The Three Functional Fluidics assays under CLIA, offered clinically, have a PLA code?
- PLA Code 012U: Flow adhesion (FA) on VCAM1 substrate on Whole Blood
- Looks for adhesion of RBC on VCAM1
- PLA 0122U: Flow Adhesion (Fa) on P-Selectin substrate on Whole Blood
- Looks at adhesion of WBC on P-Selectin, but can have a contribution from RBC as well
- PLA 0123U: Mechanical fragility (MF)
- Looks at RBC propensity to hemolysis under applied mechanical stress
Whole Blood Adhesion on VCAM1 PLA Code 012U & PSEL PLA Code 0122U Process:
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 air forces 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).
LA 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.
For any questions about our tests please complete the webform below or contact:
Functional Fluidics Laboratories
440 Burroughs Street, Suite 641
Detroit, MI 48202