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- 2. Wallace Coulter (1913-1998) 2 WallaceCoulter, an American electronics engineer, developed the Coulter Principle of the counting and sizing of microscopic particles (including blood cells) during the late 1940’s into the early 1950’s. He successfully secured a US patent for the first Coulter counter in 1953 About the same time in Kobe, Japan, scientists were developing using radio frequency to count cells.
- 3. Technologies Used in AutomatedCell Counters 3 Impedance Technology Absorbance Spectrophotometry Optical Technology Fluorescent Technology Conductivity (radio frequency) Cytochemical staining Monoclonal Antibodies are starting to be utilised.
- 4. Impedance Technology 4 Earliest formof automated particle (cell) counting. Based on cells being suspended in an electrically conductive fluid. Cells pass through an aperture of known size. Hydrodynamic focussing is applied to keep diluted specimen in the middle of the stream. Current is applied across the aperture.
- 5. Impedance Technology cont’d 5 Cellsimpede current as they pass through the aperture. Each time a cell “impedes”, a pulse is created. Number of pulses equal the number of cells passing through the aperture. Size of the pulse (resistance) is proportional to the size of the cell.
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- 7. Pitfalls of ImpedanceTechnology 7 Co-incidence Counting. 2 or more cells passing through the aperture at the same time. Non-axial Passage. Cells “swirling” back into the sensing zone Deformability of cells and different cell shapes. Vacuum applied, pulling cells through the aperture distorts the shape of the cell and hence the size of the cell.
- 8. Elliptocytes Passing throughan Impedance Transducer Aperture Elliptocytes passing through the aperture impedes the current differently depending on the orientation of the cell as it passes through the aperture. Aperture 8
- 9. Aperture However if we makethe cells uniform, we see that we remove the issue of shape and orientation having an effect on the amount of current impeded. Aperture 9
- 10. Absorbance Spectrophotometry 10 Usedto measure the quantity of Haemoglobin in blood. Complete lysis of the red blood cells is required to release Haemoglobin into solution. Haemoglobin is measured at 540nm ( an LED is the light source). Haemoglobin is calculated by measuring the absorbance readings at 540nm of both the lysed specimen and the blank Haemoglobin reagent. A Photodetector measures the amount of light that passes through the lysed specimen and the blank reagent.
- 11. Absorbance Spectrophotometry cont’d 11 Thetransmitted light signal is lower when the Haemoglobin flow cell is filled with lysed specimen than when filled with the blank reagent. This difference is due to absorption by the Haemoglobin complex and is used to calculate the concentration of Haemoglobin in the blood specimen.
- 12. Specimen- Related Interference withHaemoglobin Calculations 12 High White Blood Cell counts (refer to the manufacturer’s guidelines). High level of plasma lipids. High levels of bilirubin (obstructive jaundice). Haemolysed specimens.
- 13. Optical Technology 13 Cells arecounted and classified using flow cytometry technology. Lasers are used as the light beam source and light scatter patterns at various angles are measured and collated to classify cell types. Fluorescence technology also incorporated to enumerate RNA (reticulocytes)and DNA (nucleated red blood cells and white cell abnormalities)
- 14. Optical Technology cont’d 14 Hydrodynamicfocusing of cells is achieved by sheath reagent being injected into the flow cell at a greater rate than the diluted specimen. This causes laminar flow which positions and spaces the cells into the centre of the flow cell allowing the cells to pass into the sensing zone.
- 15. Optical Technology Cont’d 15 Thelaser beam is perpendicular to the path of the cells. Light hits the cell causing light to scatter at various angles. Hence the size of the cell, it’s internal complexity and granulation are measured. If fluorescence technology is incorporated, PMT’s will measure the amount of fluorescence emitted.
- 16. Benefits of OpticalTechnology 16 Laser light can be focused on individual cells. More than 2 measurements can be made. -more information can be gathered giving better separation of cell populations. Cells are passed in single file through the flow cell. - no issue with backflow into sensing zone More realistic results. E.g. Mean Cell Volume.
