Strategies to improve quantitative assessment of immunohistochemical and immunofluorescent labelling.
Ontology highlight
ABSTRACT:
SUBMITTER: Johnson SJ
PROVIDER: S-EPMC4464180 | biostudies-other | 2015
REPOSITORIES: biostudies-other
Similar Datasets
Project description:New methods for chemo-selective modifications of peptides and native proteins are important in chemical biology and for the development of therapeutic conjugates. Less abundant and uncharged amino-acid residues are interesting targets to form less heterogeneous conjugates and preserve biological functions. Phenylurazole (PhUr), N-methylphenylurazole (NMePhUr) and N-methylluminol (NMeLum) derivatives were described as tyrosine (Y) anchors after chemical or enzymatic oxidations. Recently, we developed the first electrochemical Y-bioconjugation method coined eY-click to activate PhUr in biocompatible media. In this work, we assessed the limitations, benefits and relative efficiencies of eY-click conjugations performed with a set of PhUr, NMePhUr and NMeLum derivatives. Results evidenced a high efficiency of NMeLum that showed a complete Y-chemoselectivity on polypeptides and biologically relevant proteins after soft electrochemical activation. Side reactions on nucleophilic or heteroaromatic amino-acids such as lysine or tryptophan were never observed during mass spectrometry analysis. Myoglobine, bovine serum albumin, a plant mannosidase, glucose oxidase and the therapeutically relevant antibody trastuzumab were efficiently labelled with a fluorescent probe in a two-step approach combining eY-click and strain-promoted azide-alkyne cyclization (SPAAC). The proteins conserved their structural integrity as observed by circular dichroism and the trastuzumab conjugate showed a similar binding affinity for the natural HER2 ligand as shown by bio-layer interferometry. Compared to our previously described protocol with PhUr, eY-click with NMeLum species showed faster reaction kinetics, higher (complete) Y-chemoselectivity and reactivity, and offers the interesting possibility of the double tagging of solvent-exposed Y.
| S-EPMC8635215 | biostudies-literature
Project description:The 123I-labeled meta-iodobenzylguanidine (MIBG) is an analogue of noradrenaline that can evaluate cardiac sympathetic activity in scintigraphy. Quantitative analysis of 123I-MIBG images has been verified in patients with heart failure and neurodegenerative diseases. However, quantitative results differ due to variations in scintigraphic imaging procedures. Here, we created and assessed the clinical feasibility of a calibration method for 123I-MIBG imaging. The characteristics of scintigraphic imaging systems were determined using an acrylic calibration phantom to generate a multicenter phantom imaging database. Calibration factors corresponding to the scintigraphic imaging procedures were calculated from the database and applied to a clinical study. The results of this study showed that the calibrated analysis eliminated inter-institutional differences among normal individuals. In summary, our standardization methodology for 123I-MIBG scintigraphy could provide the basis for improved diagnostic precision and better outcomes for patients.
| S-EPMC7736873 | biostudies-literature
Project description:It has been demonstrated that HER3 plays an important role in some human cancers and the HER3 expression is associated with worse survival in solid tumors.This study was conducted to compare HER3 expression in epithelial lining of radicular cysts (RCs), dentigerous cysts (DCs) and odontogenic keratocysts (OKCs).This was a descriptive-analytical study, which assessed all 57 paraffin blocks of RCs, DCs and OKCs (21 RCs, 16 DCs, 20 OKC) from pathological archive of Dentistry College of Zahedan, Iran. The HER3 expression in cytoplasm and membrane was examined by immunohistochemical method. The data collected was analyzed using SPSS16 by ANOVA and Chi-square. P < 0.05 was considered as statistically significant.The HER3 expression had positive results in 52.4% of OKC, 50% of DC and only 20% of RC samples. There was a significant difference between HER3 expression in OKCs and RCs.The HER3 expression in developmental odontogenic cysts was higher than that in inflammatory odontogenic cysts. The higher rate of HER3 expression in OKC may justify inherent growth potential, stimulation-independent proliferation capability, invasive growth and high recurrence rate of the cyst accepted today as a tumor.
