scholarly journals Selecting an Optimal Positive IHC Control for Verifying Antigen Retrieval

2019 ◽  
Vol 67 (4) ◽  
pp. 275-289 ◽  
Author(s):  
Seshi R. Sompuram ◽  
Kodela Vani ◽  
Anika K. Schaedle ◽  
Anuradha Balasubramanian ◽  
Steven A. Bogen
Keyword(s):  
Amino Acids ◽  
Positive Control ◽  
Formalin Fixation ◽  
Primary Antibody ◽  
Antigen Retrieval ◽  
Analyte Concentration ◽  
Peptide Epitopes ◽  
Variable Level ◽  
Definition Of ◽  
Formalin Fixed

Positive immunohistochemistry (IHC) controls are intended to detect problems in both immunostaining and heat-induced epitope retrieval (HIER). However, it is not known what features in a control are important for verifying HIER. Contrary to expectation, the fact that a tissue is formalin-fixed does not necessarily render it suitable in verifying proper HIER. Some tissue controls, for some immunostains, strongly stain even without HIER. Consequently, the control may verify the immunostain but provide little or no information regarding the HIER step. To sort this out, we used formalin-fixed peptide epitopes, a model that provides for precise definition of analyte concentration, epitope composition, and degree of fixation. Our data demonstrate that formalin fixation generates a variable level of protein epitope masking, depending on the epitope recognized by the primary antibody. Some epitopes are highly masked while others hardly at all. Furthermore, the ability of amino acids in the epitope to react with formaldehyde can, at least in part, account for this variability. Most important, we demonstrate the importance of selecting a positive control with a low or intermediate analyte concentration (relative to the immunostain’s analytic sensitivity). High analyte concentrations can be insensitive in verifying the HIER step.

National HER2 Proficiency Test Results Using Standardized Quantitative Controls: Characterization of Laboratory Failures

2008 ◽  
Vol 132 (2) ◽  
pp. 211-216
Author(s):  
Kodela Vani ◽  
Seshi R. Sompuram ◽  
Patrick Fitzgibbons ◽  
Steven A. Bogen
Keyword(s):  
Positive Control ◽  
Antigen Retrieval ◽  
Test Results ◽  
Her2 Testing ◽  
Tissue Sections ◽  
Staining Protocol ◽  
Invasive Breast Carcinomas ◽  
Microscope Slides ◽  
Formalin Fixed

Abstract Context.—An important component in fostering test standardization for HER2 testing by immunohistochemistry is an appropriate positive control. We developed a new standardized, quantitative immunohistochemical HER2 control using a HER2 peptide covalently attached to glass microscope slides. The peptide controls can be formalin fixed or unfixed, providing the new capability of distinguishing errors associated with antigen retrieval from errors associated with the staining process itself. Objective.—To investigate the causes of variability in HER2 immunohistochemistry staining performance. By comparing laboratory performance with both formalin-fixed and unfixed analyte controls, we aimed to distinguish problems associated with antigen retrieval from reagent or staining protocol deficiencies. Design.—HER2 peptide analyte controls were printed on 2 slides that also contained unstained sections of invasive breast carcinomas and were mailed with the College of American Pathologists' 2006 HER2-B proficiency testing survey. Laboratory participants were asked to stain the 2 slides and return them for central review and quantification. This study is unique in combining central review with new quantitative HER2 controls. Results.—Of 109 participants who returned evaluable stained slides, staining was suboptimal in 20 (18.3%) as judged by quantification of the peptide analyte controls and review of tissue sections. Of those, 35% failed due to antigen retrieval errors, 20% failed due solely to antibody or staining protocol problems, and the remainder failed due to a combination of the two. Conclusions.—In practice, errors in HER2 testing are caused by variables associated with antigen retrieval and the reagents and staining protocol, as well as interpretive error. Analyte controls help distinguish these different causes.

scholarly journals Antibody Identification for Antigen Detection in Formalin-Fixed Paraffin-Embedded Tissue Using Phage Display and Naïve Libraries

Antibodies ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Célestine Mairaville ◽  
Pierre Martineau
Keyword(s):  
Phage Display ◽  
Formalin Fixation ◽  
Antigen Retrieval ◽  
Alternative Strategies ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Antibody Libraries ◽  
Formalin Fixed

Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.

scholarly journals Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 889
Author(s):  
Pooja Lahiri ◽  
Suranjana Mukherjee ◽  
Biswajoy Ghosh ◽  
Debnath Das ◽  
Basudev Lahiri ◽  
...  
Keyword(s):  
Comprehensive Evaluation ◽  
Antigen Retrieval ◽  
Tissue Fixation ◽  
Ftir Microspectroscopy ◽  
Protein Secondary Structures ◽  
Associated Proteins ◽  
Cancer Tissues ◽  
Formalin Fixed ◽  
Fixation System

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

scholarly journals Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections.

