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Automated cell counters
- 2. Cell counters- • 1950s – Semi automated • Automated - screening devices. Abnormalities - verified by blood film microscopic examination INTRODUCTION
- 3. CBC specimens must be checked for -clots (visually, by applicator sticks, or by automated analyzer histogram inspection or flags), -significant in-vitro haemolysis and -interfering lipaemia CBC processing, either automated or manual, should be done within 8 hours but in no case later than 24 hours of sample collection Blood samples must be adequately mixed before analysis.
- 4. TYPES COULTER BASED RADIOFREQUENCY CONDUCTIVITY LIGHT SCATTERING CYTOCHEMISTRY • Beckman coulter instruments • Sysmex • Abbott Diagnostics • Beckman coulter instruments • Sysmex • Technicon H instruments • Coulter Maxm • Sysmex SF 3000 • Abbott CELL DYNE • Siemens automated hematology series • Technicon H • ABX Diagnostics
- 5. AUTOMATED CELL COUNTERS ADVANTAGES DISADVANTAGES • Speed • Number of samples • Increased reliability, accuracy and precision • Multiple tests simultaneously • Reduced labor requirements • Cost • Spurious results • Interfering factors • Red cell morphology • Less efficient in detecting atypical cells • Flagged samples need review
- 6. CBC: WBC, RBC, Hgb, PLT, RBC indices, RDW-SD( LH 780) WBC Differential: can be • 3 part • 5 part • 6 part Nucleated red cell count cWBC count(LH 750) Reticulocyte count Body fluid counts PARAMETERS
- 7. • Number of pulses = number of cells counted Height of pulse volume of cell SAMPLE DILUTED WITH BUFFERED SALINE CELL DRAWN THROUGH APERTURE CHANGE IN RESISTANCE DETECTION OF PULSE IMPEDANCE- COULTER PRINCIPLE
- 8. Electrolytes solutions allow current to pass from them Electrolyte Like ISOTON III
- 9. Current does not pass from cells - they are insulator
- 11. Coincident passage loss Recirculation Cell orientation( NON AXIAL FLOW) CAUSES OF POOR PULSE
- 12. 2 cells passing together- counted as single cell • Sample diluted • Lysing agents used Coincidence
- 13. Coincidence Correction Pulse to be edited Diluent stream
- 14. Sensing Zone RBCs recirculating Oscilloscope screen RBC PLT + - Recirculation
- 15. Recirculation correction-Sweep Flow technology Diluent stream RBCs swept away from sensing zone
- 16. Non central cell flow can give rise to inaccurate cell size data. Does not affect the actual count Corrected by – PULSE EDITING HYDRODYNAMIC FOCUSSING NON AXIAL FLOW
- 18. Modes- • automatic- 350 micro • manual – 200micro Closed tube system Requirements- • Bloods collected in EDTA • Bar code labels • Slides and cassettes for LH SlideMaker • Required reagents and diluents • LH Workstation with HELP • LH 780 with LH SlideMaker and LH SlideStainer BECKMAN COULTER LH 780
- 20. 4 Aliquots RBC/ PLATELET • 1.6 microltr • Impedance • Three apertures • RBCs >36fL • Platelets- 2- 20fL WBC/ Hb • 28 microltr • Lytic agent- • Impedance • WBC>35fL 5- DC/nRBC • 100 microltr • Lyse agent • Stabilise reagent • VCS • Orbital mixing chamber RETICULOCYTE • New methylene blue reagent • Acidic hypotonic solution • Light scatter • Flowcytome ter
- 21. Coulter RBC histogram It is a graphic representation of blood cells Produced from thousands / millions of signals generated by the cells passing through detector where they are differentiated by their size and frequency of occurrence in the population The Size Distribution Curve should always start on the base line and fall between the lower and the upper discriminator. Main RBC population RBC doublets
- 22. RBC FLAGS SUSPECT DEFINITIVE • nRBCs • Fragmented RBCs • H and H error • Aged sample • Anemia • Anisocytosis • Microcytes • Macrocytes
- 23. PLT histogram and curve fitting Typical platelet histograms are log normal and have two curves for accurate count: A smooth curve from 2 to 20 fL. A fitted curve from 0 to 70 fL The Platelet count is derived from the number of cells under the fitted curve from 0 fl extending up to 70 fl Fitted Curve extend up to 70 fl to report very large PLT count Raw data
