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ABC of automated CBC

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Automation in cell counting

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ABC of automated CBC

  1. 1. ABC of automated CBC Dr.Parth S.Kaneria Shanti Pathology Lab Junagadh
  2. 2. Pathology in old days Antoni van Leeuwenhoek
  3. 3. Wallace H. Coulter an electrical engineer and businessman while working with the US Navy in the late 1940s developed and first applied the Coulter Principle. Man who changed the world of counting...
  4. 4. Advantages • Speed with efficient handling of large number of samples. • Accuracy and precision in quantitative blood tests. • Ability to perform multiple tests on a single platform. • Significant reduction of labor requirements. • Invaluable for accurate determination of red cell indices.
  5. 5. Disadvantages • Flagging of a laboratory test result demands labour intensive manual examination of a blood smear • Comments on red cell morphology cannot be generated • Platelet Clumps are counted as single ,so low count. • Erroneously increased or decreased results due to interfering factors • Expensive with high running costs
  6. 6. TYPES OF AUTOMATED ANALYSER Three part Five Part Seven Part Differentiate cells into three categories 1. Granulocytes 2. Lymphocytes 3. Monocytes/mixed cells Differentiate cells into the five basic leukocyte types 1.Neutrophils 2.Eosinophils 3.Basophils 4.Lymphocytes 5.Monocytes In addition are able to distinguish 1.Nucleated RBCs, 2Abnormal and atypical cells and immature cells
  7. 7. Counting Chambers RBC/Platelet Chamber WBC Chamber Differential Chamber Reticulocyte Channel
  8. 8. General Principles • Introduction to Instrumentation – Basic principles used : • Electronic Impedance • Radiofrequency • Optical Scatter a. Laser light scatter b. Flow cytometry c. Chemical dye method
  9. 9. The Coulter Principle • The poorly conductive blood cells are suspended in a conductive diluent (liquid). • The diluent is passed through an electric field created between two electrodes. • The liquid passes through a small aperture (hole). • The passage of each particle through the aperture momentarily increases the impedance (resistance) of the electrical path between the electrodes. • The increase in impedance creates a pulse that can be measured. • The number of pulses = blood cell count • The amplitude (height) of the pulse = Volume of cell
  10. 10. Electronic Impedance: Disrupts electrical current by displacing fluid proportional to the size of the cell.
  11. 11. Computing and generation of graph • Each pulse is recorded as an oscillation , the height of which is proportional to the volume and size of the cell. • These oscillations are rearranged according to volume interval to form a histogram.
  12. 12. Optical Scatter
  13. 13. Low angle scatter 2o - 3o (Volume) High angle scatter 5o - 15o 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
  14. 14. 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 signal or an orthogonal light scatter signal.
  15. 15. Forward-angle light scatter correlates to cell volume/size Side scatter correlates to degree of internal complexity (granules and nucleus)
  16. 16. Radiofrequency ( impedance) Impedance - size cells Conductivity (RF) – proportional to cell interior density (granules and nucleus) Five-part WBC differential •Scatter plot (RF X DC) •Computer cluster analysis provides absolute counts
  17. 17. ERRORS 1. Recirculation error Cells that re circulate through the edge of an electrical field produce an aberrant impulse, which is smaller than cell passing through the aperture. 2. Coincidence error Cells that pass through the aperture simultaneously, or almost so, are counted and sized as a single large cell called coincidence. 3. Non central flow error Cell pass through the aperture off centre produce aberrant impulses and appear larger than their actual size
  18. 18. 1. Recirculation error 2. Coincidence error 3. Non central flow error How to over come such errors ?
  19. 19. Hydrodynamic focusing and Laminar flow
  20. 20. COULTER VS FLOWCYTOMETER • The Coulter-type volume signal is proportional to the volume of a particle as well as to its electrical characteristics. • In flow cytometer signal is proportional to the cross-sectional area of a particle as well as to its refractive index.
