Automated cell counters

Principles of Automated cell counters used in hematology

1. PRINCIPLES OF AUTOMATED CELL COUNTERS Dr. Sadaf Khan

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 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

10. The Coulter Principle

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

17. 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

18. 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 Flowcytometer

19. 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

20. RBC FLAGS SUSPECT DEFINITIVE nRBCs Fragmented RBCs H and H error Aged sample Anemia Anisocytosis Microcytes Macrocytes

21. 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

22. Lymp Mono Eos Neut Baso

23. 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

24. Hemoglobin Estimation

25. 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

26. 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”.

27. 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

28. WBC with all three technologies applied simultaneously: Volume - Coulter Principle using direct current. Conductivity - high frequency current. Light Scatter- Laser light.

29. 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

30. OPTICAL LIGHT SCATTER

31. 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

32. 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.

33. Non-WBC’s (Debris) Scatter V o l ume 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) EO NE MO LY BA

34. 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

35. FLAG NO FLAG

36. Immature HLR . Retic = Total Retic # Fraction High light Scatter Retics (HLR) RBCs Platelets WBCs & NRBCs Retics RETICULOCYTE Flow cytometric analysis using VCS

37. 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

38. 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.

39. 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

40. Volume vs RAMLS 1 - N eutrophil 2 - Lymphocyte 3 - Monocyte 4 -Eosinoph il

41. TLC -DIFF -WBC/BASO SYSMEX XE SERIES DIFF WBC/ BASO lyse reagent fluorescent d ye ( 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

42. IMI Selective action of reagents on lipid membranes HPC master software n RBC channel Lyse + flourescent dye reagent Sensitive 0.1 nRBC / 100 WBC c WBC count and lymphocyte count RETIC channel Flouresecnt dye( oxazine + polymethine ) Parameters -absolute reticulocyte count -reticulocyte percentage -reticulocyte Hb content -platelet count -immature platelet fraction

43. HAEMOGLOBIN Sodium lauryl sulphate ( cyanide free) ferrous ferric SLS- hemichrome (555nm)

44. 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)

45. 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

46. PARAMETER WBC RBC HGB DLC COULTER STKS Impedance Impedance Modified cyanmethemoglobin (525nm) VCS SYSMEX NE- 8000 RF, DC detection or impedance Hydrodynamic focussing , DC detection Cyanmethemoglobin (535- 545nm) RF, DC detection ABBOTT CELL DYN 3000 Optical scatter Impedance Cyanmethemoglobin (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 cyanmethemoglobin (546nm) Peroxidase staining, optical scatter and absorption

47. First hematology instruments to introduce extended RBC and reticulocyte parameters Delta neutrophil index- prognostic indicator early marker of sepsis CSF analysis SIEMENS HEALTHCARE ADVIA

48. Beckman coulter SYNCHRON LX R i725 It can perform 146 chemistry and immuno assay tests including basic critical care, metabolic ,cardiac, Thyroid ,Reproductive, tumour markers . Recent advances

49. 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

50. 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