- 25. Lymphs 35 – 90 fL Monos 90 -160 fL Granulocytes 160 - 450 fL The WBC Histogram Flagging Crtiteria • Clumped or Giant Platelets, • NRBCs, • Cryoglobulins, • intracellular parasites R1 R2 R3 R4 • Immature Granulocytes, • Abnormal Cell Populations, • Eosinophilia • Basophilia • Variant Lymphs, • Blasts, • Plasma Cells, High Absolute Granulocytes
- 29. All VCS instruments use a flow cytometer & flow cytometry principles Generic Flow Cytometer: Light Source- LASER Lens Block- focus & beam shaper Flow Cell- presents sample for analysis Light Scatter Sensor DIFFERENTIAL COUNT- VCS
- 30. ORBITAL MIXING CHAMBER: Blood from the loop on the center section of the BSV is pushed to the mixing chamber by the Erythrolyse and mixed. Erythrolyse lyses the RBC’s by creating a hypotonic environment. Stabilise is added at the right time and mixed to stop the action of the Erythrolyse. WBC’s are left in their “near native state”. SAMPLE PRESSURE SHEATH TANK VENTED WASTE SHEATH FLUID SAMPLE FLOW CELL PREPARED 7135-035
- 31. Sample Flow Sample is pushed to Flow Cell. Upper & lower sheath in ports supply flow cell with Sheath Fluid from Sheath Tank The sample stream is hydrodynamically focused. Sheath surrounds sample but does not mix. Sample Pressure > Sheath Pressure Cells are analyzed one at a time as they pass through the center of the flow cell. All three VCS technologies are applied simultaneously
- 32. WBC with all three technologies applied simultaneously: Volume- Coulter Principle using direct current. Conductivity- high frequency current. Light Scatter- Laser light.
- 33. WBC interacting with all three technologies to provide VCS characteristics for Diff analysis. Contour gating Coulter Principle Direct Current Total Cell Volume Total Cell Volume Conductivity Principle High Frequency Current Nucleus, Cytoplasm, Etc. Granularity Light Scatter Principle Laser Shape & Surface Characteristics
- 35. Forward-angle light scatter (FALS) Illuminating beam that has been bent to a small angle from direction of the original beam .It measures size or volume of cells
- 36. Side scatter (SSC) The illuminating beam that is scattered by particle to an angle of 90* from the illuminating beam. This depends on cell's surface texture and internal structure as well as to its size and shape and granularity. It is sometimes referred to as a granularity signalor an orthogonal light scatter signal.
- 37. Non-WBC’s (Debris) Scatter V o l u m e WBC’s Discriminator Lines The five WBC Differential populations are: EO- Eosinophils: EO’s are a minor population and may not appear on the scatterplot. NE- Neutrophils MO- Monocytes LY- Lymphocytes BA- Basophils (behind the Lymphs) EONE MO LY BA
- 38. Better Abnormal Cell Detection 1 Mono-Blasts 2 Myelo-Blasts 3 Immature Granulocytes 4 Band Neutrophils 5 Lympho-Blasts 6 Variant Lymphocytes 7 Low Volume Lymphocytes 7a NRBC s 8 PLT Clumps and Malaria Parasites 9 &10 Giant Platelets 1 2 3 4 5 6 7 8 9 10 7a
- 39. Automated differential performed Slide made, labeled, stained and reviewed Review with microscope Review with cell counter Automated differential reported FLAG NO FLAG
- 40. Immature HLR . Retic = Total Retic # Fraction High light Scatter Retics (HLR) RBCs Platelets WBCs & NRBCs Retics RETICULOCYTE Flow cytometric analysis using VCS
- 41. ERRORS FLUIDICS • Clean the vacuum trap • Check tubing connections and routing for leaks or disconnects REAGENTS • High background counts -contaminated -clean spills and leaks -thaw frozen reagents TRANSPORT • Debris on cassette or underside of rockerbed VLS VLS Diluent • Carryover • Insufficient rinsing of vent line • No air in vent line • Automatic mode disabled
- 42. ASPIRATION C CARRYOVER • Backwash- • diluent N NO BLOOD • Short sample or clot in tube • Obstruction in the aspiration pathway • Diluted blood • Low Hgb (approx. ≤ 4 g/L) B BUBBLE • Short sample, clot or bubble • Obstruction in the aspiration pathway • Bubble P PARTIAL • Short sample or clot in tube • Obstruction in the aspiration pathway • BSV did not rotate fully • Blood detectors turned OFF • Diff or Retic Sample Valves did not move correctly TROUBLESHOOT • Clots and sufficient sample volume • Repeat in manual mode • Clean needle, remove and replace if necessary • Inspect/Clean BSV ensure it is not leaking • Partial clogging in flow cell can be corrected by LATRON CONTROL.