  21. 21. PRINCIPLES • Principles of Measurement – Direct Measurement: • RBC – Impedance, hydrodynamic focusing • WBC - Impedance, hydrodynamic focusing • Platelets – Impedance (2-20 fl), hydrodynamic focusing • Hgb – mod. Cyanmethemoglobin (525 nm) • MCV – mean RBC volume (histogram) – Indirect Measurement: • Hct, MCHC, and MCH (calculations) – RDW and MPV (CV of respective histograms) – WBC differential • VCS – volume, conductivity, scatter • employs differential shrinkage – Reticulocyte • Supravital stain • Fluorescent detection • Flags
  22. 22. TWO SIDES OF INSTRUMENTS (THREE PART INSTRUMENTS) RBC SIDE WBC SIDE (DILu-1:50,000) (DILu-1:500) >30 fl size 2-30 fl size lysing reagent RBC PLETELET 35-90 fl 90-160 fl 160-450 fl Lymphocytes Mononuclear cells Granulocytes Monocytes Eosinophils Neutrophils Basophils
  23. 23. Erythroid Parameters • Measured parameters: Hemoglobin RBC count MCV RDW • Calculated parameters • These are calculated values and depend on the values that go into their calculation. • HCT(%) = MCV x RBC concentration • MCH(pg) = Hb concentration / RBC concentration • MCHC(g/dl) = Hb concentration / HCT
  24. 24. Hemoglobin Estimation • Estimated from a part of the blood mixed with lysing agent to lyse the RBCs.(the same can be used for WBC counts) Lyse treated blood Cyanide Solution added Cyanmeth hemoglobin method Sodium Lauryl Sulphate added Sulphmeth homoglobin method
  25. 25. HGBColorimetry for HB estimation
  26. 26. X axis: Volume of cells Y axis: No. of cells LD: 25-75 fL UD: 200-250 fL RBC Histogram Median =MCV +1SD
  27. 27. RBC- and PLT-Histograms 1. Platelets have a size between 8 and 12 fl and are counted between 2 and 30 fl. 2. Erythrocytes have a size of 80-100 fl and are counted between 25 and 250 fl.
  28. 28. The Size Distribution Curve should always start on the base line and fall between the lower and the upper discriminator.
  29. 29. Histograms FLAGS Interpretation
  30. 30. Erythrocyte histogram Flagging Possible causes: • Giant platelets • Microerythrocytes • Fragmentocytes or dysplastic RBC • Platelet clumps Mark “ RL “, abnormal height at lower discriminator LD RBC PLT LD RBC The curve does not start at the basis line.
  31. 31. Erythrocyte histogram Flagging Note: All results marked with “ RL “ should be controlled. Explanation: RBC agglutination might cause a low incorrect RBC count and effect also the parameter Hb, MCV, MCH and MCHC. In case of cold agglutinates warm the sample up to 37°C. (MCHC should drop back to normal value if the problem is solved) Mark “ RU “, abnormal height at upper discriminator. UD RBC The curve does not end at the base line. UD RBC Possible causes: • Cold agglutination • RBC agglutination • Rouleux formation
  32. 32. Possible causes: • Iron deficiency in recovery (therapy) • Dimorphic picture • Multiple RBC transfusions • Extreme leukocytosis (> 600 x 10³/µl) Mark “ MP “: multiple peak of RBC histogram RBC Note: Parameter: RBC, MCV, RDW-SD & RDW-CV are flagged. Explanation: Often: Extreme anisocytosis is found. In case of anisocytosis the RBC result is not affected. Seldom: extreme high numbers of leukocytes may cause high incorrect RBC results. Therefore all RBC parameters should be controlled.
