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Haematology Measurement Technologies

Modern automated blood cell counters use various technologies to determine the parameters of a full blood count including the method recommended by the ICSH (International Committee for Standardization in Haematology) for measuring haemoglobin concentration- the cyan-methaemoglobin method. DC sheath flow detection method and Fluorescence flow cytometry which is used to analyse physiological and chemical properties of cells. 

SLS Detection Method

Haemoglobin is a routine diagnostic parameter in each blood count. The method recommended by the ICSH (International Committee for Standardization in Haematology) for measuring haemoglobin concentration is the cyan-methaemoglobin method.

Our SLS haemoglobin detection method uses cyanide-free sodium lauryl sulphate (SLS). The reagent haemolyses red blood cells and white blood cells in the sample. The chemical reaction begins by altering the globin and then oxidising the haeme group. Now the SLS’ hydrophilic groups can bind to the haeme group and form a stable, coloured complex (SLS-HGB), which is analysed using a photometric method.

An LED sends out monochromatic light and by moving through the mixture light is absorbed by the SLS-HGB complexes. The extinction is measured by a photo sensor and is inversely proportional to the haemoglobin concentration of the sample.

Absorption photometric methods are usually influenced by the turbidity of the sample itself. In blood samples, turbidity can be caused due to lipaemia or leucocytosis. By using the SLS-HGB method these interferences can be minimised due to the effect of the reagent.

DC Sheath Flow Detection Method

Sysmex analysers use the DC sheath flow detection method to count red blood cells and platelets, RBVC and PLT. A portion of blood is separated from the aspirated whole blood and mixed with the diluent in a pre-set ratio.

Of this dilution a defined amount is sent to the detection chamber and passed through a small opening, known as the aperture. There are also electrodes on each side of the aperture – and direct current passes through these electrodes. The direct current resistance between the electrodes changes as blood cells suspended in the diluent pass through the aperture. This resistance causes an electrical pulse change proportional to the size of the blood cell.

These electrical data are converted into graphical displays of volume distribution curves, or histograms.

Once the cells leave the sample nozzle exit they are surrounded by a sheath flow of diluent. Here, they are aligned and moved to the centre of the orifice. This reduces interference errors and the possibility of abnormal cell pulse detection, which could be caused by cells passing through the transducer off-centre. As soon as the cells have passed the orifice, they are seized by another, inverse flow and immediately led to the drain. This prevents renewed circulation and a change in the platelet count.

Fluoresecence Flow Cytometry

Fluorescence flow cytometry is used to analyse physiological and chemical properties of cells. It can also be used to analyse other biological particles in urinalysis analysers. It provides information about cell size, structureand interior

In flow cytometry, we examine cells and particles while they are flowing through a very narrow flow cell. First a blood sample is aspirated and proportioned, then diluted to a pre-set ratio and labelled with a proprietary fluorescence marker that binds specifically to nucleic acids.

Next the sample is transported into the flow cell. The sample is illuminated by a semiconductor laser beam, which can separate the cells using three different signals:

  • forward scattered light (forward scatter or FSC)
  • side scattered light (side scatter or SSC)
  • side fluorescence light (side fluorescence or SFL)

 

The intensity of the forward scatter indicates the cell volume. The side scatter provides information about the cell content, such as nucleus and granules. The side fluorescence indicates the amount of DNA and RNA present in the cell. Cells with similar physical and chemical properties form a cluster in a graph known as a scattergram.

The principle of fluorescence flow cytometry is used in different analysers for haematology and urinalysis. For blood cell counts we use fluorescence flow cytometry, e.g. for the WBC and differential, for NRBC counting and reticulocyte measurement.  

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