- 43. Beckman Coulter Unicel DxH 800 VCSn module- DLC and nRBCs Multitransducer module for flow cell analysis Light scatter -LMALS and UMALS: cells granularity and topography -AL2 : cellular transparency -LALS: cellular complexity index NRBCs- DxH diluent+ DxH cell lyse
- 44. Volume vs RAMLS 1- Neutrophil 2- Lymphocyte 3- Monocyte 4-Eosinophil
- 45. TLC -DIFF -WBC/BASO SYSMEX XE SERIES DIFF WBC/ BASO • lyse reagent • fluorescent dye (polymethine) • optical detection laser block( 6333nm) • forward scatter- size • side scatter-cell complexity • fluorescent intensity- amount of cellular DNA and RNA • IG count • special lyse reagent • FS vs SS • TWBC count and Basophil count
- 46. IMI • Selective action of reagents on lipid membranes • HPC master software nRBC channel • Lyse + flourescent dye reagent • Sensitive 0.1 nRBC/ 100 WBC • cWBC count and lymphocyte count RETIC channel • Flouresecnt dye(oxazine+ polymethine) • Parameters • -absolute reticulocyte count -reticulocyte percentage -reticulocyte Hb content -platelet count -immature platelet fraction
- 47. HAEMOGLOBIN Sodium lauryl sulphate( cyanide free) ferrous ferric SLS- hemichrome (555nm)
- 48. XE -5000 Additional erythrocyte parameters Body fluid mode- RBC(impedance) WBC 2- part DC XN- 10 Adaptive flagging algorithmm Accurate DC for WBC< 0.5 X1O^3 Body fluid-high fluorescent intensity body cells ADVANCES (Fluorescence flow cytometry)
- 49. RBC/ PLT WBC HB One dilution Hydrodynamic Optical focusing and flow cell Impedance • Internal quality control Second dilution • Reticulocyte count • Flourescent dye(flourescein isothiocyanate) • Optical flow cell • Optical light scatter • WBC reagent + propidium iodide • MAPSS O degree-size 7 degrees- complexity 90 degree- nuclear lobularity 90 degree D- granularity • TLC unaffected by nRBCs • Hemoglobin reagent- dilutes, destroys RBCs and leukocytes • Chromogen with imidazole • Absorption spectrophotometry at 540nm Abbott CELL- DYN Sapphire Immuno T- cell assay(CD3/4/8) and Immunoplt(CD 61) assay
- 50. PARAMETER • WBC • RBC • HGB • DLC COULTER STKS Impedance Impedance Modified cyanmethemoglo bin(525nm) VCS SYSMEX NE- 8000 RF, DC detection or impedance Hydrodynamic focussing, DC detection Cyanmethemogl obin(535- 545nm) RF, DC detection ABBOTT CELL DYN 3000 Optical scatter Impedance Cyanmethemogl obin(540nm) Multiangle(0, 7, 90, 90 degree depolarized) polarized scatter) MILES/ TECHNICON H SYSTEMS Hydrodynamic focussing, optical scatter and absorption Hydrodynamic focussing -Laser low angle (2-3 degree) -High angle(5-15 degrees) scatter Modified cyanmethemoglo bin(546nm) Peroxidase staining, optical scatter and absorption
- 51. First hematology instruments to introduce extended RBC and reticulocyte parameters Delta neutrophil index- prognostic indicator early marker of sepsis CSF analysis SIEMENS HEALTHCARE ADVIA
- 52. Beckman coulter SYNCHRON LX Ri725 It can perform 146 chemistry and immuno assay tests including basic critical care, metabolic ,cardiac, Thyroid ,Reproductive, tumour markers . Recent advances
- 53. 5million sample analysis /year 100 parameters in patient samples, including blood, urine and CSF They are linked to Novel laboratory information system MOLIS (Sysmex) OLYMPUS OLA2500
- 54.
- 55. References Clinical Laboratory Hematology- Shirlyn B. McKenzie, 3rd edition Clinical And Laboratory Methods- Henry Todd Recent advances in Hematology –M.K.Brenner, A.V Hoffbrand. Manual of Beckman Coulter Hematolgy 2012