scholarly journals Strategies for H-score Normalization of Preanalytical Technical Variables with Potential Utility to Immunohistochemical-Based Biomarker Quantitation in Therapeutic Reponse Diagnostics

2011 ◽  
Vol 34 (3) ◽  
pp. 159-168 ◽  
Author(s):  
William E. Pierceall ◽  
Michele Wolfe ◽  
Jessica Suschak ◽  
Hua Chang ◽  
Yan Chen ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Immunohistochemical Analysis ◽  
Average Intensity ◽  
Positive Staining ◽  
Breast Cancers ◽  
Potential Utility ◽  
Quantitative Expression ◽  
Broad Dynamic Range ◽  
Score Normalization ◽  
Biomarker Quantitation

Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.

scholarly journals Multiplex Assays of Luminex for Identification of COVID-19 Antibodies in Patient Serum: Performance Evaluation and Comparison to ELISA

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S92-S92
Author(s):  
M S Shapiro ◽  
X Wang ◽  
D R Mendu ◽  
A Firpo
Keyword(s):  
Dynamic Range ◽  
High Titer ◽  
Mount Sinai Hospital ◽  
Antibody Testing ◽  
Luminex Assay ◽  
Multiplex Assays ◽  
Negative Results ◽  
Broad Dynamic Range ◽  
Circulating Antibodies ◽  
Mount Sinai

Abstract Introduction/Objective Mount Sinai Hospital has received emergency use authorization (EUA) from the FDA for Coronavirus Disease 2019 (COVID-19) antibody testing using ELISA. This serological assay detects and titrates the presence of circulating antibodies to COVID-19. Other platforms have aimed to achieve the credentials of the ELISA instrument, including the multiplex assays of Luminex. The platform is known to have a greater throughput (384 wells vs. 96 wells per microplate) and faster processing speed (8 hours vs. 17 hours). Methods Luminex utilizes beads that couple to the same COVID-19 antigens (mRBD and mSpike) which were utilized for the ELISA assay. The beads are read determining the mean fluorescence intensity (MFI). In order to compare the two methods, our study included 61 patients with COVID-19 at Mount Sinai Hospital, to screen and titrate their sera using Luminex, and to correspond the MFI values with the ELISA titers. Results The Luminex assay has achieved the same level of confidence as ELISA. The 61 patients, representing 30 negatives and 31 positives, are consistently identified as such on both platforms. Our data highlights 32% of patients with a low titer (<1:160), 42% of patients with a high titer (1:160 ~ 1:320), and 26% of patients with a very high titer level (>1:320). These titers correlated well with the MFI values. Based on a cutoff of 80,000 MFI, the sensitivity and specificity of the assay is 98% and 85%, respectively, with no overlapping of MFI between positive and negative results. Conclusion Overall, the study has demonstrated that the Luminex is a strong alternative for the ELISA platform. The Luminex highlights the broad dynamic range with no overlapping between positives and negatives. Migration from ELISA to Luminex, a platform with faster and greater throughput, is therefore, highly desirable.

scholarly journals A tandem giant magnetoresistance assay for one-shot quantification of clinically relevant concentrations of N-terminal pro-B-type natriuretic peptide in human blood

2021 ◽  
Author(s):  
Fanda Meng ◽  
Weisong Huo ◽  
Jie Lian ◽  
Lei Zhang ◽  
Xizeng Shi ◽  
...  
Keyword(s):  
Detection Limit ◽  
Giant Magnetoresistance ◽  
Dynamic Range ◽  
Point Of Care ◽  
Sample Volume ◽  
Small Sample ◽  
Broad Dynamic Range ◽  
Gmr Sensors ◽  
Gmr Sensor ◽  
Sample Volume Requirement

AbstractWe report a microfluidic sandwich immunoassay constructed around a dual-giant magnetoresistance (GMR) sensor array to quantify the heart failure biomarker NT-proBNP in human plasma at the clinically relevant concentration levels between 15 pg/mL and 40 ng/mL. The broad dynamic range was achieved by differential coating of two identical GMR sensors operated in tandem, and combining two standard curves. The detection limit was determined as 5 pg/mL. The assay, involving 53 plasma samples from patients with different cardiovascular diseases, was validated against the Roche Cobas e411 analyzer. The salient features of this system are its wide concentration range, low detection limit, small sample volume requirement (50 μL), and the need for a short measurement time of 15 min, making it a prospective candidate for practical use in point of care analysis.

