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Automated Hematology: An Overview

Automated Hematology: An Overview. Session Outline. Principles of Automation Initial Setup Calibration Quality Control Flagging Troubleshooting Case Studies. Objectives. Describe the electrical impedance and light scatter principle for performing cell counts

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Automated Hematology: An Overview

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  1. Automated Hematology: An Overview

  2. Session Outline • Principles of Automation • Initial Setup • Calibration • Quality Control • Flagging • Troubleshooting • Case Studies

  3. Objectives • Describe the electrical impedance and light scatter principle for performing cell counts • Explain the underlying causes of invalid automated hematology results. • Utilize quality control procedures to determine if patient results are acceptable • Recognize the significance of flagging and take appropriate actions

  4. Range of Technologies in Zambia ABX Micos 60

  5. ABX Pentra 60 C+

  6. Hematology Automation • Two General Principles • Electronic resistance ( impedance) • Light scattering

  7. Coulter Principle Dilution Vacuum and pressure Electrical impedance Reagent systems Cell counting

  8. Electronic Resistance (Impedance) • Utilizes non-conductive properties of blood cells • as blood cell passes through orifice of aperture it displaces its own volume • increased resistance between electrodes results in an electrical pulse • RBCs and Platelets counted together, separated by pulse heights • hydrodynamic focusing forces cells to pass single file through sensing zone

  9. Light scattering • Cells counted as passed through focused beam of light( LASER) • Sum of diffraction(bending around corners), refraction (bending due to change in speed) and reflection (light rays turned back by obstruction). • Multi angle polarized scatter separation (M.A.P.S.S) • 0° : indicator of cell size • 10° : indicator of cell structure and complexity • 90° polarized: indicates nuclear lobularity • 90° depolarized: differentiate eosinophils

  10. ABX Technologies

  11. Initial Setup

  12. Out of the BoxFor the service Engineer • Check instrument for visual damage • Check for any loose parts or connections • Make sure all computer boards are properly sealed • Check the socket to verify proper voltage outlet • Plug instrument power cord into (voltage stabilizer) electrical supply • Confirm the correct voltage on instrument • Main power supply • Photometric voltage • Any other voltage supply that is pertinent to instrument functions

  13. Out of the BoxFor the service Engineer • Permit instrument to stabilize/equilibrate • Let all components reach proper temperature • Set in any parameters that may be required • Ranges • Temps

  14. 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

  15. Calibration • Calibration fine tunes your hematology analyzer and provides the most accurate results possible. • In the normal process of tracking data for an extended period of time, your laboratory can make a specific decision to recalibrate a given parameter. 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.

  16. Performing Reproducibility Check – CBC (N=10) Sample Requirements - For reproducibility studies, ensure the patient for the sample that is being tested: • Is receiving no medication • Has normal hematologic parameters, with a WBC count of 5.0 ± 1.0. • Has normal erythrocyte, leukocyte, and platelet morphology and, if you want to check the Diff parameters, with Diff values • Neutrophils 40 to 72% • Lymphocytes 17 to 45% • Monocytes 4 to 12% • Eosinophils 0 to 10% • Basophils 0 to 1% Ensure you have enough normal whole blood from a single donor for 11 cycles.

  17. Daily startup procedures(Daily Maintenance) • Daily cleaning • Background counts • Electronic checks • Compare open and closed mode sampling (use a normal patient sample) • Run controls Must be within specified limits

  18. Ensure the Instrument is Functioning Properly 1. Check the reagent containers for: • Sufficient quantity • Not beyond expiration date • No precipitates, turbidity, particulate matter, or unusual color • Proper connections between the instrument and the reagent containers

  19. Ensure the Instrument is Functioning Properly 2. Check the waste container for: • Sufficient capacity • Proper connections • Perform daily startup • In addition to verifying daily startup results, verify acceptable: • Reproducibility • Carryover • Control Results

