Automation in hematology
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The document discusses various automation technologies in hematology, including automated hematology analyzers, flow cytometry, and automated coagulometers. It details the procedures for cell counting, hemoglobin estimation, and coagulation testing, emphasizing the advantages of reducing human errors and improving efficiency. Additionally, it covers sample preparation and the principles of technologies like high-performance liquid chromatography for diagnosing hemoglobinopathies.
Automation in hematology
- 1. Automation In Haematology Dr. K.Siddartha Mod: Dr. Faiq Ahmed Dept Of Lab Medicine Basavatarakam Indo American Hospital And Research Institute
- 2. Automation in hematology includes Cell counts (Automated hematology analysers) Diagnosis of hemoglobinopathies by HPLC Flow cytometry Automated coagulometers Automated ESR
- 3. Automation in Cell Counters
- 4. Basic parameters – 3 part counter • Haemoglobin • RBC, PLT count • Red cell indices • RDW • WBC with 3-part differential 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 LYMPH% 31,2 % MXD% 6,8 % NEUT% 62,0 % LYMPH# 1,8 x103/µl MXD# 0,4 x103/µl NEUT# 3,6 x103/µl 250 RBC RDW-SD 40,0 fl 40 PLT PDW 13,1 fl MPV 10,4 fl P-LCR 28,1 % WBC 300
- 5. Hemoglobin estimation V07063-part Diff technology Haemoglobin molecule RBC Ammonium salts Fe2+ Fe2+ RBC Fe2+ Fe2+ Lysis of RBC Fe2+ Fe2+ Fe2+ Fe2+
- 6. V07063-part Diff technology Haemoglobin molecule Methemoglobin-complex Stable coloumetric complex – directly proportional to Hb Absorbance of solution is measured Fe3+ Fe3+ Fe2+ Fe2+ O2 Oxidation Fe2+ Fe2+ Fe2+ Fe2+
- 7. V07063-part Diff technology As samples are passing through the centre of the aperture with sheath flow solution DC for RBC & PLT Hydrodynamic Focusing Recirculation and coincidence are prevented Enhanced linearity & accuracy
- 8. V07063-part Diff technology 2-6 fl 12-30 fl fixed at 12 fl WL WU 100% 20% WBC detection: between 30 and 300 fL Leukocytes are separated in 3 parts: lymphocytes, mixed cells (mono, eo, baso) and neutrophils by discriminators: T1, T2 T1 T2 Leukocyte (WBC) Histogram
- 9. 5-part differential counters Various technologies :- Fluorescence Flowcytometry Volume Conductivity Scatter Peroxidase Staining
- 10. VOLUME MEASUREMENT Volume of the cell is done by using Direct Current (DC) across the two electrode in a flow cell. Beckman Coulter VCS technology
- 11. CONDUCTIVITY MEASUREMENT The Radiofrequency (RF) energy penetrates into cell and reveal information about its size and internal structure.
- 12. SCATTER MEASUREMENT As cells are passed in single stream (flow cell) they are struck by laser 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
- 13. 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) Eosinophils Neutrophils Monocytes LUC Lymphocytes + Basophils Perox Activity Volume ADVIA TECHNOLOGY
- 14. 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. Basos are subtracted from the lymph/baso cluster in the perox channel to calculate the lymphocytes. ADVIA TECHNOLOGY
- 15. Fluorescence Flow Cytometry The intensity of scattered lights (FSC and SSC) reflects cell surface structure, particle shape, nucleus form, refractive index and reflectivity of the cells. The intensity of the side fluorescent light (SFL) mainly reflects the type and amount of nucleic acids and cell organelles.
