WBC and Differential measurement The XE-2100 WBC and Differential measurement

Principles of cell detection Wbc and differential counts are determined using: Flow Cytometry Method Semiconductor Laser. Blood cell information is obtained using: Forward light scatter Lateral light scatter Lateral fluorescent light

Optics

Specific Measurements

The Stain • Staining Reagent: – Polymethine Dye (Group of staining reagents): X = O, S, Se, N-alkyl or C(Ch 3 ) n R 1,2 = alkyl (1-6C) ; R 3,4 = Alkyl, Methoxy or NO n = 0-1

The Wavelength The new stain opens up the possibility of using a different laser wavelength on a compact laser system with little need of increases in power  = 633nm Red laser beam

Methodologies

WBC/BASO Channel Blood sample is aspirated, diluted to a ratio of 1:50 with STOMATOLYSER-FB. Diluted sample is sent to WBC/BASO detection chamber. In this channel, forward and side scatter signals are used to derive the WBC and BASO count.

Measurements Forward Scatter - Measurement of size Side Scatter - Measurement of internal structure i.e size of nucleus

The WBC/BASO Channel Lysing Surfactant BASO Other WBC RBC

WBC/BASO Scattergram

WBC Classification Blood sample is aspirated and diluted with: STOMATOLYSER-4DL and STOMATOLYSER -4DS. Diluted sample is sent to the detector chamber Cells are classified according to side scatter and fluorescence signals in the Diff channel.

Measurements Side scatter - measurement of internal structure. Fluorescence - measurement of nucleic acid content.

The DIFF Channel Abnormal Lymph/ Lymphoblast 1.Lysing 2.Staining High Fluorescence Intensity High Fluorescence Low Fluorescence Blast, I.G. Normal Cell STROM-4DL STROM-4DS Lysing surfactant Polymethine Dye +Organic Acid

Diff Scattergram

Neutrophil Count This is a calculated parameter # Neut = Neut/Baso count(diff channel) - Baso count (WBC/Baso channel)

IMI Channel discriminates between immature and mature white blood cells. Detection is performed using RF/DC detection method.

Methodology Blood is aspirated and diluted to a ratio of 1:250 with STOMATOLYSER-IM. Diluted sample is sent to the IMI detector for analysis. A two-dimensional distribution of cell size and cell structure is drawn according to RF/DC detection method.

Measurements Direct current - measurement of cell volume Radio frequency - measurement of internal cell structure.

IMI Scattergram Platelet Clumps

ACAS & Advanced Discrimination

Adaptive Cluster Analysis System Cells are classified by the process of cluster analysis which is based on learning algorithms. (An astronomical technique!) This allows optimal adaptation to biological differences between samples including abnormal cells

Advanced Discrimination DIFF-CH WBC/BASO-CH 150+-1 Gst/NLME Bas/NLME NLME:WBC except BASO Ly/Ne2 Ly/Mo2 150+-1 Mo/Eo Ly/Mo1 Mo/NE Ly/Ne1 Ne/Eo Ly/Ba Ly/Gst Ba/Gst Ne/Gst

Extended ACAS Principle High adaptation to individual changes Moving clusters Moving discriminators Enhanced separation in abnormal cases Low “Vote Out” rate