  33. 33. RDW-CV (%) = 100 x s/µ µ = L2 + L1 / 2 s = L2 – L1 / 2 100 % L1 µ Turning points Normal value: 11 - 16 % 100 % 20 % RDW-SD is calculated in 20 % of the total height of the distribution curve. Normal value: 37 - 46 fl Clinical relevant > 60 fl Erythrocyte histogram Distribution width (RDW) RDW-CV RDW-SD L2 Note: RDW-CV can be used as a marker for anisocytosis Note: RDW-SD can be used as a marker for anisocytosis
  34. 34. Mark “DW “: abnormal histogram distribution (distribution width) (only RDW-SD or RDW-CV is flagged) RBCRBC Explanation: The flag “DW” is shown in case of abnormal histogram curve. The overall height of the curve represents 100%. The width is calculated on the 20% line of the curve. If the histogram curve does not match the 20% line either on the lower (RL) or upper (RU) discriminator. The flag “DW” is generated for RDW-SD or RDW- CV and these results can not be calculated. Possible causes: • same as RL or RU Histogram curve does not match the 20% line twice
  35. 35. 100 fl 100 fl Left shift in Microcytic Anaemia Right shift in Macrocytic Anaemia
  36. 36. Cold agglutinins Because in this case erythrocytes have passed through the detector as clusters of several cells, the RBC, HCT,MCH, MCV, MCHC and RDW values are abnormal. The RBC histogram shows a second peak.
  37. 37. Calculating reported parameters • MPV Mean of 2D-PLT Vol histogram (Mean Platelet Volume) • LargeP- LCR Platelets with volumes greater than 20 fL (Large Platelets) The Platelet Method
  38. 38. • The histogram curve should lay within the lower and upper platelet discriminator (PL & PU) and start and end on the base line. • PLT counted between 2 fl and 30 fl. 1 flexible Discriminator PL 2 to 6 fl. 1 flexible Discriminator PU 12-30 fl. 1 fixed Discriminator at 12 fl Thrombocyte histogram
  39. 39. Parameter of the thrombocyte histogram MPV = mean PLT volume reference range: 8 - 12 fl P-LCR = ratio of large platelets Reference range 15 - 35 %  Increase could be a sign for: • PLT Clumps • Giant PLT • Microerythrocytes 12 fl LD UD PLT P-LCR 100 % 20 % Pct (%) PLT (x 103/µl) MPV (fl) = PDW = platelet distribution width calculated at 20 % of peak height Reference range: 9 - 14 fl Increase could be a sign for: • PLT Clumps • Microerythrocytes • Fragments
  40. 40. Note : Check blank (background check)! Initiate auto rinse or check sample. Explanation: In case of high background numbers (blank), check reagent for contamination (bacteria). Check expiry date. In order the background check is within range, the patient sample should be checked – platelet results might be incorrect high due to cell fragments or bacteria's. In some cases also platelet aggregates might cause the problem. In this case the histogram curve would also show an abnormal distribution at the upper discriminator. Platelet aggregation might cause low incorrect platelet results. Flag display: mark „PL“ is shown with higher priority than „PU“. Mark “ PL “, abnormal height at lower discriminator Possible cause: • High blank value • Cell fragments • High numbers of bacteria • Contaminated reagent • Platelet aggregation PLT The curve does not start at the basis line. Thrombocyte histogram Flagging
  41. 41. Mark “ PU “: abnormal height at upper discriminator Explanation: In case of platelet aggregation, the PLT count is false low. Check EDTA incombatibility – e.g. re- collect the sample and use citrate as anticoagulant to avoid clogging caused by EDTA. In case of giant platelets, the PLT count might be incorrect low. PLT results should be confirmed with alternative methods: e.g. chamber counting. Possible Cause : • PLT clumps EDTA-incombatibility Clotted sample • Giant Platelets (False low) • Microerythrocytes (False High) • Fragmentocytes or dysplastic RBC
  42. 42. Possible Cause: • Platelet anisocytosis • Recovery after chemotherapy • Platelet aggregation • Platelet transfusion Mark “ MP “: multi peaks in PLT histogram Note: Parameter: PLT, MPV, PDW and P-LCR is flagged. Explanation: In case of platelet anisocytosis the PLT result is not affected. Seldom: Multiple peaks can be seen in some cases of platelet aggregation (jagged curve). In case of PLT aggregation the PLT result might be incorrect low. Therefore recollect the sample, In case of EDTA incompatibility sodium citrate as anticoagulant can prevent platelet clumping.
