pankhi dutta md dm haematopath consultant haematopathologist sevenhills hospital mumbai n.
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  1. PankhiDutta MD DM (haematopath) Consultant haematopathologist SevenHills Hospital, Mumbai. Automated cell counters- THE BASICs

  2. Introduction • Automated cell counter-backbone of the haemat lab • Wallace Coulter in 1956 – impedance method • Various technologies today • More accurate, more precise reports at a faster rate • Basic CBC + newer parameters • Inherent technological limitations

  3. NO. 4 DATUM: 9/10/95 15:11 MODE: VOLLBLUT WBC 5,8 x 103/µl RBC 4,84 x106/µl HGB 13,7 g/dl HCT 42,0 % MCV 86,8 fl MCH 28,3 pg MCHC 32,6 g/dl PLT 257 x103/µl WBC 300 LYMPH% 31,2 % MXD% 6,8 % NEUT% 62,0 % LYMPH# 1,8 x103/µl MXD# 0,4 x103/µl NEUT# 3,6 x103/µl RBC 250 RDW-SD 40,0 fl PLT 40 PDW 13,1 fl MPV 10,4 fl P-LCR 28,1 % Basic parameters – 3 part counter • Haemoglobin • RBC, WBC, PLT count • Red cell indices • RDW • 3-part differential • Histograms

  4. DC supply Registor(constant current) Vacuum Internal electrode External electrode Aperture Blood cell suspension Blood cell Transducer chamber 3-part differential analyser Two chambers • Hb + WBCs • RBCs + PLTs

  5. Haemoglobin estimation 1. Lysis of RBC Haemoglobin molecule     Fe2+ Fe2+ Fe2+ Fe2+ Ammonium salts     Fe2+ Fe2+ Fe2+ Fe2+ RBC RBC 3-part Diff technology

  6.  Fe2+ Fe2+   RBC 2. Change of conformity Haemoglobin molecule   Fe2+ Fe2+   Fe2+ Fe2+ Fe2+ Fe2+ RBC 3-part Diff technology

  7.  Fe3+ Fe3+   3. Oxidation Haemoglobin molecule O2   Fe2+ Fe2+ Fe2+ Fe2+ Fe2+  Fe2+  • Methemoglobin-complex • Stable coloumetric complex – directly proportional to Hb • Absorbance of solution is measured against standard 3-part Diff technology

  8. DC detection method Particle counting • DC - direct current - impedance principle - volumetric measurement • WBC count and 3-part differential • RBC count • PLT count 3-part Diff technology

  9. DC Detection Method internal electrode aperture external electrode vacuum U = R x I Impulse 3-part Diff technology

  10. Impedance Principle External Electrode Internal Electrode Aperture V = Voltage C = Current R = Resistance V = R x C

  11. Impedance Principle Aperture External Electrode Internal Electrode V = Voltage C = Current R = Resistance V = R x C

  12. A C B cells aperture pulse A pulse B pulse C Problems- recirculation and coincidence

  13. Hydrodynamic Focusing for RBC & PLT Samples are passing through the centre of the aperture with sheath flow solution  Recirculation and coincidence are prevented  Enhanced linearity & accuracy 3-part Diff technology

  14. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DC Detection Method From pulse to histogram: pulse diagram time pulse height 3-part Diff technology

  15. 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 DC Detection Method 30 cells 20 Cumulative Distribution Curve 10 4 1 0 0 0 1 2 3 4 5 4 3 2 1 Histogram 3-part Diff technology

  16. NO. 4 DATUM: 9/10/95 15:11 MODE: VOLLBLUT WBC 5,8 x 103/µl RBC 4,84 x106/µl HGB 13,7 g/dl HCT 42,0 % MCV 86,8 fl MCH 28,3 pg MCHC 32,6 g/dl PLT 257 x103/µl WBC 300 LYMPH% 31,2 % MXD% 6,8 % NEUT% 62,0 % LYMPH# 1,8 x103/µl MXD# 0,4 x103/µl NEUT# 3,6 x103/µl RBC 250 RDW-SD 40,0 fl PLT 40 PDW 13,1 fl MPV 10,4 fl P-LCR 28,1 %

  17. Erythrocyte (RBC) Histogram RU RL PLT RBC 200-250 fl 25-75 fl • RBC detection: between 25 and 250 fL • Distribution curves are separated by flexible discriminators: RL & RU 3-part Diff technology

  18. Erythrocyte (RBC) Histogram RU RL PLT RBC 200-250 fl 25-75 fl • The histogram curve should start and end at the base line within the discriminators 3-part Diff technology