- 17. Optical Technology –Flow Cytometry Laser Beam Focussed on individual cells. Scattered laser light, which is picked up by a series of detectors, allowing us to classify cells on their scatter characteristics. Sheath Fluid Surrounding sample stream Cells pass through flow cell in single file due to HYDRODYNAMIC FOCUSSING 17
- 18. Fluorescent Technology 18 Fluorescence isobtained when light is emitted at a wavelength that is higher than that of the light source RNA fluorescence measured Reticulocytes DNA fluorescence measures white cell viability, nucleated red blood cells and fragile white blood cells (eg. Smudge cells)
- 19. Analyser Running toManufacturer’s Specifications 19 Analyser background limits essentially zero. No carryover following high cell counts. Good precision. Commercial Quality Controls running within limits. Patient Moving Averages within acceptable limits
- 20. Cell Dyn Sapphire 20 Processes 106 specimens per hour in CBC mode, and only requires 117ul of blood (both open and closed modes). If there are a lot of cytopenic patients processed, there will be fewer processed per hour Processes 76 specimens per hour in CBC/Reticulocyte mode, and still only requires 117ul of blood (for both modes). WBC linearity is up to 250 x 109/L. Platelet linearity is up to 2,000 x 109/L. Utilises impedance, absorbance spectrophotometry, optical and fluorescence technologies (3 colour fluorescence). Nucleated red cells counted as part of an FBC analysis (no reflex testing required) utilising DNA fluorescence
- 21. Cell Dyn SapphireCont’d 21 Has an Argon ion solid phase (blue) laser with a wavelength of 488nm. Ability to run specimens in an extended lyse mode if lyse resistant red cells present. Red cells are “sphered” to enable consistent MCV measurement. Fully automated and intergrated Monoclonal Antibody testing for CD61 platelet counts and CD3,4 and 8 for T-cell lymphocyte typing. Kits are available for CD64 Neutrophil sepsis marker and Hb F for foetal cell enumeration. CD34 stem cell counting is at the developmental stage. White cell viability measured using DNA fluorescence.
- 22. Multi Angle Polarised ScatterSeparation Hydrodynamic focusing 22
- 23. Red Blood Celland Platelet Counting 23 Red cell count and platelet counts are obtained by both impedance and optical methods. Optical platelets are enumerated using 2 dimensional light scatter at 7°and 90º using the complexity of the platelets to effectively separate the platelets from small or fragmented red blood cells
- 24. Benefits Of Reportingthe Optical Platelet Count 24 Interfering substances NOT included in the optical platelet count. These include: circulating blast fragments, lipids, some protein complexes and cryoglobulins. Already stated improved separation between platelets and fragmented and/or microcytic red cells. The lower the platelet count, the longer the count period to give a more accurate count.
- 25. Impedance Red Celland Platelet plots vs. Optical Platelet plots 25
- 26. New Software Upgrades 26 Reticulatedplatelet counts processed when a specimen is processed in CBC/Reticulocyte mode Reportable extended White cell Differential Extended red cell parameters including red cell cytograms Extended Reticulocyte parameters
- 27. Normal Patient Reportsfrom the Cell Dyn Sapphire 27
- 28. Sysmex XE-5000 28 Haemoglobinis measured by absorbance spectrophotometry (cyanide free)using the SLS method (reagent is sodium-lauryl-sulphate). Red cells are measured by impedance technology Platelets are measured by impedance and optical light scatter technology. White cell count and differential using 3 technologies. Nucleated red blood cells and Reticulocytes are measured using Fluorescence technology. The laser is a semiconductor type.