  43. 43. WBC Histogram
  44. 44. The distribution curve should be within the discriminators. The curve should start and end at the basis line. • The LD is flexible, but can not be lower than 30 fl. • The WBC-channel shows Leukocytes and Thrombocytes ( Erythrocytes are lysed). • The volume of the Thrombocyts is usually between 8 - 12 fl, therefore the LD at the WBC-Histogramm seperates the Leukocytes from the Thrombocytes. (Thrombocytes were not counted).
  45. 45. Flag “ WL “, Curve does not begin at the basis line Possible causes : • PLT Clumps EDTA-Incombatibility coagulated Sample • High osmotic resistant (Erythrocytes not lysed) • Erythroblasts • Cold agglutinate
  46. 46. Flag “ WU “, Curve does not end at the base line. High leukocyte count
  47. 47. T1 and T2 are valley discriminators defined by the plateau. This discriminators separates the Leukocytes populations. • The discriminators are flexible and will be set automatically according the sample. • In special cases is a separation from the valley discriminators not possible.
  48. 48. T1 could not be detected High no. of large or atypical lymphocytes T1 was detected but not T2 Neutrophilic hypolobation eg. Pelger Huet and pseudo Pelger Huet anomaly.
  49. 49. This is a case of WBC agglutination, which occurs rather rarely. The histogram does not show a clear tri-modal pattern, with particles present in the region above 250 fl . The count of leukocytes is likely to be falsely low. Depending on the nature of leucocytes antibodies, agglutination may be dissolvable and measurement may become possible upon incubation the at 37 o C or upon washing the samples with isotonic saline.
  50. 50. The valley between the erythrocytes ghost area and the small leucocytes area exceeds the limit, and WL flags are given. NRBC are likely to contribute significantly to the population on the WBC histogram, therefore most of them are counted as leukocytes. corrected by the following equation: corrected WBC Count = measured WBC Count x 100 (100 + NRBC count ) NRBC Count: The number of NRBC per 100 leukocytes.
  51. 51. ABN / INDICATOR PROBABLE CAUSE COMMENT WBC histogram (lymph peak) does not start at baseline Giant platelets, NRBC, Plt clumping Review smear, correct WBC for NRBC Elevation of left portion of granulocyte Left Shift Review smear Elevation of right portion of granulocyte peak Neutrophilia Review smear WBC Histogram
  52. 52. Five groups of leukocytes (stained) What’s 5AND 7-part diff? Neutrophil Eosinophil Basophil Lymphocyte Monocyte
  53. 53. VCS Technology
  54. 54. COULTER TECHNOLOGY
  55. 55. VOLUME As opposed to using 0ø light loss to estimate cell size,VCS utilizes the Coulter Principle of (DC) Impedance to physically measure the volume that the entire cell displaces in an isotonic diluent. This method accurately sizes all cell types regardless of their orientation in the light path.
  56. 56. CONDUCTIVITY Alternating current in the radio frequency (RF) range short circuits the bipolar lipid layer of a cell's membrane allowing the energy to penetrate the cell. This powerful probe is used to collect information about cell size and internal structure, including chemical composition and nuclear volume.
  57. 57. LASER LIGHT SCATTER When a cell is struck by the coherent light of a LASER beam, the scattered light spreads out in all directions. Using a proprietary new detector, median angle light scatter (MALS) signals, are collected to obtain information about cellular granularity, nuclear lobularity and cell surface structure
  58. 58. 3-D Cellular Analysis- VCS
  59. 59. Gate Software Technology
  60. 60. NORMAL DATAPLOT
  61. 61. DOT plot or Scattergram
  62. 62. Better Abnormal Cell Detection
  63. 63. LEUKOPENIA & THROMBOCYTOPENIA
  64. 64. NEW PARAMETERS • Nucleated RBCs • Immature granulocyte • Haematopoitic progenitor cells • Immature reticulocyte fraction • RBC fragments (schistoytes) • Reticulated platelets • Reticulocyte indics • Malarial parasites
  65. 65. Newer Parameters(contd..) • Cellular Hb Concentration Mean(CHCM): Uses Light scatter technology. True estimate of hypochromia in IDA. • Hb Distribution Width: Degree of variation in red cell hemoglobinization. Range-1.82 to 2.64. • Nucleated Red Cells: nRBCs identified,separated & corrected count obtained. WBCs have high fluorescence & forward scatter.
