Sawako KAWAUCHI, Mari KONO, Yuri TAKAGI, Atsushi WADA, and Takashi MORIKAWA
Scientific Affairs, Sysmex Corporation
The newly launched XN-Series ( Sysmex Corporation, Kobe, Japan ) multi-parameter automated hematology analyzers contain a new channel - the WPC channel, which detects blasts and abnormal cells of the lymphocyte class. In this channel, dedicated WPC reagents penetrate the cell membranes of leukocytes and stain the intranuclear DNA. The forward scattered light ( FSC ), side scattered light ( SSC ), and side fluorescent light ( SFL ) of the cells are then measured and expressed as two 2D scattergrams, the WPC and the WPC ( SSC-FSC ) scattergram.
Here, we examined the WPC scattergram distribution of the 3 major subtypes of leukocytes ( T-lymphocytes, monocytes, and neutrophils ) isolated from healthy volunteers. We also performed some molecular analyses to elucidate the underlying reasons for lymphocyte distribution. We confirmed T-lymphocytes, monocytes, or neutrophils from healthy volunteers were located in the left, middle, or right cluster of the WPC scattergram. Moreover, T-lymphocytes were separated in 2 clusters in the WPC ( SSC-FSC ) scattergram, and T-lymphocytes had the lowest FSC intensity, followed by monocytes and neutrophils. Confocal laser scanning fluorescence microscopy showed that all 3 leukocyte subtypes had the same stain intensity level following incubation with WPC reagents. Transmission electron microscopy after treatment with WPC reagents revealed that the intracellular complexity was the lowest in lymphocytes, followed by monocytes, and it was the highest in neutrophils; this order was consistent with the order of SSC intensities. Additionally, the intracellular structure of T-lymphocytes showed 2 types: some cells had bulging nuclei and little cytoplasm, whereas others retained most of their cytoplasm with organelles and had dense nuclei. Scanning electron microscopy after treatment with the reagents showed that T-lymphocytes were the smallest cell type, followed by monocytes and neutrophils. Surface morphology of T-lymphocytes also showed 2 types: some cells were much smaller than control and had abnormally smooth surfaces, and others had the size and surface-structures equivalent to control cells. Finally, by using a general-purpose flow cytometer, we investigated why T-lymphocytes exhibited 2 kinds of morphology, and found that most small lymphocytes lost their surface antigen. This result suggests that T-lymphocytes with highly damaged cell membranes appear to be smaller post treatment.
In summary, the 3 major leukocyte subtypes form 3 major clusters in the WPC scattergram. From the left of the scattergram, the appearance was in the order of T-lymphocytes, monocytes, and then neutrophils. The results also show that T-lymphocytes appear in 2 clusters in the WPC ( SSC-FSC ) scattergram according to the level of cell membrane damage.
Multi-parameter Automated Hematology Analyzer, XN Series, WPC channel, Scattergram