- 16. WDF
- 18. WPC Scattergram SSC SFL WBC WBC Blast Abn Lymph Blast Abn Lymph WPC channel FSC
- 19. RET CHANNEL Reticulocyte count Reticulocyte fractions (LFR, MRF, HRF) Immature reticulocyte fraction Ret-He (reticulocyte Hb content) Platelet –O (fluorescent platelets) Fragmented red cells (FRC)
- 21. SLS-Hemoglobin Hemolytic reaction between SLS and RBC menbrane 1 2 3 4
- 22. • Photometrical analysis at 555 nm • Lambert Beer Law: Absorbance α Hgb concentration HGB Photometer lens Sample stream Cellpack Flow cell photosensorLED
- 23. Hematocrit (Hct) • Cumulative Pulse Height Detection • Direct measurement, not calculated. • Resistance change is detected as the height of the pulse, proportional to size of RBC. V t Total Volume RBC Volume RBC RBC Volume Total Volume X 100 = Hct Cumulative sum of all RBC pulses RBC Volume
- 24. RBC indices • Calculated from three basic parameter. RBC, HCT, Hb Hct (%) RBC (x 106/µl) MCV (fl) = Mean Corpuscular Volume (MCV) Mean Corpuscular Hemoglobin(MCH) Hb (g/dl) RBC (x 106/µl) MCH (pg) = Hb (g/dl) Hct (%) MCHC (g/dl) = Mean Cellular Hemoglobin Concentration (MCHC)
- 25. RBC- and PLT-Histograms Platelet MPV-8-12 fl Red blood cells 80-100 fl
- 26. Normal RDW Increased RDW The RDW is determined by calculating the width of RBC histogram in fl and is expressed as CV(12-14)or SD(40-46)
- 27. The Mean of the platelet volume between the lower discriminator PL and the upper discriminator PU Pct (platelet crit) is equivalent to the sum of platelet impulses which are individually detected by means of the impedance measurement principle Mean Platelet Volume (MPV)
- 28. From PLT histogram to PDW and P-LCR Platelet Large Cell Ratio (P-LCR) Percentage of large platelets with a volume >12 fL (N= 15-35%) Platelet Distribution Width (PDW) The platelet distribution width measured at 20% relative height of the total height of the curve (9-14fl)
- 29. Summary Principles RBC/PLT HGB WNR WDF WPC RET/ PLT-O PLT-F ChannelsTechnology Sheath Flow DC Detection Method Histograms/ Scattergrams SLS- Hemoglobin Method Hemoglobin Flow Cytometry method Flow Cytometry method Flow Cytometry method Flow Cytometry method Flow Cytometry method
- 31. Curve not ending at base line
- 41. Screening test: PT, APTT, Fibrinogen and D- dimer. Tests which look for abnormalities in coagulation & gives direction for selecting the specific test and help in the diagnosis and treatment Specific test: Platelet Aggregation studies, Specific coagulation factor assay, Antithrombin ,Protein C activity etc.,
- 42. Manual Methods Reagents and samples are added manually. Temperature is maintained by a water bath. Values are measured manually by using stopwatch. human error and are difficult to do when there is large workload.
- 43. Semi Automated Analysers Has mechanism to automatically initiate timing device upon addition of final reagent and internal mechanism for detecting Clot formation. All reagents and samples are added manually by the operator. Semi automated analyzer may or may not internally maintain the temperature . E.g., Start® (Stago Diagnostica)
- 44. Automated analyser Human errors are eliminated(sample identification, addition of sample and reagents and end point detection) Maintains and check the temperature. Perform auto dilutions mainly for the single factor assays.
- 45. ¶ Storage of large no. of patient data and control result ¶ Generates flagging for sample abnormality and instrumental malfunctioning. ¶ Batch processing of multiple samples for single test or multiple tests on a single sample can be performed
- 46. Clotting assay Functional assay In this sample and reagent are mixed together to form a clot. End point of the reaction i.e., clot is detected by Mechanical Optical detection Electrochemical
- 47. Mechanical method Magnetic steel ball method The sample is introduced to a cuvette that has a small steel ball inside The cuvette continuously moves when reagent and sample is added The fibrin strands begin to form and attach to the moving ball There is a break in contact with the magnetic sensors when the steel ball becomes incorporated into a fibrin clot as the cuvette Clotting time is recorded.
