Functional Fluidics proprietary biomarkers are unique diagnostic platforms that assess red blood cell health by replicating the environment that red blood cells experience in our bodies.
Whole Blood Adhesion on VCAM1 PLA Code 0121U:
- A peripheral (venous) blood sample is drawn in a sodium citrate tube (1.7 ml, 2,9% sodium citrate, blue top vacutainer).
- Small volume of the sample (~ 100 uL) is extracted and diluted 2-fold with the buffer containing Mg2+, Ca2+ and with about 30 uL of the diluted blood used per microfluidic channel to measure the adhesion.
- Microfluidic channels are coated with the selected ligand (substrate, i.e., VCAM-1, P-Selectin or collagen). The flow is regulated by an external control unit consisting of an air compressor and electro-pneumatic regulator. Compressed airforces blood samples through micro-fluidic channels across a typical viewing window (350 um width x 75 um height). Samples are flowed through the channels under the pulsative flow (1 Hz) at 1 dyne/cm2 at 37 oC for 3 minutes flow time.
- Channels are then washed with reverse buffer flow at 1 dyne/cm2 for 10 minutes to eliminate non-adhered cells. FA-Cell adhesion is then visualized, in real-time, through bright-field or fluorescent microscopy.
- Digital images of the viewing arear are acquired with a high-resolution CCD camera using an automated process under the manual guidance and supervision. 3 images are acquired from 3 different regions of the viewing area of each channel with 4 channels used for each sample.
- Acquired images represent the final cell adhesion under the selected flow rate.
- FA-Images are analyzed with Montage imaging software (Molecular Devices, Downingtown, PA), and adhered cells are counted using a combination of automatic process and manual editing and counting within each regions of the viewing area with the results averaged per channel.
- An adhesion index (in cells/mm2) is established based on the cell adhesion quantification in the four channels used for the sample.
- Laboratory staff then documents the AI in laboratory report form, a laboratory director certifies the results, and the report is transmitted to a provider FAA-Remaining adherent cells are quantified, followed by a sequential increase in shear (5, 10, and 20 dyne/cm^2) to generate an avidity adhesion index (AAI).
- FAC-Adhered cells are fixed with 4% formalin and stained following an adhesion assay (FF-FA, FF-FAR, FF-FAA). Fluorescence microscopy is utilized to differentiate between and quantify specific cell populations.
- FAD- Time-lapse images are acquired to measure cell rolling / sliding along the channel surface. Mean velocity for rolling objects and cell flux are measured to generate a dynamic adhesion index (dAI).
PLA Code 0123U: Mechanical Fragility Process:
- Whole blood is diluted to 0.5 g/dL hemoglobin concentration is subjected to a predefined mechanical shear stress within a single use cuvette. Each sample is measured 3 time (with 3 cuvettes are used).
- The stress is applied using a proprietary electromagnetic bead mill that is causing oscillation (at 5Hz) of a magnetic cylindrical bead with biocompatible coating contained within the cuvette with the sample.
- The sample is then probed optically with follow-up spectrophotometric analysis using proprietary algorithms.
- The analysis allows to non-invasively determine the amounts of extra- and intra-cellular hemoglobin with the ration of the values used to calculate the amount of stress-induced hemolysis.
- Measurements are done at progressively increasing stress duration to obtain Mechanical Fragility Profiles (Hemolysis vs Time). Mechanical Fragility Indices (MFI) for RBC fractions with different stability are calculated using an automated process after the manual curve fitting performed by the operator.
- Laboratory staff then documents the MFI values in the laboratory report form, a laboratory director certifies the results, and the report is transmitted to a provider.
Why Choose Functional Fluidics?
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Proprietary Lab Tests
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CLIA Certified Lab
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Complex Tests Experience
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Leadership Access
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Specialized Staff
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Strong Industry Relationships
Functional Fluidics Assays
Our suite of proprietary cell function assays can help validate assumptions or support clinical claims.
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Flow Adhesion of whole blood on VCAM-1 (FA-WB-VCAM)
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Flow Adhesion of whole blood on P-Selectin (FA-WB-Psel)
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Mechanical Fragility – Normoxia (MF)
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Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS)
Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) that produce severe pain, organ damage, and early death. Lack of reliable biomarkers to objectively define VOEs, hinders the development of clinically useful interventions to improve the care for these patients.
Functional Fluidics recently participated in a ground-breaking study involving sickle cell patients. This non-interventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization.
The study data suggest that Functional Fluidics FA-WB-VCAM assay may serve as a predictive biomarker for impending VEEs, and a monitoring biomarker to assess response to SCD-modifying therapies.
LEARN MORE Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by Electronic Patient-Reported Outcomes, Actigraphy, and Biomarkers
Functional Fluidics Biomarker Assay Featured in ELIPSIS article in American Society of Hematology (ASH) Blood Magazine
Key Points
- Feasibility of monitored out-of-hospital pain and patient-reported VOC days as endpoints for clinical trials in SCD is demonstrated.
- ePROs, actigraphy, and laboratory biomarkers enable improved identification and assessment of in-hospital and out-of-hospital VOCs.
Publications
Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by Electronic Patient-Reported Outcomes, Actigraphy, and Biomarkers
This non interventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization.
Red Blood Cell Mechanical Fragility as Potential Metric for Assessing Blood Damage Caused by Implantable Durable Ventricular Assist Devices: Comparison of Two Types of Centrifugal Flow Left Ventricular Assist Devices.
Implantable Ventricular Assist Devices (VADs) have become a treatment of choice for patients with end-stage heart failure or cardiogenic shock, significantly increasing both survival rates and the quality of life of patients.
Individual Variability in Response to a Single Sickling Event for Normal, Sickle Cell, and Sickle Trait Erythrocytes
Hemoglobin S (Hb-S) polymerization is the primary event in sickle cell disease causing irreversible damage to red blood cell (RBC) membranes over repeated polymerization cycles.
Sevuparin blocks sickle blood cell adhesion and sickleleucocyte rolling on immobilized L-selectin in a dose dependent manner
Adhesion of sickle red blood cells (SSRBC) to the vascular endothelium may initiate and propagate vascular obstruction in sickle cell disease (SCD)
Impact of Environment on Red Blood Cell ability to Withstand Mechanical Stress.
Susceptibility of red blood cells (RBC) to hemolysis under mechanical stress is represented by RBC mechanical fragility (MF), with different types or intensities of stress potentially emphasizing different perturbations of RBC membranes
An Approach to Measuring RBC Haemolysis and Profiling RBC Mechanical Fragility
Red blood cells (RBC) can be damaged by medical products, from storage or from disease. Haemolysis (cell rupture and haemoglobin release) is often a key indicator, with mechanical fragility (MF) offering the potential to assess sub-haemolytic damage as well
Low Molecular Weight Heparin Inhibits Sickle Erythrocyte Adhesion to VCAM-1 through VLA-4 Blockade in a Standardized Microfluidic Flow Adhesion Assay
The vaso-occlusive events in sickle cell disease (SCD) begin in early childhood, warranting the need for more preventative and therapeutic interventions for those affected
Impact of the Oscillating Bead Size and Shape on Induced Mechanical Stress on Red Blood Cells and Associated Hemolysis in Bead Milling
While in circulation, red blood cells (RBC) need to elastically undergo large deformations without lysing, an ability that may be compromised by cell membrane damage.