Sickle Cell Imaging Flow Cytometry Assay (SIFCA)

Fertrin, Kleber Yotsumoto; Samsel, Leigh; Beers, Eduard J. van; Mendelsohn, Laurel; Kato, Gregory J.; McCoy, J. Philip

doi:10.1007/978-1-4939-3302-0_20

Cited by 15 publications

(14 citation statements)

References 7 publications

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“…Postfixation cells were washed and analyzed for shape change using the Amnis ImageStream X Mark II Imaging Flow Cytometer (MilliporeSigma). Shape change was quantitated using IDEAS application software (MilliporeSigma) using a modified protocol (SIFCA) provided by Gregory J. Kato (Vascular Medical Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA) (49,50).…”

Section: Methodsmentioning

confidence: 99%

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

Krishnamoorthy¹,

Pace²,

Gupta³

et al. 2017

JCI Insight

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Section: Methodsmentioning

confidence: 99%

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

Krishnamoorthy¹,

Pace²,

Gupta³

et al. 2017

JCI Insight

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“…The Range mask was created using the System 80, Range Area 350-1,200 pixels and Range Aspect Ratio 0.3-1 masks to reach a tighter fit to the actual contour of the BF region, and thus providing an accurate measurement of cellular information for the individual cell. This range is slightly different as compared to the RangeSystem mask at van Beers and coworkers (18) which indicates that Area varies between 350 and 5,000 pixels, and Aspect Ratio is in the range of 0-1. The combination of Shape Ratio (that is, minimum thickness of the masked object divided by the length) and Area features as a Sickle Index shape feature (= Shape Ratio×1,000/Area) enhances the selection of the "true" sickle cell region boundary from other delineated SS-RBC phenotypes (Fig.…”

Section: Finding An Optimal Shape Quantification Factor For Deoxygenamentioning

confidence: 69%

“…This has not been shown to be practical for the assessment of large populations of RBC with a highly heterogeneous morphology that is different from sample to sample. Imaging flow cytometry (IFC) combines the single cell image‐based screening of microscopy with the high‐throughput speed of conventional FCs . IFC can provide information regarding cellular structures, such as cell size, shape, orientation, signal intensity, location, and texture for each cell with high statistical validity through bright‐ and dark‐field channels, which require no staining procedures, and fluorescence channels.…”

mentioning

confidence: 99%

Image‐Based Flow Cytometry and Angle‐Resolved Light Scattering to Define the Sickling Process

Goktas

Sukharevsky

Larkin³

et al. 2019

Cytometry Pt A

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Red blood cells (RBCs) from sickle cell patients exposed to a low oxygen tension reveal highly heterogeneous cell morphologies due to the polymerization of sickle hemoglobin (HbS). We show that angle‐resolved light scattering approach with the use of image‐based flow cytometry provides reliable quantitative data to define the change in morphology of large populations of RBCs from sickle cell patients when the cells are exposed for different times to low oxygen. We characterize the RBC morphological profile by means of a set of morphological and physical parameters, which includes cell shape, size, and orientation. These parameters define the cell as discocyte, sickle, elongated, as well as irregularly or abnormal RBC shaped cells, including echinocytes, holly‐leaf, and granular structures. In contrast to microscopy, quick assessment of large numbers of cells provides statistically relevant information of the dynamic process of RBC sickling in time. The use of this approach facilitates the understanding of the processes that define the propensity of sickle blood samples to change their shape, and the ensuing vaso‐occlusive events in the circulation of the patients. Moreover, it assists in the evaluation of treatments that include the use of anti‐sickling agents, gene therapy‐based hemoglobin modifications, as well as other approaches to improve the quality of life of sickle cell patients. © 2019 International Society for Advancement of Cytometry

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“…Sickling of samples was assessed using imaging flow cytometry in air. 33 However, glutaraldehyde must be removed from each sample prior to flow cytometry, preventing application of this assay to a high-throughput setting.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Assay to Screen Small Molecules for Their Ability to Prevent Sickling of Red Blood Cells

et al. 2022

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Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic “sickled” shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput “sickling assay” that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z ′-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD.

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Sickle Cell Imaging Flow Cytometry Assay (SIFCA)

Cited by 15 publications

References 7 publications

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

Image‐Based Flow Cytometry and Angle‐Resolved Light Scattering to Define the Sickling Process

High-Throughput Assay to Screen Small Molecules for Their Ability to Prevent Sickling of Red Blood Cells

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