Automation/Instrumentation

• historically cell counts were performed using a counting chamber, now they are fully automated
• full blood count is collected in a purple bottle coated in potassium EDTA an anticoagulant which stops blood clotting as it removes the calcium
• the full blood count is the first test when screening anaemia, platelets and number of blood cells
• full blood count is a combination of:

  • electrical impedance
  • direct current (DC) sheath flow detection
  • laser light scatter
  • fluorescence flow cytometry
  • haemoglobin quantification
• RBC 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 as the centre 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
• DC sheath flow detection is closed sampling - better for the scientist as there is no direct contact with the sample
• block of the current in DC sheath flow detection shows a cell - the amount of current blocked tells cell type (e.g. big = RBC, small = Platelets)
• laser light scatter is used to count the number of WBC that pass and their approximate nucleus size
• the intensity of forward-scattered light indicates the cell volumes
• side-scatter of light provides information about the cell content such as nucleus and granules
• side fluorescence indicates the amount of DNA and RNA present in the cell - the fluorescent molecule fluoresces at 633nm causing it to then release energy at a higher wavelegnth
• the semiconductor laser used in laser light scatter has a wavelength of 633nm
• in a DIFF channel a lysis reagent perforates the cell membrane while leaving the cells largely intact and intracellular DNA and RNA are labelled with a fluorescence marker - the cells are differentiated according to their fluorescence and internal structure. the intensity of the fluorescence is directly proportional to the nucleic acid content of the cell
• in a DIFF scattergram monocytes lymphocytes and eosinophils are all able to be differentiated however neutrophils and basophils must be done by hand
• for haemoglobin quantification, the SLS-haemoglobin detection method is used as it uses a cyanide-free reagent containing sodium lauryl sulphate (SLS)
• sodium lauryl sulphate lyses red blood cells and white blood cells in a 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
• to test blood coagulation, the sample is centrifuged to get plasma then clotting is initiated and when a clot starts to form the reaction solution becomes more turbid and a change in optical density is observed - time is measured from initiation to a stable end point
• blood coagulation test samples are taken in light blue capped bottles containing sodium citrate which removes calcium
• the time is recorded in seconds with the normal range to form a clot being 26-34s, and a higher APTT value indicates haemophillia
• an immunoassay is similar to ELIZA microparticles are coated with antibodies/antigens to target analyte/antibody in the sample. they are bound an acridinium labelled conjugate is added then an activation solution to generate light. the amount of light generated is proportional to the amount of analyte/antibody present
• an immunoassay is used to measure serum ferritin, serum vitamin B12 and serum folate levels
• light generated in an immunoassay os proportional to the number of sample molecules