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  • Scientific Calendar 2014
    Scientific Calendar 2014

Measurement principles


In a two-stage reaction, the cell membrane of the white blood cells is perforated in the first stage, during which the cells remain largely intact. In the second stage, the nucleic acids in the cells are labelled with a special fluorescence marker. The cell membrane of nucleated red blood cells (NRBC) is completely lysed and only their nuclei are labelled. In the scattergram, the cells are differentiated based on their fluorescence and size. Because fluorescence is now also used in the WNR channel, interferences from lipids and lyse-resistant RBC are reduced to a minimum.


The unique lysis reagent initially perforates the cell membranes while leaving the cells largely intact. Subsequently, intracellular DNA and RNA are labelled with a fluorescence marker. The improved formulation of the two new reagents results in a mild reaction, leaving most of the blood cell structure intact. As a result, even better separation is achieved, particularly of the lymphocytes and monocytes. In the scattergram, the cells are differentiated according to their fluorescence and internal structure. The intensity of the fluorescence signal is directly proportional to the nucleic acid content of the cell. Some of the strongest fluorescence signals are shown by immature and activated cells, due to their high RNA content. Therefore, these are successfully detected and can even be counted


The lysis reagent initially perforates the cell membranes while leaving the cells largely intact. In a second step, the fluorescence marker labels the intracellular nucleic acids and the resulting fluorescence signal is directly proportional to the nucleic acid content. Reticulocytes emit a higher fluorescence signal than mature red blood cells, which no longer contain RNA, and a considerably lower fluorescence signal than white blood cells. Since the RNA content decreases during the maturation process of the reticulocytes, one can determine three parameters that reflect these maturation stages: low fluorescence ratio (LFR), medium fluorescence ratio (MFR) and high fluorescence ratio (HFR).


In a two-stage reaction, the cell membranes of the platelets are first perforated, leaving the cells largely intact. Subsequently, the newly developed fluorescence marker specifically labels the platelets and in doing so almost completely masks interfering particles such as RBC fragments. The measured fluorescence signal is directly proportional to the degree of maturity of the platelets.


The first stage of the reaction is affected by the composition of the cell membranes: the lysis reagent specifically affects lipids and therefore the lipid content of the cell membranes determines cell perforation. This affects the permeability of the membranes for the novel fluorescence marker that labels the cells in the second stage of the reaction. For example, the membranes of abnormal lymphocytes have a high lipid content and are therefore labelled to a greater extent than normal lymphocytes. This allows reliable differentiation of these two populations. In contrast, blasts show some of the lowest fluorescence signals due to their low membrane lipid content. Combining the information of cell size, intracellular complexity and fluorescence intensity also allows further differentiation and reliable identification of conspicuous cells like neoplastic lymphocytes (Abnormal Lympho? flag), which previously were difficult to distinguish from normal or reactive lymphocytes (Atypical Lympho? flag). In addition, large blasts can be reliably identified due to their combination of low fluorescence signal and a high forward-scattered signal (Blasts? flag).


The SLS-haemoglobin detection method uses a cyanide-free reagent containing sodium lauryl sulphate (SLS). The reagent lyses red blood cells and white blood cells in the sample. The chemical reaction begins by altering the globin and then oxidising the haem group. This allows the hydrophilic groups of the SLS molecule to bind to the haem group and form a stable, coloured complex (SLS-HGB), which is analysed using a photometric method. Monochromatic light is emitted by an LED and absorbed by the SLS-HGB complexes in the reaction mixture. The absorbance is measured by a photo sensor and is proportional to the haemoglobin concentration of the sample.


Red blood cells and platelets are counted in the RBC/PLT channel using the sheath flow DC (direct current) detection method. A diluted sample is brought into a sheath flow and passed through a detector. The sheath flow directs the cells into a specified path at the center of the detector unit. While passing through the aperture the blood cells cause an electrical resistance proportional to their volume which is recorded as a pulse. These electrical data are converted into graphical displays of volume distribution curves, or histograms.

Scientific Calendar 2015

Advanced clinical parameters

Peer-reviewed literature list

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