Background

Since the foundation of Biocytex in the early 90's in the close proximity of the University and Hospital Complex of La Timone / La Conception in Marseille (France), the need of standardization in flow cytometry analysis still remains a topic of concern. Inter-laboratory reproducibility, day to day reproducibility and positivity and/or negativity threshold determination are a real need that can be addressed by the use of quantitative flow cytometry application kits. This is the expertise offered by Biocytex. We spent 7 years developing and tuning our quantitative flow cytometry technology. After the success of our first generation of products with the QIFIKIT, we introduced our PLATELET kits, a comprehensive line of no wash whole blood flow cytometry kits for the monitoring of antiplatelet drugs (PLATELET VASP/P2Y12) as well as the diagnosis of platelet disorders (PLATELET Gp/Receptors).
The same quantitative technology was applied to the development of kits for leukocyte and red cell receptor analysis (CELLQUANT / REDQUANT kits). A major application is offered for the standardized diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH).
In the past two years, the aim of BioCytex was to increase its range of products in the field of Thrombosis and Haemostasis.

Quantitative flow cytometry technology

On a small sample volume containing few cells and heterogeneous cell populations, flow cytometry allows to analyze with high sensitivity the cell size, its contents and the intensity of these stained cells.
Although in current practice of immunophenotyping flow cytometers are mainly used to differentiate positive and negative staining of cells, BioCytex uses these same instruments to quantitate molecules bound on cells, receptors, ligands or drugs as well as intracellular targets.

How does quantitative flow cytometry work ?

Quantitative flow cytometry is achieved by comparing fluorescence intensity and a calibration standard.
BioCytex uses a calibrator made of a mix of bead populations . These calibration beads are coated with known and increasing numbers of immunoglobulins (IgG) bound to their surface thus mimicking the binding of specific monoclonal antibodies to the biological sample cells.
Then, this calibrator and the target stained cells are incubated with polyclonal antibodies labeled with FITC fluorochrome. The beads bear specific number of sites and will allow to draw a calibration curve relating mean fluorescence intensity into a number of receptors per cell. The mean fluorescence intensity (MFI) of the tested sample is reported on the calibration curve to determine the number of molecules expressed per cell. Non specific staining is evaluated using an irrelevant antibody (negative isotypic control).

The critical parameters to succeed in antigen quantitation include :

	Reaching saturation of antigen sites with the appropriate IgG monoclonal antibodies
	Using appropriate isotypic controls to evaluate specific binding, especially for low level detection
	Using beads which mimic cells bearing different amounts of antibody. The beads must offer specific features such as size and background fluorescence to correctly match the features of the cells of interest
	Calibrating the fluorescence response of the instrument
	Standardizing and calibrating the antibody binding capacity. Even in the same cluster differentiation (CD) specificity, several monoclonal antibodies may reveal different numbers of antigen sites since they will recognize different epitopes
	Finally, pre-analytical variables have to be established and controlled for any specific application.

These requirements has led BioCytex to the development of kits including all necessary reagents, not only calibrators but also antibodies, buffers, agonists and a written procedure for in vitro diagnostic use of these applications.

Why using Biocytex quantitative flow cytometry technology ?

Numerous biological molecules present an interest in the measurement of their expression level. Indeed any modulation of receptor expression level associated to physiological differentiation, pathological state or therapeutic treatment brings valuable information to the biologists and the physicians.
Such measurements in Clinical Biology require full reliability in term of day to day, inter instrument and lab-to-lab reproducibility.
As an example, an ambulatory patient treated with a new generation of antiplatelet drugs will be easily monitored wherever he travels.

What are the major applications for the BioCytex quantitative flow cytometry technology ?

BioCytex focus is Thrombosis and Haemostasis diagnosis and drug monitoring especially arterial thrombosis and platelets.
The R&D efforts of the major pharmaceutical companies to generate new and safer antiplatelet aggregation drugs offer a growing market for the BioCytex technology applied to platelet receptor analysis.
Indeed platelet analysis typically illustrates the interest of quantitative flow cytometry. On a clinical point of view, there will be more interest to measure the efficacy of a drug to modulate the functional expression of platelet receptor rather than attempting to interpret the relative frequency of a particular subset of platelet population.
Because flow cytometry offers the capability of platelet analysis directly on whole blood, it can also serve as an easy alternative to old reference functional assays such as platelet aggregometry.
Although platelets are one of the most accessible blood players in thrombosis and haemostasis disorders, platelets are just starting to be explored for the diagnosis and drug monitoring purposes.
Platelets are also a frequent target of auto-immune drug induced adverse reactions. Finally, much effort is devoted to the design and pharmacology studies of new antiplatelet drugs.

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