| S-EPMC4698317 | biostudies-other
Project description:Despite great interest in the use of silica mesoporous nanoparticles (MSNs) in drug delivery little is known on their biological fate. Positron emission tomography (PET) studies of radiolabelled MSNs face a major difficulty due to the degradation of the MSNs during circulation as it is difficult to assign activity values to either the MSNs or their degradation products. Here, a PET study is conducted using two strategies of labelling. MSNs are either radiolabelled in the core by complexation with silanols from the MSNs with 89Zr, or on the MSN coating through attachment of 131I radiolabelled Lin-TT1 (AKRGARSTA), a homing peptide for targeting cancer tissue. Results from the biodistribution of MSNs with the two labels are compared, obtaining meanful information on the fate of MSNs. While MSNs accumulate in liver and spleen, MSN degradation products 89Zr or silicate bearing the radioisotope, are found in the bones and probably in lungs. A partial detachment of the peptide from the surface of the MSN is also observed. This work highlights the importance of choosing an appropriate labelling strategy for nanoparticles since core or surface labelling may result in different particle biodistribution if the labelled component degrades or the label detaches.
| S-EPMC9417343 | biostudies-literature
Project description:Purpose: PD-L1 expression in the pretreatment tumor microenvironment enriches for response to anti-PD-1/PD-L1 therapies. The purpose of this study was to quantitatively compare the performance of five monoclonal anti-PD-L1 antibodies used in recent landmark publications.Experimental Design: PD-L1 IHC was performed on 34 formalin-fixed paraffin-embedded archival melanoma samples using the 5H1, SP142, 28-8, 22C3, and SP263 clones. The percentage of total cells (including melanocytes and immune cells) demonstrating cell surface PD-L1 staining, as well as intensity measurements/H-scores, were assessed for each melanoma specimen using a computer-assisted platform. Staining properties were compared between antibodies.Results: Strong correlations were observed between the percentage of PD-L1(+) cells across all clones studied (R2 = 0.81-0.96). When present, discordant results were attributable to geographic heterogeneity of the melanoma tissue section rather than differences in PD-L1 antibody staining characteristics. PD-L1 intensity/H-scores strongly correlated with percentage of PD-L1(+) cells (R2 > 0.78, all clones).Conclusions: The 5H1, SP142, 28-8, 22C3, and SP263 clones all demonstrated similar performance characteristics when used in a standardized IHC assay on melanoma specimens. Reported differences in PD-L1 IHC assays using these antibodies are thus most likely due to assay characteristics beyond the antibody itself. Our findings also argue against the inclusion of an intensity/H-score in chromogenic PD-L1 IHC assays. Clin Cancer Res; 23(16); 4938-44. ©2017 AACR.
| S-EPMC6175606 | biostudies-literature
Project description:AimsBreast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs.Methods and resultsTwenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2).ConclusionsIHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs.
| S-EPMC7225035 | biostudies-literature
Project description:In psittacine birds, round cell neoplasms that originate from lymphocytes, plasma cells, histiocytes, or mast cells are sporadic and poorly described. The lack of morphological and immunohistochemical diagnostic criteria or grading schemes make specific diagnoses and prognoses challenging. We assessed cases of psittacine birds diagnosed with round cell neoplasia from 3 North American veterinary diagnostic laboratories to describe the diagnostic features of these tumors. For all cases, demographic data, anatomic distribution, histological features, and immunoreactivity for T (CD3) and B (Pax5 and MUM-1) cell markers were assessed using tissue microarrays and whole slide mounts. Thirty-eight psittacine birds representing 14 species were included. Tumors were mainly infiltrative and multicentric, were composed of homogenous sheets of round to polygonal cells, and commonly presented with a high mitotic count (average 21 mitoses per high-power field). Based on Pax5 immunoreactivity, B-cell lymphoma was most common (19/38 [50%]), and was significantly associated with involvement of the gastrointestinal and urogenital systems. Of the 38 cases, 6 (16%) were consistent with T-cell lymphoma, 3 (8%) with plasma cell tumor, and 3 (8%) were double-reactive for both B- and T-lymphocyte markers. This is the first study to describe morphologic and immunohistochemical features of round cell neoplasia in a large number of psittacine birds, and provides benchmark data for future studies aimed at elucidating the diagnosis and prognosis of these neoplasms. These data also provide useful information about reactivity of commercially available antibodies as lymphocyte markers in tissues of multiple psittacine species.
