microns), pulp (from 107.43± 4.12 microns to 120.38±5.26 microns), as well as in the ameloblast density (from 6471.39± 272.18 units/mm2 to 6849.06±253.84 units/mm2). The outcomes expand the understanding of the structural changes affecting rat dental tissues at various times through modeling of Type 1 diabetes, as well as results could be used for developing pharmacological correction methods aiming at the reduction of the pathology intensity.

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Effects of Hydrochloric Acid and Formic Acid Decalcification on Breast Tumor Biomarkers and HER2 Fluorescence In Situ Hybridization

Clark

Yoest

Oniśko

et al. 2019

Applied Immunohistochemistry &Amp; Molecular Morphology

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Biomarker analysis of metastatic breast carcinoma (MBC) is routinely recommended by ASCO/CAP guidelines, and establishing a diagnosis of MBC often requires immunohistochemistry (IHC). The reliability of breast tumor biomarkers and breast-specific markers on decalcified tissues has not been extensively studied. We performed IHC studies on breast tumors exposed to hydrochloric acid (HCl) and formic acid (FA) decalcification solutions, and HER2 fluorescence in situ hybridization on a subset of these tumors to establish a protocol for handling bone specimens with suspicion for MBC. Fifteen fresh cases of primary breast carcinoma and 8 HER2+ paraffin-embedded core biopsy cases were studied. Fresh tissue was divided into 5 fragments to approximate a bone core biopsy. One fragment (control) was fixed in 10% neutral buffered formalin. The remaining fragments were also exposed to FA or HCl decalcification for 1 or 5 hours. All fragments were embedded in 1 block and tested with an IHC panel. The known HER2+ cases were exposed to either 1 or 5 hours of FA, and HER2 fluorescence in situ hybridization was also performed. Results were interpreted as follows: H-scores for estrogen receptor, progesterone receptor, and GATA-3 were assigned from 0 to 300; HER2, cytokeratin 7, gross cystic disease fluid protein-15, Pax-8, TTF-1, cytokeratin 20, and mammaglobin were scored from 0 to 3+; and Ki67 from 0% to 100%. Mean scores were compared using the t test or Wilcoxon test for paired samples. No significant differences in mean score were seen between NF and 1 hour FA for any IHC immunoreactivity. After 5 hours of FA, only Ki67 average score was significantly less than NF. Mean scores for estrogen receptor, progesterone receptor, HER2, Ki67, and GATA-3 were significantly lower than NF in the tissue after either 1 or 5 hours of HCl. Mean scores for gross cystic disease fluid protein-15, mammaglobin, and cytokeratin 7 staining were not significantly lower than NF after 1 or 5 hours of HCl.

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One-Step Preservation and Decalcification of Bony Tissue for Molecular Profiling

Cited by 4 publications

References 27 publications

Genetic characteristics and molecular diagnostics of bone tumors

Genetic characteristics and molecular diagnostics of bone tumors

Morphological Features of Dental Tissues in Streptozotocin-Induced Diabetes Mellitus Model

Effects of Hydrochloric Acid and Formic Acid Decalcification on Breast Tumor Biomarkers and HER2 Fluorescence In Situ Hybridization

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