- 49. Optical detection method • As the plasma sample clots, it becomes more optically dense and the amount of light falling on a photosensitive detector decreases. • The drop or change in light is determined as the endpoint = turbidometric method
- 50. Nephelometric method The nephelometer uses a light -emitting diode at a high wavelength (>600 nm) to detect variations in light scatter as fibrin clot is formed. When the light rays encounter insoluble complexes such as fibrin strands, they are scattered at 900 angle.
- 51. Chromogenic Method • Chromogenic or amidolytic methodology is based on the use of specific color producing substance = chromophore. • Normally used chromophore is para-nitroaniline (405nm)
- 52. Immunologic Method • Immunologic assays are based on Ag-Ab reactions. • Latex Micro particles are coated with a specific Ab directed against Ag. • A beam of monochromatic light is then passed through the suspension of micro latex particles. • Formation of agglutination lead to increase in turbidity of the test solution and increase in Absorbance which, in turn, is proportional to the antigen level present in the sample, which is read from a standard curve.
- 53. Electro chemical • The INRatio single-use test strip has a sample well where blood is applied, three channels through which the blood sample flows to reach the testing areas. • Reagents start the coagulation process. • The device detects a change in electrical resistance when blood clots. INRatio meter
- 54. Uses stimulators of platelet adhesion and aggregation in an environment that stimulates an injured blood vessel wall. More sensitive screening test than the bleeding time method Nonspecific test- not diagnostic for any single disorder Platelet Function Analyser
- 55. The instrument adds citrated blood to a reservoir with collagen/epinephrine on a bioactive membrane A pressure sensor detects the formation of a platelet plug on the membrane The time it takes to close the aperture in the membrane with the platelet plug is recorded. The result is a function of platelet count, platelet activity, VWF activity. Platelet Function Analyser
- 56. Platelet Aggregometry • Platelet aggregometry involves a series of tests performed on whole blood or platelet-rich plasma, using several agonists (platelet activators). • The agonist is added to the suspension and a dynamic measure of platelet clumping is recorded. • Simultaneously to platelet aggregation, luminometry test can be performed. In that case, ATP release is assayed using a luminescent marker.
- 57. Thrombelastography (TEG) •Sample of citrated whole blood is placed in a cup which has a pin carefully connected to a torsion wire. •As the cup rotates in a back and forth movement, the aggregates formed within the cup cause the wire to become more rigidly placed and reflects the strength of the aggregates formed within the cup. •The movement or lack of movement is reflected via either an optical or magnetic detector •A graphic presentation is produced
- 58. TEG Graphic Result R=Time of latency from start of test to initial fibrin formation K=Time taken to achieve certain level of clot strength Alpha angle =measures the speed at which fibrin build up and cross linking takes placeMA=maximum amplitude
- 59. Typical TEG Graph Patterns
- 60. Illustrates function and dysfunction in the Hemostatic system Allows physicians to give appropriate amounts of FFP, Cryo, and platelets to control hemorrhage Reduces unnecessary use of blood products Allows effective management of hypercoaguability Differentiates surgical from pathological bleeding Uses of TEG
- 61. Flowcytometry
- 62. Flow cytometry • Immunophenotyping of cells is one of the most important clinical application of Flow cytometery. • Flow cytometery is the methodology used to detect cell surface antigens using monoclonal antibodies conjugated with different fluorochromes.
- 63. Uses of flowcytometry • Diagnosis. • Assess response to therapy. • Prognosis and detection of minimal residual disease.
- 64. Indications • Acute leukemias • Chronic lymphoproliferative disorders • Myelodysplastic syndrome • Plasma cell dyscrasias • Red cell disorders • Platelet disorders PNH Fetal cell estimation Hereditary spherocytosis Reticulocyte estimation
- 65. BD FACSCalibur
- 66. The common elements in all Flowcytometers • A light source • Fluid lines and controls to direct a liquid stream containing particles through the focused light beam • An electronic network for detecting the light signals coming from the particles as they pass through the light beam and then converting the signals to numbers that are proportional to light intensity • A computer for recording the numbers derived from the electronic detectors and then analyzing them
- 67. The specimen must be in a monodisperse suspension. In this, isotonic fluid is forced under pressure where a fluid column with laminar flow and a high flow rate is generated (so- called sheath fluid). The sample is introduced into the flow cell in the center of the sheath fluid, creating a coaxial stream so that they are presented to the light beam one at a time.