| S-EPMC8290990 | biostudies-literature
Project description:BACKGROUND: Computer image analysis techniques have decreased effects of observer biases, and increased the sensitivity and the throughput of immunohistochemistry (IHC) as a tissue-based procedure for the evaluation of diseases. METHODS: We adapted a Cyan/Magenta/Yellow/Key (CMYK) model for automated computer image analysis to quantify IHC stains in hematoxylin counterstained histological sections. RESULTS: The spectral characteristics of the chromogens AEC, DAB and NovaRed as well as the counterstain hematoxylin were first determined using CMYK, Red/Green/Blue (RGB), normalized RGB and Hue/Saturation/Lightness (HSL) color models. The contrast of chromogen intensities on a 0-255 scale (24-bit image file) as well as compared to the hematoxylin counterstain was greatest using the Yellow channel of a CMYK color model, suggesting an improved sensitivity for IHC evaluation compared to other color models. An increase in activated STAT3 levels due to growth factor stimulation, quantified using the Yellow channel image analysis was associated with an increase detected by Western blotting. Two clinical image data sets were used to compare the Yellow channel automated method with observer-dependent methods. First, a quantification of DAB-labeled carbonic anhydrase IX hypoxia marker in 414 sections obtained from 138 biopsies of cervical carcinoma showed strong association between Yellow channel and positive color selection results. Second, a linear relationship was also demonstrated between Yellow intensity and visual scoring for NovaRed-labeled epidermal growth factor receptor in 256 non-small cell lung cancer biopsies. CONCLUSION: The Yellow channel image analysis method based on a CMYK color model is independent of observer biases for threshold and positive color selection, applicable to different chromogens, tolerant of hematoxylin, sensitive to small changes in IHC intensity and is applicable to simple automation procedures. These characteristics are advantageous for both basic as well as clinical research in an unbiased, reproducible and high throughput evaluation of IHC intensity.
| S-EPMC1810239 | biostudies-literature
Project description:Changing climatic conditions and unsustainable land use are major threats to savannas worldwide. Historically, many African savannas were used intensively for livestock grazing, which contributed to widespread patterns of bush encroachment across savanna systems. To reverse bush encroachment, it has been proposed to change the cattle-dominated land use to one dominated by comparatively specialized browsers and usually native herbivores. However, the consequences for ecosystem properties and processes remain largely unclear. We used the ecohydrological, spatially explicit model EcoHyD to assess the impacts of two contrasting, herbivore land-use strategies on a Namibian savanna: grazer- versus browser-dominated herbivore communities. We varied the densities of grazers and browsers and determined the resulting composition and diversity of the plant community, total vegetation cover, soil moisture, and water use by plants. Our results showed that plant types that are less palatable to herbivores were best adapted to grazing or browsing animals in all simulated densities. Also, plant types that had a competitive advantage under limited water availability were among the dominant ones irrespective of land-use scenario. Overall, the results were in line with our expectations: under high grazer densities, we found heavy bush encroachment and the loss of the perennial grass matrix. Importantly, regardless of the density of browsers, grass cover and plant functional diversity were significantly higher in browsing scenarios. Browsing herbivores increased grass cover, and the higher total cover in turn improved water uptake by plants overall. We concluded that, in contrast to grazing-dominated land-use strategies, land-use strategies dominated by browsing herbivores, even at high herbivore densities, sustain diverse vegetation communities with high cover of perennial grasses, resulting in lower erosion risk and bolstering ecosystem services.
| S-EPMC8931791 | biostudies-literature
Project description:In the United States, colorectal cancer (CRC) is the second-leading cause of cancer-related deaths. The most effective strategy for prevention of CRC is to screen for and remove precancerous polyps. There are various ways to screen for CRC. In the United States, the most common method is colonoscopy. Polyps are classified primarily through pathology. Size is the primary risk factor for malignancy. In general, the bigger the polyp, the greater the risk for malignancy. There are 3 basic options for removal: standard polypectomy; advanced resection techniques, known as endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD); and surgical removal. In the past 10 years, the use of surgical removal has significantly decreased. Noninvasive, nonmalignant polyps can be removed endoscopically. EMR and ESD are very effective and achieve similar clinical outcomes. Both procedures begin with a submucosal injection. The submucosal lift is one of the most significant advances that have been made in polypectomy. Traditionally, the approach to achieving a submucosal lift has relied on the use of a saline solution. Saline is inexpensive and widely available, but it dissipates quickly. Various viscous agents have been added to saline to maintain mucosal lifting throughout the procedure. Although most are effective, they are used off-label. The only solution approved by the US Food and Drug Administration for the submucosal lift procedure is Eleview. In a clinical trial, Eleview decreased the mean total injected volume and the mean total injected volume per lesion as compared with saline. Other advances in polypectomy techniques include the use of cold snare polypectomy and high-definition colonoscopes. The quality of the colonoscopy can also be improved if a physician knows and monitors his or her adenoma detection rate.
| S-EPMC5721691 | biostudies-other