Flow Cytometry whole blood differential
DRAQ5™ is the open gate to flow cytometry on whole blood ....
DRAQ5™ labels all nucleated cells in a blood sample: - as the first gate in a flow cytometry analysis
DRAQ5™ can be excited at 488 nm and detected in the far-red: - combines compensation-free with FITC, R-PE fluorochromes
Recent reports have described methods to get a full and reliable differential cell count from whole peripheral blood on a single 488nm laser flow cytometer (see our review article):
- Most recently Bjornsson et al. have presented a refined one-tube methodology which directly compares the use of flow cytometry, microscopy and cell counter. The use of DRAQ5 along with 4 groups of antibodies (CD36, CD203/CD138, CD45, CD16/CD56) on the 5 fluorescence channel Beckman Coulter FC500 permitted the classification of 10 classes of nucleated cells. The key components of the methodology are an optimised RBC lysis, no-wash antibody and nuclear dye labeling and FlowCount Beads for cell enumeration. Additionally, following further dilution the DRAQ5-, CD36+ events were counted as platelets.
- An earlier method by Maples et al. used the following antibody panel (FITC-CD3, -CD16, -CD20, PE-CD64, -CD33 and PC5-CD45) and DRAQ5™ on a 488 nm laser cytometer (Fl1-Fl4). They proposed this as a reference method for differential leukocyte counting and that this CD combination (with FSC & SSC) would allow segregation of lymphocytes, monocytes, granulocytes, activated / immature granulocytes, basophils, eosinophils, nucleated red blood cell precursors and potentially other nucleated (but CD45 negative) cells such as stem cells and blood parasites. Fluorescent beads could be added to provide absolute counts.
- Flye et al. (GLIIFCA 2003) described a similar strategy to go beyond gating of a CD45+ population. Using density gradient separated unfixed bone marrow aspirates they found a combination of CD45-FITC, CD71-PE and CD34-ECD with DRAQ5™ to be suitable. Where necessary CD41 was included for megakaryocytes. By this method they were able to ascertain nucleated cell populations in fresh material, their DNA S-phase and the myeloid / erythroid ratio on a single laser cytometer with standard filter set up.
- At its simplest, for a rapid screening a washed whole blood sample can be stained with FITC conjugated antibodies against CD45 & CD4, PE conjugated antibodies against CD14 & CD8 and 10µM DRAQ5™. Using this approach, gating on DRAQ5™, it would be possible to get relative enumeration of neutrophils, lymphocytes (incl. T-cell subsets) and monocytes. With the addition of a RBC lysis step it may be possible to retain all SSC and FSC light scatter information. An alternative might be to use a sphering agent to narrow the distribution of non-nucleated cells.
References
Bjornsson et al, Cytometry Part B 2008; 74B:91-103
Maples et al, Cytometry Part A 2004; 59A(1): Abstract 97027 (page 139 of pdf).
Flye et al, GLIIFCA 12, Milwaukee October 2003 Abstract 7 (page 21 of pdf).
Pers. Comm. Dr Terence Hoy
See an alternative approach using the Laser Scanning Cytometer (LSC™) here.
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