Stabilised cellular immuno-fluorescence assay: CD45 expression as a calibration standard for human leukocytes

We first confirmed the precision of the quantitative indirect immunofluorescence (QIFI) test by demonstrating that blood lymphocytes from different individuals expressed CD45 leucocyte antigens at a very similar level (mean: 201 x 10(3) antibody binding capacity (ABC)/lymphocyte) with only minimal variation (CV% 2.5%). These values were maintained for 4 days in blood samples when kept at 20 degrees C, and for up to 14 days in samples fixed with TransFix. Using long-term stabilisation, after an initial drop of 10-15% the CD45 ABC/lymphocyte values remained stable at an 85-90% level for >1 year. These biological standards were used to check other quantitative IF techniques. The quantum simply cellular (QSC) method showed variable results (85-240%), and the QuantiBRITE method gave values as low as 30-40% of the expected values, indicating that biological standards such as CD45 ABC/lymphocytes are absolutely essential to check the performance of methods that claim to quantify immunofluorescence (IF). Next, these standards were used to establish the stabilised cellular immuno-fluorescence assay (SCIFA) as follows. The ABC x 10(3)/cell values established by QIFI on leucocyte populations such as lympho-, mono- and granulocytes were used to create calibration curves for the CD45 antigen and its isoforms CD45RA, -R0 and -RB. The same cell populations were then stained with monoclonal antibodies (MAbs) directly conjugated to different fluorochromes in order to translate the mean fluorescence intensity (MFI) values seen with the directly labelled reagents to values of ABCx10(3)/cells. Using SCIFA with a triple-colour direct IF, the display of CD45 and its isoforms were quantitated on the 'virgin' or 'unprimed' (CD45RA+) and 'primed' (CD45R0+) subsets in both the CD4+ and CD8+ T cell lineages. We also observed that the CD4+ and CD8+ T cells in transit between the 'virgin' and 'primed' subsets frequently displayed different levels of the CD45RA and -R0 molecules, pointing to the physiological variability of the CD45RA-R0 switching process. In conclusion, internal biological standards, with known stable expression of ABC/cell, should be used to evaluate IF staining patterns in a quantitative manner during routine investigations.