- 68. • The emitted light is focused by a lens onto fiber optic cables and transmitted to octagonal detectors. • The sensors convert the photons to electrical impulses that are proportional to the number of photons received and to the number of fluorochrome molecules bound to the cell.
- 69. Sample preparation and processing Types of samples Anticoagulants EDTA (stable upto 24 hrs) Acid citrate dextrose (upto 72 hrs) Sample storage ideally within 48 hrs <40C and >300C should be avoided Peripheral blood BMA Body fluids Lymphnode aspirates
- 70. 100 l of sample Add 8l of antibody (30 min) 2ml of lysing sol (15 min) Centrifuge 1500 rpm for 5min Supernatant fluid discard 2ml of sheath fluid Centrifuge 1500 rpm for 5min Supernatant fluid discard Add 0.5 ml of sheath fluid Acquire into flow machine Sample preparation and processing
- 71. For cytoplasmic antibodies 100 l of sample Add 8l of antibody (30 min) 2ml of lysing sol (15 min) Centrifuge 1500 rpm for 5min Supernatant fluid discard Permealisation sol 0.5ml (15 min) 1ml of sheath fluid Centrifuge 1500 rpm for 5 min Supernatant fluid discard 10l of antibody (15 min) 1ml sheath fluid Centrifuge 1500 rpm for 5min Supernatant fluid discard 0.5ml of sheath fluid Acquire
- 72. • A pair of light scatter channels provides an approx. measure of cell size {Forward scatter(FS)} and granularity {Side scatter(SS)}.
- 75. Fully automated analyzer No reagents required – no waste. Direct use of primary EDTA tubes – no contact with blood. Therefore the EDTA tube can be used unchanged for further analyses after erythrocyte sedimentation. Compatible with all the commonly used EDTA tubes in the market. Photometric infrared (950nm) reading prevents interference caused by lipids or bilirubin in the sample.
- 76. High Performance Liquid Chromatography
- 77. • HPLC is a chromatographic technique that can separate a mixture of compounds. • Type of liquid chromatography where the sample is forced through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer.
- 78. Principle:- Depends on interchange of charged groups on the ion exchange material with charged groups on Hb molecule.
- 79. Carboxyl groups attached to a resin base Direction of flow Detector Cation Exchange Cartridge
- 80. Whole blood + hemolysate Introduction Positively charged hemoglobin fragments in the hemolysate attach to the carboxyl groups at varying strengths.
- 81. Starting Gradient: Low Ionic Strength Buffer The gradient starts with a low % of Buffer At this gradient, hemoglobin fragments with an ionic strength lower than the buffer gradient, such as HbF, are displaced from the cartridge and pass into the detector
- 82. Ending Gradient: High Ionic Strength Buffer • As the % of the Ionic Strength of Buffer increases, the more hemoglobin fragments will be displaced • Once the gradient is 100% all remaining hemoglobin fragments, including any variant hemoglobins such as S, D and C, will be removed
- 83. • Separated components pass through dual wavelength detectors. • Absorbance is measured at 415 nm and the data is displayed as chromatogram. • Finally it is converted into peaks as per the retention time • Separated Hb’s with % is displayed. Retention time: Time in min from the sample injection to the max point of elution peak of Hb
- 84. Retention time for Hb HPLC of abnormal Hb
- 85. Conclusion • The automation in hematology is efficient, lack inter observer variability and size distribution errors. • They give an estimate of many variables which are manually not possible and produce data with increased reliability, precision and accuracy. • Data can be stored in automated analysers.
Editor's Notes
- #27: RDW-cv = (One Standard deviation of red cell volume ÷ mean cell volume) x 100 and is reported as a %.