The origin and identification of unknown events associated with low-level leucocyte counting by flow cytometry

Flow cytometric enumeration of residual leucocytes (WBC) in leucocyte-depleted components occasionally reveals a population of events that stain with propidium iodide, but which are outside the main counting region. These extraregional events may result in discrepancies in residual WBC counts.

To identify the origin of these unknown events, separate populations of mononuclear cells and polymorphonuclear neutrophils (PMNs) (prepared by density-gradient centrifugation) were spiked into leucocyte-depleted red cell concentrates (RCC) to a concentration of 150 cells/ microl and assessed, using LeucoCount and DNA Prep reagents, by flow cytometry. In addition, isolated WBC nuclei, DNA in saline, or enzymatically digested free DNA, was spiked into diluted whole blood (final concentration 5 microg/ml).

Isolated WBC nuclei fell in the main counting region of the dot plot. Extraregional events were generated in leucocyte-depleted RCC spiked with PMN (17/ microl by day 4), but not with mononuclear cells, and increased with sample storage (4 days). A significantly (P < 0.01) greater number of extraregional events was observed using the LeucoCount reagent, compared with the DNA Prep reagent. Enzymatically digested WBC nuclei or free DNA, added to diluted whole blood, generated extraregional events using LeucoCount, but not the DNA Prep reagent. When free DNA was enzymatically digested, no extraregional events were observed.

These previously unidentified events are probably fragmented nuclei or free DNA originating from PMN owing to a combination of ageing and reagent addition. Currently, in our protocols, the region used for WBC enumeration counts intact WBC nuclei. To achieve WBC counting consistency, flow cytometric gating protocols must be standardized, and a decision taken as to whether to include extraregional events in the count.