  19. Abnormal Erythrocyte(RBC) Histogram RU RL 100% Example: RL flag message PLT RBC 20% 200-250 fl 25-75 fl • In case of abnormal histogram curves the flag messages: RL; RU or MP are generated and results must be checked • RL : Abnormal height at lower discriminator • RU : Abnormal height at upper discriminator • MP : (Multi Peak) RBC Anisocytosis 3-part Diff technology

  20. Platelet (Plt) Histogram PL PU 100% PLT RBC 20% fixed at 12 fl 2-6 fl 12-30 fl • PLT detection: between 2 and 30 fL • Fixed discriminator at 12 fL 3-part Diff technology

  21. Abnormal Platelet (Plt) Histogram PL PU Example:abnormal PLT curvePU message 100% RBC PLT 20% 2-6 fl 12-30 fl • In case of abnormal histogram curves the flag messages: PL; PU or MP are generated and results must be checked • PL : Abnormal height at Lower discriminator • PU : Abnormal height at Upper discriminator • MP : (Multi Peak) Platelet Anisocytosis 3-part Diff technology

  22. Leukocyte (WBC) Histogram Lysing reaction to the WBCs Structure of WBS Lysing reaction on the WBC Mitochondria Nucleus Nucleolus Cell membrane Ribosome Cytoplasm

  23. Lymphocyte After lysing reaction Cell volume in fl Neutrophile Lymphocyte Monocyte Basophile Eosinophile Neutrophile Monocyten Basophile Eosinophile 30 - 80 60 - 120 70 - 130 80 - 140 120 - 250 0 50 100 150 200 250 300 Leukocyte (WBC) Histogram Lysing reaction and WBC Cell size in µm Before lysing reaction Neutrophile Basophile Eosinophile Monocyte Lymphocyte 10 - 15 9 - 14 11 - 16 12 - 20 7 - 12 0 2 4 6 8 10 12 14 16 18 20 22

  24. Leukocyte (WBC) Histogram WU WL T1 T2 100% 20% fixed at 12 fl 2-6 fl 12-30 fl • WBC detection: between 30 and 300 fL • Leukocytes are separated in 3 parts:lymphocytes, mixed cells (mono, eo, baso)and neutrophilsby discriminators: T1, T2 3-part Diff technology

  25. Abnormal Leukocyte (WBC) Histogram WU WL T1 T2 Example:abnormal WBC curveWL message in case of Lyse resistant RBC 100% 20% -300 fl ~30 fl • The histogram curve should start within the lower and upper discriminator at the base line • Abnormal curves are flagged with WL, WU, T1, T2, F1, F2  results must be checked 3-part Diff technology

  26. QUESTIONS? about 3-part differential counters

  27. 5-part differential counters Various technologies :- • Fluorescence flowcytometry • Volume Conductivity Scatter • Peroxidase staining

  28. Beckman Coulter VOLUME MEASUREMENT VCS utilises the Coulter Principle of counting and sizing to measure the volume of the cell by using Direct Current (DC) across the two electrode in a flow cell.

  29. CONDUCTIVITY MEASUREMENT Cell exposed to RF, the RF energy penetrates into cell and reveal information about its size and internal structure.

  30. SCATTER MEASUREMENT As cells are pass in single stream (flow cell) they are struck by laser strike which gets scattered. The light scatter at angles between 10 and 70 deg is used by VCS instruments. The scattered light gives information about cell surface and granularity

  31. Neuts Monos Eos Lymphs Basos NRBCs 3D Data Analysis

  32. WBC and Differential • Peroxidase Channel Stain Cells With Peroxidase :Eosinophils- Strong Staining :Neutrophils- Medium Staining :Monocytes- Weak Staining :Lymphocytes and Basophils- No Staining :Large Unstained Cells (LUC) No staining • Also Measure Cell Size Using Low Angle ScatterPlot 2D • Scattergram To Give 4 Part Differential ADVIA TECHNOLOGY Volume Monocytes Neutrophils LUC Lymphocytes + Basophils Eosinophils Perox Activity

  33. ADVIA TECHNOLOGY The ADVIA WBC differential is calculated from a 3 step process. • Cells are stained by peroxidase reagent and analyzed for size and peroxidase stain intensity. • Cell specific lysis reagents are used to separate basophils from all other white cells. • Basosare subtracted from the lymph/baso cluster in the perox channel to calculate the lymphs.

  34. Sysmex X-class analyzers-Fluorescence flow cytometry

  35. Fluorescence flow cytometry-(light scatter and fluorescent dyes)

  36. Differential- SFL vs SSc (diff channel) FSc vs SSc (baso channel)

  37. SFL SSC ACAS / Centroids • 1. The firstcentroidsare provided: • The starting position of centroids has been determined from thousands of samples. These values are stored in the instrument and are used as the starting position for cluster analysis. Mono Lymph Ghost Neut + Ba Eo