(7) Inexpensive Red Diode FCM.  

Can red diode lasers (635nm) operate on their own without assistance from an other light source?
In 1993 and 1994 Doornbos et al. first showed that flow cytometers with diode lasers could differentiate between lymphocytes, monocytes and granulocytes by scatter. Thereafter a 3mW 635 nm diode laser was shown to produce good immunofluorescence using CD8 MAbs conjugated to allophycocyanin (APC). Now APC and Cy-5 (indodicarbocyanin) conjugated CD4 and CD8 antibodies are available.
Legend: The scatter discrimination between lymphocytes, monocytes and granulocytes using red diode laser (RDL) light is excellent (a). When compared to FACSCalibur (b) and to other FCM, similar leucocyte differentials are obtained.

APC and Cy-5 conjugated CD4 and CD8 antibodies provide good staining using primary gating.

Legend: When CD4-APC fluorescence intensity (y) and side scatter (x) were displayed using a flow cytometer equipped with a red diode laser, the CD4++ lymphoid (smaller) and CD4+ monocytic (larger) cells were well distinguished. Similar findings were seen with CD4-Cy-5 as well as the CD8-APC and CD8-Cy-5 reagents.
 

Red diode laser flow cytometry is compatible with affordable CD4 counts [18].
Double-platform CD4 counts were performed using CD4% lymphocyte values obtained with a red diode laser FCM; absolute lymphocyte counts were obtained on a haematological analyser. Calculated absolute CD4 counts were compared to values obtained on the volumetric absolute counter Cytoron (c). These results are good (r=0.93) but can still be improved by volumetric absolute counters equipped with red diode laser (studies in progress).  
 

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