  66. 66. Newer Parameters(contd..) • Reticuloctes: Various dyes & flurochromes bind with RNA RNA content- 3 Maturation stages; LFR,MFR & HFR  Immature reticulocyte Fraction(IRF): Sum of MFR & HFR. Early and sensitive index for erythropoisis.  Reticulocyte Hb Equivalent(RET-He): Hb content of freshly prepared RBCs. Real time information on Fe supply to erythropoiesis. Early detection of Fe deficiency. Differentiate IDA & ACD. Monitoring of erythropoietin & Fe therapy.
  67. 67. Newer Parameters(contd..) • P-LCR(Platelet Large Cell Ratio): % of platelets with a vol >12fl. Due to platelet aggregates,microerythrocytes,giant platelets. • Reticulated Platelets /Immature Platelet Fraction(IPF): Newly produced platelets that have remains of RNA in their cytoplasm. Reflects rate of thrombopoiesis.
  68. 68. WBC Research Population Data Case Study – Malaria Parasites(Normal plot and Research Population Data compared to a patient infected with malaria type Plasmodium falciparum. Note the increased size and variation of the lymph's and Monocyte's.) NORMAL Normal Normal MO Noral LY Macrophage Parasitized RBC MALARIA MP Positive Reactive LY
  69. 69. Peroxide based counters: MPO is used to count neutrophils.Lymphocytes not stained Fluroscence based: Retic and platelet count.Immature platlets detected best Immunological based: Accurate platelet count using CD41/CD61 antibodies
  70. 70. The Retic Method
  71. 71. Must to do before running the sample • 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 before reporting results. • CBC processing, either automated or manual, should be done within 8 hours but in no case later than 24 hours of sample collection, as storage beyond 24 hours results in erroneous data on automated / semi- automated Haematology analyzers • Blood samples must be adequately mixed before analysis.
  72. 72. Quality Control
  73. 73. Quality Assessment: Adequate control of the pre & post analytical from sample collection to report dispatch. Quality Control: Measures that must be included during each assay run to verify the test working properly. Proficiency Testing: Determines the quality of results generated by lab. Terminologies
  74. 74. Accuracy & Precision • Accuracy: Refers to closeness to the true value • Precision: Refers to reproducibility of test 1 2 3 4
  75. 75. Internal Quality control: Continuous evaluation of the reliability of the daily works of the lab with validation of tests. External Quality Control: Evaluation by an outside agency of between- laboratory & between-method comparability. TYPES
  76. 76. CBC Quality Control • Commercial Controls: • 3 levels (low, normal, high) • Values stored in instrument computer • Levey-Jennings graph generated and stored for each parameter • Delta Checks • When the Laboratory Information System (LIS) and the instrument are interfaced (connected) delta checks are conducted by the LIS on selected parameters. –Current values compared to most previous result –Differences greater than the limits set within the LIS are flagged
  77. 77. Controls & Calibrators • Controls: Substances used to check the precision . Analyzed either daily or along each batch. Should have same test properties as blood samples. Stabilized anticoagulated whole blood or pooled red cells. 3 conc.-high,normal ,low • Levey-Jennings graph generated and stored for each parameter • Calibrators: Check the accuracy. Value assigned to them by a reliable ref. center.