- 29. White Cell Countand Differential on the Sysmex XE-5000 29 Uses Impedance Technology, direct current (DC). Radio frequency technology (RF). Optical and fluorescent light scatter. Neutrophils, Lymphocytes and Monocytes initially sorted by size and light scatter patterns following lysis of red cells and platelets. Eosinophils are classified by a different light scatter due to their granulation. Basophils are counted using low angle forward scatter and side scatter at 90º after further lysis of all other white cells except Basophils.
- 30. Sysmex XE-5000 30 Capable ofprocessing 150 specimens per hour on CBC, CBC+NRBC, CBC+ NRBC+WBC5DIFF modes. 113 specimens per hour in CBC+NRBC+WBC5DIFF+RET and CBC+RET modes. Approximately 38 body fluid analysis per hour. Requires 200uL of blood in the closed mode. Requires 130uL of blood in the open mode
- 31. Radio Frequency andDirect Current Methods 31
- 32. White Cell Countand Differential - Optical Values for white cell differential derived from the scatterplots. Scatterplots are a graphical representation of electronic data generated by the laser /cell interaction. Each coloured area represents a reportable parameter (the dotted regions indicate flagging areas). 32
- 33. Special Features ofthe XE- 5000 33 Ability to quantitate the RNA content of platelets when processing specimens in the reticulocyte mode. Measurement of stem cells (Human Progenitor Cells) utilising the DC/RF white cell counts and differentials. Body Fluid analysis High fluorescent Lymphocytes – reactive B Lymphocytes Additional red cell parameters ie % microcytes and % macrocytes
- 34. Technicon Advia 2120i 34 CBC CBC/Diff CBC/Diff/Retic CBC/Retic Retic 120Samples/hr 120 Samples/hr 120 Samples/hr 74 Samples/hr 74 Samples/hr Reagents used specific to request
- 35. Advia 2120i -Improved Efficiency 35 Enhanced Linearity WBC RBC Hb Plt Retic 0.02 - 400 x 10^9/L 0-7.5 x 10^12/L 0 - 225 g/L 5 - 3500 x 10^9/L 0 - 24.5% 175uL of blood required for processing in both the open and closed modes.
- 36. White Blood CellTechnology Dual WBC counts and Differentials. Optical Light Scatter. Peroxidase Staining. Ce ll Size Peroxidase Activity Nuclear Complexity 36 Ce ll Size
- 37. Red Blood CellTechnology Cellular Haemoglobin and Volume of rbc’s. Dual Haemoglobin. Accurate Morphology - rbc differential. Macro 120fL 60fL 28g/dL 41g/dL Micro HgbHyCpooncentration( g H / d y p L ) e r 37 RBC Volume (fL)
- 38. 38 Red Blood CellCytogram: Visual Analysis Normal -Thalassemia Trait Iron Deficiency Anemia Sickle Cell Anemia
- 39. 2 Dimensional PlateletAnalysis Linearity - 5 to 3500 x109 PLT/L Size range - 1 to 60 fL Refractive Index - 1.35 to 1.40 Large PLTs included RBC fragments excluded 39
- 40. Reticulocytes and NucleatedRed Blood Cells 40 Nucleated red blood cells are automatically counted when specimens are run in the CBC/DIFF mode (based on size and intensity of the nucleus). Reticulocytes are measured in a specific reticulocyte channel, and additional reticulocyte parameters are derived such as the cellular haemoglobin reticulocyte.
- 41. Special Features ofthe Advia 2120i 41 CSF Assay - FDA approved cell count and differential Cellular Haemoglobin cross checked with the colorimetric Haemoglobin method. Red cell differential - %hypo, %hyper, % micro and % macro. Cross checked WCC - Peroxidase and Baso channel.