scholarly journals Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Adam Jordan ◽  
Jenna Chandler ◽  
Joshua S. MacCready ◽  
Jingcheng Huang ◽  
Katherine W. Osteryoung ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Morphology ◽  
Dynamic Range ◽  
Enhanced Recovery ◽  
Downstream Processing ◽  
Crop Species ◽  
Content Type ◽  
Alternative Crop ◽  
Cell Harvesting ◽  
Broad Dynamic Range

ABSTRACT Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an ∼20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level. IMPORTANCE We show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives.

scholarly journals Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range

2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Scott M. Tabakman ◽  
Lana Lau ◽  
Joshua T. Robinson ◽  
Jordan Price ◽  
Sarah P. Sherlock ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Protein Microarrays ◽  
Broad Dynamic Range

A FRET based pH probe with a broad working range applicable to referenced ratiometric dual wavelength and luminescence lifetime read out

2015 ◽  
Vol 51 (28) ◽  
pp. 6145-6148 ◽  
Author(s):  
Robert J. Meier ◽  
Johann M. B. Simbürger ◽  
Tero Soukka ◽  
Michael Schäferling
Keyword(s):  
Dynamic Range ◽  
Dual Wavelength ◽  
Luminescence Lifetime ◽  
Ph Sensing ◽  
Ph Probe ◽  
Broad Dynamic Range ◽  
Europium Chelate ◽  
Ratiometric Ph Sensing

A FRET system composed of a europium chelate and carboxynaphthofluorescein enables ratiometric pH sensing with an exceptionally broad dynamic range.

scholarly journals Effect of Trehalose Supplementation on Autophagy and Cystogenesis in a Mouse Model of Polycystic Kidney Disease

Nutrients ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 42 ◽  
Author(s):  
Li-Fang Chou ◽  
Ya-Lien Cheng ◽  
Chun-Yih Hsieh ◽  
Chan-Yu Lin ◽  
Huang-Yu Yang ◽  
...  
Keyword(s):  
Drinking Water ◽  
Kidney Disease ◽  
Mouse Model ◽  
Polycystic Kidney Disease ◽  
Renal Cyst ◽  
Pure Water ◽  
Immunohistochemical Analysis ◽  
Positive Staining ◽  
Wild Type ◽  
Polycystic Kidney

Autophagy impairment has been demonstrated in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) and could be a new target of treatment. Trehalose is a natural, nonreducing disaccharide that has been shown to enhance autophagy. Therefore, we investigated whether trehalose treatment reduces renal cyst formation in a Pkd1-hypomorphic mouse model. Pkd1 miRNA transgenic (Pkd1 miR Tg) mice and wild-type littermates were given drinking water supplemented with 2% trehalose from postnatal day 35 to postnatal day 91. The control groups received pure water or 2% sucrose for the control of hyperosmolarity. The effect on kidney weights, cystic indices, renal function, cell proliferation, and autophagic activities was determined. We found that Pkd1 miR Tg mice had a significantly lower renal mRNA expression of autophagy-related genes, including atg5, atg12, ulk1, beclin1, and p62, compared with wild-type control mice. Furthermore, immunohistochemical analysis showed that cystic lining cells had strong positive staining for the p62 protein, indicating impaired degradation of the protein by the autophagy-lysosome pathway. However, trehalose treatment did not improve reduced autophagy activities, nor did it reduce relative kidney weights, plasma blood urea nitrogen levels, or cystatin C levels in Pkd1 miR Tg mice. Histomorphological analysis revealed no significant differences in the renal cyst index, fibrosis score, or proliferative score among trehalose-, sucrose-, and water-treated groups. Our results demonstrate that adding trehalose to drinking water does not modulate autophagy activities and renal cystogenesis in Pkd1-deficient mice, suggesting that an oral supplement of trehalose may not affect the progression of ADPKD.

Fluorescence lifetime-based biosensing of zinc: Origin of the broad dynamic range

1995 ◽  
Vol 5 (2) ◽  
pp. 123-130 ◽  
Author(s):  
Richard B. Thompson ◽  
Marcia W. Patchan
Keyword(s):  
Fluorescence Lifetime ◽  
Dynamic Range ◽  
Broad Dynamic Range

scholarly journals Histopathology findings of non-mass cancers on breast ultrasound

2018 ◽  
Vol 7 (6) ◽  
pp. 205846011877495 ◽  
Author(s):  
Hye Rin Kim ◽  
Hae Kyoung Jung
Keyword(s):  
Ductal Carcinoma ◽  
Immunohistochemical Analysis ◽  
Breast Ultrasound ◽  
Her2 Overexpression ◽  
Tumor Type ◽  
Breast Cancers ◽  
Mammographic Finding ◽  
Minimal Invasion ◽  
Mass Lesions