1993 ◽  
Vol 41 (11) ◽  
pp. 1599-1604 ◽  
Author(s):  
S R Shi ◽  
B Chaiwun ◽  
L Young ◽  
R J Cote ◽  
C R Taylor
Keyword(s):  
Androgen Receptor ◽  
Enzymatic Digestion ◽  
Antigen Retrieval ◽  
Urea Solution ◽  
Paraffin Sections ◽  
Buffer Solution ◽  
Solution Ph ◽  
Citrate Buffer ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed

We developed a staining protocol for demonstration of androgen receptor (AR) in formalin-fixed, paraffin-embedded tissue sections. The method is based on the antigen retrieval microwave (MW) heating technique. Results are compared with different types of enzyme digestion pre-treatments. The strongest immunostaining signal and clearest background were obtained by MW heating of dewaxed paraffin sections in 5% urea or citrate buffer solution (pH 6); pure distilled water gave less consistent results. Enzymatic digestion with pepsin (0.05% in 2 N HCl) for 30 min at room temperature, or trypsin followed by pronase, or pronase digestion alone, also produced enhanced staining of AR in some cases, but there was more nonspecific background, and specific reactivity was less intense. The antigen retrieval MW method can be used to demonstrate AR epitope in prostate tissue after fixation in formalin for as long as 7 days. AR immunolocalization was also compared in frozen and paraffin sections processed from the same specimen of prostate carcinoma tissue and was found to be qualitatively and quantitatively similar. This study also provided new information concerning the basic principles of the antigen retrieval MW method that may be helpful in further development of this technique.

scholarly journals IMMUNOHISTOCHEMICAL LOCALIZATION OF HUMAN CHORIONIC GONADOTROPIN

1962 ◽  
Vol 115 (2) ◽  
pp. 289-294 ◽  
Author(s):  
A. R. Midgley ◽  
G. B. Pierce
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Hydatidiform Mole ◽  
Immunohistochemical Localization ◽  
Formalin Fixation ◽  
Cell Of Origin ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Immunohistochemical Techniques ◽  
Formalin Fixed

Through the use of immunohistochemical techniques, human chorionic gonadotropin has been localized to syncytiotrophoblastic cells of immature placenta, hydatidiform mole, chorioadenoma destruens, and choriocarcinoma. No gonadotropin has been detected in cytotrophoblast. Evidence is discussed which suggests that syncytiotrophoblast is the cell of origin of human chorionic gonadotropin. The observation that formalin fixation did not alter the ability of human chorionic gonadotropin to react with its specific antibody permitted the study of formalin-fixed paraffin-embedded tissues stored in the tissue collection. In addition, the excellence of histologic preparations following formalin fixation facilitated cytologic identification.

scholarly journals A Molecular Mechanism of Formalin Fixation and Antigen Retrieval

2004 ◽  
Vol 121 (2) ◽  
pp. 190-199 ◽  
Author(s):  
Seshi R. Sompuram ◽  
Kodela Vani ◽  
Elizabeth Messana ◽  
Steven A. Bogen
Keyword(s):  
Molecular Mechanism ◽  
Formalin Fixation ◽  
Antigen Retrieval

scholarly journals Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the ‘RED’ family)

1994 ◽  
Vol 304 (1) ◽  
pp. 95-99 ◽  
Author(s):  
G Labesse ◽  
A Vidal-Cros ◽  
J Chomilier ◽  
M Gaudry ◽  
J P Mornon
Keyword(s):  
Amino Acids ◽  
Sequence Alignment ◽  
Active Site ◽  
Tertiary Structure ◽  
Binding Specificity ◽  
Single Domain ◽  
Divergent Evolution ◽  
Cofactor Binding ◽  
Sequence Identity ◽  
Definition Of

Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution.

scholarly journals Human GATA-3 trans-activation, DNA-binding, and nuclear localization activities are organized into distinct structural domains.

