Comparison of the Leukocyte differentiation Scattergrams Between the XN-Series and the XE-Series of Hematology Analyzers

• Comparison of the Leukocyte differentiation Scattergrams Between the XN-Series and the XE-Series of Hematology Analyzers

AUTHOR(S)

Sawako KAWAUCHI, Yuri TAKAGI, Mari KONO, Atsushi WADA and Takashi MORIKAWA

Scientific Research Division, Scientific Affairs, Sysmex Corporation

SUMMARY

The newly launched XN-Series multiparameter, automated, hematology analyzer features a new channel named the WDF channel. Like the DIFF channel of the XE-Series, this channel can differentiate leukocytes from cells treated with specific reagents containing detergents and fluorescent stains, by using the 2-parameter flowcytometric method. The scattergrams of the 2 channels have different patterns due to the differences in the reagents used as well as differences in the hardware and software. In particular, the WDF channel differentiates between lymphocytes and monocytes and enhances the separation capacity, thereby distinguishing it from the DIFF channel. In this study, we morphologically examined the reasons for the positional appearance of each subtype of the leukocytes on the scattergram. Additionally, we also assessed the reason why lymphocytes and monocytes separated evidently on the WDF scattergram, in terms of the influence of the reagents.

First, using the XN- and XE-analyzers, we confirmed that the lymphocytes and monocytes separated better on the WDF scattergram than on the DIFF scattergram. Next, the separation of leukocytes was assessed following treatment with the WDF or DIFF reagents by the same method using the analyzers. Fluorescence staining was performed, and the intensity of the stained area in the leukocytes was observed under the Confocal Laser Scanning Microscope ( CLSM ) ; the intracellular structure of the leukocytes was observed under the Transmission Electron Microscope ( TEM ) ; and the size and surface structures of the leukocytes were observed under the Scanning Electron Microscope ( SEM ) . Each leukocyte appeared at the same position as they are measured in whole blood. Analysis under the CLSM showed that of all the leukocytes, the staining intensity after treatment with the reagents was highest in the monocytes, followed by that in the T lymphocytes, B lymphocytes, neutrophils, and eosinophils, which correlated with the side fluorescence intensity on the WDF scattergram. In addition, observation by TEM revealed that of all the subtypes of leukocytes, the intracellular complexity after treatment with reagents was simplest in the lymphocytes, followed by that in monocytes, neutrophils, and eosinophils, which correlated with the side-scattered intensity on both the scattergrams. Moreover, observation by SEM demonstrated that after treatment with reagents, the size of the lymphocytes was the smallest, followed by that of monocytes, neutrophils, and eosinophils, which correlated with the forward-scattered intensity on the WDF ( FSC and SSC ) scattergram. These observations indicated that each leukocyte cluster was different in terms of the amount of its organelles as well as the detergent tolerance of cell membranes.

From each electron microscope observation, it was clear that the intracellular structures of the leukocytes were retained after treatment with WDF reagents compared with that after treatment with DIFF reagents. In conclusion, the separation of lymphocytes and monocytes was better demonstrated by a WDF scattergram than by a DIFF scattergram.

KEY WORDS

XN-Series, WDF Channel, Separation Ability, Blood Cell Morphology

NOTE(S)

This article is translated and republished from the Sysmex Journal Web Vol.14 No.2, 2013. (Japanese)