Cell-based Fluorescence Assays Research Articles

Screening of Eight Biofortified Rice Varieties Based on Total Content of Zinc and Iron as Well as Its Uptake by Caco-2 Cells: An In Vitro Cellular Model

Zinc (Zn) and iron (Fe) are two essential micronutrients that help the body to maintain its normal physiology and metabolism. Rice is the most important staple crop because it nourishes approximately half of the world’s population. The present study was intended to screen a set of eight rice varieties for total grain Zn and Fe content along with their uptake in differentiated Caco-2 cells. The total grain Zn and Fe content, as well as their bioaccessibility, was determined using inductively coupled plasma mass spectrometry (ICP-MS) techniques. The Caco-2 cell-based fluorescence assays and ferritin synthesis assays were used to determine Zn and Fe uptake, respectively. The transcriptional expression of micronutrient transporters has been analyzed in Caco-2 cells in response to the uptake of Zn and Fe from in vitro digested rice samples. The findings of this study revealed a comparative analysis of two prominent micronutrient contents in eight different varieties of polished rice. DRRDhan 48 (4.93 ± 0.10 mg/kg), Zinco Rice (4.65 ± 0.01 mg/kg), and Protozin (4.43 ± 0.03 mg/kg) were shown to have low phytate content, and high grain Zn content (32.49 ± 2.8, 27.9 ± 1.8, 26.7 ± 1.2 mg/kg, respectively), as well as significantly greater Zn uptake in differentiated Caco-2 cells. The outcome of this study provides an easy and convenient approach for the screening of food items or cereals based on micronutrient uptake.

AQP4 autoantibody assay performance in clinical laboratory service.

Objective:To compare performance of contemporary aquaporin-4–immunoglobulin (Ig) G assays in clinical service.Methods:Sera from neurologic patients (4 groups) and controls were tested initially by service ELISA (recombinant human aquaporin-4, M1 isoform) and then by cell-based fluorescence assays: fixed (CBA, M1-aquaporin-4, observer-scored) and live (fluorescence-activated cell sorting [FACS], M1 and M23 aquaporin-4 isoforms). Group 1: all Mayo Clinic patients tested from January to May 2012; group 2: consecutive aquaporin-4-IgG–positive patients from September 2011 (Mayo and non-Mayo); group 3: suspected ELISA false-negatives from 2011 to 2013 (physician-reported, high likelihood of neuromyelitis optica spectrum disorders [NMOSDs] clinically); group 4: suspected ELISA false-positives (physician-reported, not NMOSD clinically).Results:Group 1 (n = 388): M1-FACS assay performed optimally (areas under the curves: M1 = 0.64; M23 = 0.57 [p = 0.02]). Group 2 (n = 30): NMOSD clinical diagnosis was confirmed by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20; and M1-ELISA, 18. Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2. Group 3 (n = 31, suspected M1-ELISA false-negatives): results were positive for 5 sera: M1-FACS, 5; M23-FACS, 3; and M1-CBA, 2. Group 4 (n = 41, suspected M1-ELISA false-positives): all negative except 1 (positive only by M1-CBA). M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.Conclusion:Aquaporin-4-transfected CBAs, particularly M1-FACS, perform optimally in aiding NMOSD serologic diagnosis. High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

ICSH/ICCS practice guidelines special issue

ICSH/ICCS practice guidelines special issue

Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: Proposed practical method

Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell-based fluorescence assays performed on flow cytometers are currently lacking. Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined. For all assays and most instrument platforms, <5 replicates were found adequate to validate assay imprecision levels below the 5-10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95% CI and the cumulative mean for replicates, termed variance factor (VF), provide a data-driven means for determining optimal replicate numbers. The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate that three to four replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes.

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society.

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part V – assay performance criteria

Multi‐color flow cytometry is a unique technology, which enables the analysis of heterogeneous cellular systems and provides multiparametric information on a cell‐by‐cell basis. A variety of factors contribute to the complexity of validating cell‐based flow cytometric methods, including the lack of fully characterized cellular reference materials and the difficulty in obtaining, or creating, samples with varying levels of a given cell type or varying levels of expression of a given antigen. This document summarizes validation requirements and describes validation strategies for quasi‐quantitative and qualitative cell‐based flow cytometric assays. © 2013 International Clinical Cytometry Society

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part IV – postanalytic considerations

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations.

Determination of optimal replicate number for validation of imprecision using fluorescence cell based assays: Proposed practical method.

Background: Assay validation includes determination of inherent imprecision across the reportable range. However specific practical guidelines for determinations of precision for cell based fluorescence assays performed on flow cytometers are currently lacking. Methods: Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical IVD use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 (hENT1) quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell (HSC) enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals of the mean (95%CI), standard deviation and coefficient of variation (CV) of sequential replicates were determined. Results: For all assays and most instrument platforms <5 replicates were found adequate to validate assay imprecision levels below the 5-10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95%CI and the cumulative mean for replicates, termed variance factor (VF), provide a data driven means for determining optimal replicate numbers. Conclusions: The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate 3-4 replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes. © 2013 Clinical Cytometry Society.

Development and Validation of a High-Throughput Screening Assay for Human Long-Chain Fatty Acid Transport Proteins 4 and 5

Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to-background ratios were between 3- and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC(50)) of 0.21 and 0.63 microM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z' factor of 0.6 +/- 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC(50) of 2.4 and 0.22 microM, respectively. To increase throughput, a single time point measurement in a 384-well format was developed using the Analyst HT, and the results are comparable with the 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, whereas differentiated cell lines are useful for a secondary drug screen.

Segmentation of heterogeneous blob objects through voting and level set formulation

Blob-like structures occur often in nature, where they aid in cueing and the pre-attentive process. These structures often overlap, form perceptual boundaries, and are heterogeneous in shape, size, and intensity. In this paper, voting, Voronoi tessellation, and level set methods are combined to delineate blob-like structures. Voting and subsequent Voronoi tessellation provide the initial condition and the boundary constraints for each blob, while curve evolution through level set formulation provides refined segmentation of each blob within the Voronoi region. The paper concludes with the application of the proposed method to a dataset produced from cell based fluorescence assays and stellar data.