1994 ◽  
Vol 14 (3) ◽  
pp. 2201-2212 ◽  
Author(s):  
Z Yang ◽  
L Gu ◽  
P H Romeo ◽  
D Bories ◽  
H Motohashi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Zinc Finger ◽  
Nuclear Localization ◽  
Structural Domains ◽  
Cellular Genes ◽  
Dna Binding Site ◽  
Discrete Domains ◽  
Definition Of ◽  
Site Recognition

GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors.

scholarly journals Amino acid requirements of pigs. 3. Requirement for apparent digestible threonine of pigs in different stages of growth.

1990 ◽  
Vol 38 (3B) ◽  
pp. 609-622
Author(s):  
N.P. Lenis ◽  
J.T.M. van Diepen
Keyword(s):  
Amino Acids ◽  
Growth Performance ◽  
Control Diet ◽  
Maximum Growth ◽  
Positive Control ◽  
Negative Control ◽  
Net Energy ◽  
Finishing Pigs ◽  
Amino Acid Requirements ◽  
Limiting Amino Acids

Individual and group housed crossbred pigs 45 to 105 kg and 65 to 95 kg in experiments 1 and 2, respectively, were given basal diets with L-threonine 0.6, 1.2 and 1.8 g/kg. Positive and negative control diets contained total threonine 5.7 and 4.5 g/kg, respectively. To prevent other amino acids being limiting, the negative control diet was supplemented with lysine, methionine, tryptophan, isoleucine, histidine and valine. The positive control diet was supplemented with lysine and methionine. The requirement for total threonine of growing-finishing pigs for maximum growth performance was about 5.6 g/kg in a diet containing net energy 9.4 MJ/kg. This figure corresponds with about 4.7 g/kg apparent faecal digestible threonine and 4.3 apparent ileal digestible threonine. There was no difference between the growing and the finishing pigs. The requirement for ileal digestible threonine, relative to ileal digestible lysine requirement, was about 64%. It is concluded that dietary protein can be reduced by 2 percentage units without any adverse effect on growth performance, if limiting amino acids are sufficiently supplemented. (Abstract retrieved from CAB Abstracts by CABI’s permission)

scholarly journals Comparison of Heart Valve Circumference Examined Before and After 10% Formalin Fixation

2021 ◽  
Vol 73 (7) ◽  
pp. 478-484
Author(s):  
Watcharit Anantakal ◽  
◽  
Somboon Thamtakerngkit ◽  
Vijarn Vachirawongsakorn ◽  
◽  
...  
Keyword(s):  
Mitral Valve ◽  
Aortic Valve ◽  
Heart Valve ◽  
Tricuspid Valve ◽  
Heart Valves ◽  
Formalin Fixation ◽  
Pulmonic Valve ◽  
Fresh State ◽  
Before And After ◽  
Formalin Fixed

Objective: To compare the heart valve circumference before and after 10% formalin fixation. Materials and Methods: The study analyzed 63 Thai human cadaveric hearts. Each heart valve circumference was separately measured in the fresh state by specifically designed equipment. After that, the hearts were fixed in 10% formalin for 3 days. Then each heart valve circumference was measured by the same equipment and by the thread and ruler technique. The results were analyzed using SPSS package to find the association between the heart valve circumference before and after formalin fixation. Results: This study showed that the average circumferences of the heart valve measured in the fresh state were 13.329 cm in the tricuspid valve, 10.617 cm in the mitral valve, 8.416 cm in the pulmonic valve, and 7.122 cm in the aortic valve. The average circumferences of the heart valve measured after 10% formalin fixation were 11.019 cm in the tricuspid valve, 8.714 cm in the mitral valve, 6.751 cm in the pulmonic valve, and 6.089 cm in the aortic valve. The average ratios of the heart valve circumference measured fresh and after 10% formalin fixation were 0.8267 in the tricuspid valve, 0.8235 in the mitral valve, 0.8050 in the pulmonic valve, and 0.8573 in the aortic valve. There were significant differences in the heart valve circumference between the fresh state and after formalin fixation (p < 0.001). Conclusion: This study revealed important information on the dimensional changes of all the formalin-fixed heart valves. We found that the heart valve shrank after formalin fixation, with the formalin-fixed hearts an estimated 0.8 times smaller than the fresh cadaveric hearts.

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