Haematology Analyzer

A brief discussion about the instrument. The purpose of it. the way of handling..

1. HAEMATOLOGY ANALYZER Automated Cell Counting Instrumentation Presented By: Sukanya Halder M.Tech, Biotechnology KIIT School Of Biotechnology

2. Hematology analyzers are computerized , highly specialized and automated machines that count the number of different kinds of white and red blood cells in a blood sample. Definition:

3. History:

4.   Cell identification errors in manual counting : M ostly associated with distinguishing lymphocytes from monocytes B ands from segmented forms and abnormal cells (variant lymphocytes from blasts) L ymphocytes overestimated Monocytes underestimated Slide cell distribution error Increased cell concentration along edges Disadvantages of manual cell counting

5. Advantages

6. Advantages..

7. The results they provide are collectively known as complete blood counts (CBCs) . complete complete blood with differentiation of cells (CBCs with diff). Some cell counters can process 120-150 samples per hour . Results:

8. Hematology analyzer has revolutionised

9. Automated techniques of blood counting: Semi-automated instruments Require some steps, as dilution of blood samples. Often measure only a small number of variables. Fully automated instruments Require only that an appropriate blood sample is presented to the instrument . They can measure 8-20 variables including some new parameters which do not have any equivalent in manual methods. Types :

10. Hematology analyzers count cells by one of five general cell techniques : Flow C ytometry The use of fluorescent dyes The electrical impedance method The light scatter method Radiofrequency Working

11. Flow C ytometry is based on ejecting cells from a nozzle at high speed in a fluid. Each cell passes through several laser beams so that different optical properties can be measured. 1:Flow Cytometry/Cell sorting

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13. Biochemical or antigenic properties are usually classified using fluorescent dyes. 2:Use of flourescent dyes

14. Steps : Labeling with flourescent dye. A laser excites these fluorescent molecules. They emit light at various wavelengths . Amount of fluorescence can give an indication as to what percentage of various cell types are present in the sample. Continue….

15. Works on coulter principle: A stream of cells in suspension passes through a small aperture across which an electrical current is applied. Each cell that passes alters the electrical impedance and can thus be counted and sized. 3:Electrical impedance method

16. Particles such as blood cells are nonconductive BUT are suspended in an electrically conductive diluent. As a dilute suspension of cells is drawn through the aperture, the passage of each individual cell momentarily increases the impedance (resistance) of the electrical path between two electrodes that are located on each si.de of the aperture Continue

17. A blood cell's size , surface charge , concentration of the cells , shape of cells can be determined Continue…

18. The application of light scatter means that as a single cell passes across a laser light beam diffraction(bending around corners ), refraction (bending due to change in speed) and reflection (light rays turned back by obstruction) Light scatter correlates to cell volume/size Side angle/orthogonal light scatter correlates to degree of internal complexity (granules and nucleus) 4:Optical scatter method

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20. The patterns of scatter are measured at various angles . Multi angle polarized scatter separation (M.A.P.S.S) 0° : indicator of cell size 10° : indicator of cell structure and complexity 90° polarized: indicates nuclear lobularity 90° depolarized: differentiates eosinophils Continue …

21. Scattered light provides information about cell structure , shape , Reflectivity These characteristics can be used to differentiate the various types of blood cells and to produce scatter plots with a five-part differential. Continue…

22. 5:Radiofrequency

23. Examples

24.     Fluoro -chromes combine with the RNA of the reticulocytes . Fluorescent cells can then be enumerated using a flow cytometer . An automated retic counter also permits the assessment of retic maturity since the more immature reticulocytes have more RNA  An automated retic count

25. Coulter Instrumentation

26. Coulter hematology analyzer

27. Principles of Measurement Direct Measurement: RBC – DC detection (Impedance), hydrodynamic focusing WBC – DC detection (Impedance), hydrodynamic focusing Platelets – DC detection (Impedance), hydrodynamic focusing (2-30 fl ) Hgb – SLS- Hb (555 nm) ( oxyhemoglobin + sodium lauryl sulfate) HCT – cumulative pulse height detection (see oscilloscope) Indirect Measurement: MCV, MCHC, and MCH (calculation) WBC differential DC/RF detection employs differential lysis Reticulocyte Fluorescent detection

28. Symax hematology analyzer

29. Automated and portable hematology analyzers

30. Some problems which could be faced : Two cells passing through the orifice at the same time, counted as one cell . RBC agglutination(clump of cells) Counting bubbles or other particles as cells. Drawbacks

31. Questions????

32. Thank you