Background There is little research done on non-mass cancers (NMCs) on breast ultrasound (US). Purpose To evaluate large-sectional histopathology findings of NMCs on breast US. Material and Methods The mammographic and histopathology features of biopsy proven 36 breast cancers which showed pure non-mass lesions on US were retrospectively reviewed. Results The most common mammographic finding was microcalcification (23/35, 65.7%); fine pleomorphic microcalcification was predominant (18/23, 78.3%). The main tumor type was pure ductal carcinoma in situ (DCIS) (14/36, 38.9%) and DCIS with micro- or minimal invasion (11/36, 30.6%). Among the 25 DCIS, histologic grade was high in 15 (60.0%) and intermediate in nine (36%); comedo necrosis was seen in 17 (68%). Immunohistochemical analysis was available in 27 lesions and showed HER2-overexpression in 12 (44.4%) and triple-negative in two (7.4%). Conclusion According to our limited patient sample, NMCs on breast US were mainly associated with high-grade DCIS.

Validation of an immunohistochemical assay, CEACAM5 IHC 769, under development for use with the antibody-drug conjugate tusamitamab ravtansine (SAR408701).

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e21030-e21030
Author(s):  
Nichole LaPointe ◽  
Nick Hertle ◽  
Shu Chi Hsu ◽  
James Kellis ◽  
Nita King ◽  
...  
Keyword(s):  
Validation Studies ◽  
Dynamic Range ◽  
Phase 1 ◽  
Point Estimate ◽  
Positive Staining ◽  
Antibody Drug Conjugate ◽  
Nsclc Tissue ◽  
Membrane Staining ◽  
Drug Conjugate ◽  
Antibody Drug

e21030 Background: Despite recent advances in non-small cell lung cancer (NSCLC) treatment, a need remains for treatment options at disease progression. Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate of a humanized CEACAM5-specific monoclonal antibody linked to the maytansinoid DM4, a potent anti-tubulin agent. In a Phase 1/2 study, tusamitamab ravtansine showed promising antitumor activity in pretreated advanced non-squamous NSCLC (NSQ NSCLC) patients with high CEACAM5 expression, defined as ≥ 50% of the tumor cells per specimen with CEACAM5 positive staining at ≥ 2+ intensity (Gazzah A et al. J Clin Oncol 2020;38[15]: 9505). Sanofi and Agilent have partnered to develop an immunohistochemical (IHC) assay, CEACAM5 IHC 769, for detection of CEACAM5 protein in formalin-fixed paraffin-embedded (FFPE) NSQ NSCLC tissue using the EnVision FLEX visualization system on Dako Omnis. This assay is in development for the assessment of patients for whom tusamitamab ravtansine is being considered. Methods: CEACAM5 IHC 769 is a qualitative IHC assay using mouse monoclonal anti-CEACAM5 clone 769. Murine anti-CEACAM5 clone 769 is based on tusamitamab ravtansine, with the same specificity as the drug for CEACAM5. The clone was developed by Sanofi and used to evaluate CEACAM5 expression in a Phase 1/2 trial. The CEACAM5 IHC 769 scoring algorithm defines positive staining as any partial or complete tumor cell plasma membrane staining at ≥ 2+ intensity. Membrane staining may be linear or punctate. Staining may appear as complete or partial staining involving the basal, lateral, or basolateral aspects of the membrane, or as apical staining of the luminal aspect of the cell. CEACAM5 IHC 769 is being validated by Agilent Technologies for NSQ NSCLC at the cutoff of ≥ 50% at ≥ 2+ intensity. Internal studies included sensitivity, specificity, intra-block heterogeneity, observer precision, laboratory repeatability/precision, and robustness. Results: Sensitivity studies demonstrated detection across a dynamic range of CEACAM5 expression. Specificity studies indicated that the antibody is specific for CEACAM5. An intra-block heterogeneity study showed good overall diagnostic agreement for sections from the same tumor (point estimate ≥ 85%). Inter- and intra-observer precision, laboratory repeatability/precision, and robustness validation studies met acceptance criteria; the lower bound of the two-sided 95% confidence interval was ≥ 85% for positive, negative, and overall percent agreement. Conclusions: Internal validation studies demonstrated that CEACAM5 IHC 769 is sensitive, specific, precise, reproducible and robust in evaluating CEACAM5 expression in NSQ NSCLC tissue at the cutoff of ≥ 50% at ≥ 2+ intensity. CEACAM5 IHC 769 is being used for patient selection/stratification in ongoing Phase 2 and 3 clinical trials of tusamitamab ravtansine.

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