Application note describes use of assay system which overcomes limitations of manual counting by staining and counting only the nucleated white blood cells
A new application note describing the ViaCount assay and its use in counting white blood cells in unlysed whole human blood is now available from the Guava Technologies.
Counting peripheral white blood cells in whole blood by traditional methods can often be a tedious process.
The overwhelming number of red blood cells often complicates the process and leads to large variability in counts, particularly with microscopy counting methods where debris from lysed red blood cells can obscure the hemacytometer field.
The new application note describes how using the Guava ViaCount assay overcomes these limitations by staining and counting only the nucleated white blood cells in numbers sufficient to be statistically reproducible.
In addition, these absolute cell counts can be achieved without the typical red blood cell lysis step.
The protocol described was developed for analysing human peripheral blood samples.
However, the protocol may be extended to analysing blood from non-human sources by making adjustments in sample preparation and instrument settings to account for natural differences in cell characteristics and cell concentration.
Rather than looking at the generalised staining of cells based solely on membrane integrity, Guava ViaCount employs a combination of dyes that specifically bind to DNA.
The first of these dyes penetrates membranes, entering and staining the DNA of all nucleated cells.
The second is a viability dye that penetrates and stains DNA in dead and dying cells with compromised membrane integrity, and is excluded by cells with intact membranes.
Cells that take up intermediate amounts of the viability dye also react with Annexin V, indicating they have entered the apoptotic pathway leading to cell death.
Moreover, dye that is not bound to DNA does not produce a fluorescent signal, further improving resolution of the ViaCount assay.