- 42. Beckman Coulter UniCelDxH 800 42 Utilises Impedance technology for White Cell Counts, platelet counts and red cell counts. Utilises Volume, Conductivity and Scatter (VCS) principles for white cell differential and NRBC(5 angles of scatter) Random access reticulocyte testing using new methylene blue staining of reticulocytes and analysis using VCS technology Monoclonal capabilities
- 43. UniCel DxH 800 43 Processes100 specimens per hour in CBC and CBC/Diff modes (can take longer if there are a lot of cytopenic patients). Processes 45 specimens per hour in CBC/Reticulocyte mode. White cells linear from 0 – 400 x109/L Platelet counts linear from 0-3,000x109/L Sample path the same in open and closed modes and requires 165uL of blood Pre-dilute mode available - 50uL of blood to 200uL of diluent
- 44. VCS Technology -Volume V is for Volume and is obtained when the impedance white cell count is performed and the cells are grouped by size. 44
- 45. VCS Technology -Conductivity Conductivity is a measurement of cellular internal content using a high-frequency electromagnetic probe. The information gained by this method looks at the cell size, the nucleus to cytoplasm ratio as well as cell granularity and chemical composition. 45
- 46. VCS Technology -Scatter Scatter is the measurement of the light scattering characteristics of a cell , which gives valuable information on the granularity and the nuclear structure of a cell. The laser is a Helium-Neon Laser. 46
- 47. 47 DxH 800 –Multi-transducer module – 29 data measurements for each cell Direct Current Radio Frequency : Opacity Stretched Opacity 5 angles of Light Scatter : Axial Light loss Low angle light scatter Median angle light scatter Lower Median angle light scatter Upper Median angle light scatter
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- 49. VCS Technology -cont’d 49 Eosinophils separated from other populations. Monocytes identified as a separate population and then classified. Neutrophils are separated from Lymphocytes and Basophils. Finally Lymphocytes and Basophils are classified. 2-Dimensional separation completed. Actual 3-Dimensional display.
- 50. Example of aNormal Scatterplot 50
- 51. UniCel DxH 800 51 BodyFluid analysis for CSF, serous and synovial fluids. White cell differentials by monoclonal flow cytometry technology (for white cells flagged as being abnormal). Red cell counts automatically corrected in the presence of high White cell counts and other interfering substances.
- 52. Normal Neonate onthe CD Sapphire Neonatal specimens are run in an extended lyse mode. Note high MCV and Hb and low number of NRBC’s 52
- 53. Patient with RedCell Agglutination - RT RCC 2.16x1012/L Hb 111g/L MCV 81.0 Fl MCH 51.4pg MCHC 634g/L RDW 16.3%CV WCC 8.9x109/L Platelets 392x109/L 53
- 54. Patient with RedCell agglutination – warmed to 37°C RCC 4.21x1012/L Hb 107g/L MCV 82.9fL MCH 25.5pg MCHC 308g/L RDW 15.0%CV WCC 10.5x109/L Platelets 361x109/L 54
- 55. Patient with PlateletClumping Patient with known Platelet clumping with EDTA collection Platelet count of 57 x 109/L 55
- 56. Patient with lyseresistant red cells – in CBC mode WBC 31.4 x 109/L Absolute Lymphocyte 22.0 x 109/L Analyser gave a “resistant red cell” flag 56
- 57. Patient with lyseresistant Red Cells – ran in extended lyse mode WBC is now 15.2 x 109/L Lymphocyte Absolute count is 6.0 x 109/l Resistant red cell flag has now disappeared 57
- 58. Acquired Education 58 Spend asmuch time in the laboratory as possible. Familiarise yourself completely with analyser capabilities (and limitations) and information given by the analyser. Analyser flagging needs to be as accurate as possible – incorrect flagging (either false positive or negative flags) have a drastic effect on blood film review rates. Talk to the staff responsible for running and maintaining the analysers.
- 59. Where to Next? 59 Integrationof multiple analysers including automated slide makers and stainers with a “tracking” system especially in institutions with an extremley heavy workload. Already in use in the largest Private Laboratories Further development of use of monoclonal antibodies – reducing the workload for Flow Cytometry Depts. ?????????? And that’s an exciting aspect.
- 60. The true starsof this presentation 60