  78. 78. Levey-Jennings Chart Mean 1 SD 2 SD 3 SD 1 SD 2 SD 3 SD
  79. 79. Control Values and Decision  Consider using Westgard Control Rules  Use premise that 95.5% of control values should fall within ±2SD  Commonly applied when two levels of control are used  Use in a sequential fashion
  80. 80. 12S Rule = A warning to trigger careful inspection of the control data 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 12S rule violation
  81. 81. 13S Rule = Reject the run when a single control measurement exceeds the +3SD or -3SD control limit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 13S rule violation
  82. 82. 22S Rule = Reject the run when 2 consecutive control measurements exceed the same +2SD or -2SD control limit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 22S rule violation
  83. 83. 10x Rule = Reject the run when 10 consecutive control measurements fall on one side of the mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 10x rule violation
  84. 84. What to do when Control Value is out of limit? • In these situations, precision of routine work can be monitored by performing duplicate tests on patient samples. • SD of differences between results on 20 duplicate samples is determined and +2SD limits specified. Subsequent duplicate values should be within these defined limits. • Patient data can also be used to monitor precision in a laboratory performing >100 samples a day. Day-to-Day variation in MCV, MCH and MCHC should be analyzed using Bull's algorithm. This facility is available in the software of many auto analyzers. • The use of stable controls, however, is the method of choice.
  85. 85. • OUT OF CONTROL!!! –Repeat the assay ( One time occurrence ) –Check for trends (Delta check) (from Levy Jennings chart) –Check integrity of material –Troubleshoot –Verify instrumentation
  86. 86. Specimen-Related Problems • An instrument problem is differentiated from a specimen- related problem by running a control. • If the control results are acceptable, the problem is probably specimen-related. • Check for: – clots – hemolysis – lipemia
  87. 87. Instrument Problems • If the control shows similar problems, it indicates an instrument problem. – Electronic? – Pneumatic / Hydraulic? – Reagent? • Because it is easiest to detect a problem in the electronic subsystem and hardest to detect a problem in the reagent subsystem, the subsystems are usually checked in the following order: electronic, pneumatic / hydraulic, reagent.
  88. 88. Reagent Troubleshooting • A reagent problem can be as obvious as precipitate in the reagent tubing. • In the less obvious cases, the most effective way of detecting a problem is by keeping a log of the lot numbers with the opening and expiration dates of the reagents in use, and knowing how each reagent affects the data. • Refer to the labeling information with your reagents for details.
  89. 89. Calibration 1.It is done to compensate for any inaccuracies of the pneumatic hydraulic and electric systems 2. Calibration fine tunes your hematology analyzer and provides the most accurate results possible. 3. Automated Haematology analyzers should be calibrated using calibrators„ that have traceability to standard reference material or methods. 4.Controls are not used for calibration
  90. 90. Calibration • Never adjust to a specific value for an individual sample. • For best performance, calibrate all the CBC parameters. The WBC differential is calibrated at the factory. They do not require calibration in the laboratory.
  91. 91. When to Calibrate You should calibrate your instrument: • At installation. • After the replacement of any component that involves dilution characteristics or the primary measurements (such as the apertures). • When advised to do so by your service representative.
  92. 92. Blood Sample for Calibration • 4 ml specimen are obtained from three hematological normal volunteer in k2 edta
  93. 93. Hb Estimation • By Cyan meth hemoglobin method mean value is taken
  94. 94. ICSH Reference methods for PCV The reference PCV is Standard whole blood haemoglobin concentration ----------------------------------------------------------------- Packed red cell haemoglobin concentration following centrifugation in a microhaematocrit centrifuge 1.The measurement on packed red cells is performed on cells obtained from the middle of the column of red cells 2. where there is little trapping of plasma or white cell contamination. 3.It therefore produces a measurement that does not include trapped plasma.
  95. 95. RBC AND WBC TOTAL COUNT • The reference method for the RBC and WBC employs a semi-automated single-channel aperture-impedance method with accurate coincidence correction being achieved by extrapolation from counts on serial dilutions.
  96. 96. PLATELET 1.The platelet count can be determined by flow cytometry using a fluorochrome – labelled monoclonal antibody, mixture of CD41, CD42a or CD61, 2.That binds specifically to platelets and dilution errors do not affect the count 4.when there is an inherited platelet membrane defect with absence of one of the platelet glycoproteins, 5.The relevant monoclonal antibody will not bind to platelets, hence the use of two antibodies
  97. 97. CARRY HOME MESSAGE Automated analyzers provide rapid and useful information of the cell count, morphology and cell function. Automation is a supplement and not a substitute to manual methods

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