  20. Quality Control

  21. Quality Control • Purpose of QC • Assure proper functionality of instrumentation • Means of assuring accuracy of unknowns • Monitoring the Integrity of the Calibration • When controls begin to show evidence of unusual trends • When controls exceed the manufacturer’s defined acceptable limits

  22. Quality Control Methods • Assayed or Unassayed stabilized material (Commercial) • Previously analyzed patient samples • Easily obtained • Cost effective • Results and samples readily available

  23. QC Methods: Assayed or Unassayed Stabilized Material • Commercially available • Known values (Assayed only) • Analyze low, normal and high control • Results stored in the instrument computer (Pentra only) • Monitored with Levy-Jennings charts • - Easily illustrates trends and shifts

  24. Quality Control • OUT OF CONTROL!!! • Repeat the assay ( One time occurrence ) • Check for trends (from Levy Jennings) • Check integrity of material • Troubleshoot • Verify instrumentation

  25. Remedial Action to Take When a Control is Outside its Expected Range 1. Ensure the control • Material was mixed properly. If not, mix it according to the package insert. • Identification information was entered correctly. If using the Numeric Keypad, ensure you typed the correct information. • Setup information (assigned values and expected ranges) matches the control package insert for the current lot number being used. If they do not match, change the control’s information to match the package insert.

  26. Remedial Action to Take When a Control is Outside its Expected Range • If any of the problems still exist, rerun the control; otherwise, proceed to the next step • Rerun the control to ensure the problem was not a statistical outlier. • Ensure the control material was not contaminated by running another vial or level of control.

  27. Automated Hematology Problem Solving – Troubleshooting Specimen – Related OR Instrument

  28. Data Review • A review of instrument data, such as background, control, and blood sample results, is helpful in detecting problems. • Sometimes a questionable blood sample result is the only symptom of subtle reagent or pneumatic problems.

  29. 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

  30. 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.

  31. Electronic Troubleshooting • Detecting a problem in the electronic subsystem – or eliminating the electronic subsystem as the source of the problem – is simplified by indicators and electronic tests. • Correcting Electronic Problems: • minor problems,such as loose cables • most electronic problems require the assistance of your instrument service representative.

  32. Pneumatic / Hydraulic Troubleshooting • Most pneumatic / hydraulic problems are detected by observing the Diluter section in operation. • When you identify a symptom of a malfunction, try to isolate the malfunction to the specific part of the cycle, for example, during preparation, counting, or cleanup. • Then, try to isolate the malfunction to the specific components and tubing. • Next, look for one of four possible problems – pinched tubing, plugs, leaks, or defective components.

  33. Correcting Pneumatic/Hydraulic Problems You can correct most pneumatic / hydraulic problems, including defective components. • Tubing, (Need other examples) • Follow manufacturer’s instructions • Sticking float in moisture chamber

  34. 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.

  35. Correcting Reagent Problems • You can correct most reagent problems by: • changing the container of reagent • priming the instrument with the new reagent.

  36. Flagging • Condition flags • Describes cell population • normal • abnormal • WBC Suspect flags • Blasts • Imm Grans/Bands 1 • Imm Grans/Bands 2 • Variant lymphs • Review Slide

  37. More Flagging • RBC Suspect flags • NRBCs • Macrocytic RBCs • Dimorphic RBC population • Micro RBCs/RBC fragments • RBC agglutination • Definitive Flagging • Based on predetermined lab limits • Provide information for review

  38. Review ofHistogramsas aQualityControlTest

  39. RBC, PLT, and WBC plotted on histogram X-Axis Cell size in femtoliters (fL) Y-Axis # of cells Histograms

  40. Histogram • WBC: Distribution with three individual peaks and valleys at specific regions representing the lymphocytes, monocytes, and granulocytes. • All curves normally start and end at baseline

  41. RBC Histogram As A Quality Control Tool

  42. WBC Histogram As A Quality Control Tool

  43. WBC Histogram As A Quality Control Tool

  44. Platelet Histogram As A Quality Control Tool

  45. Interpreting Test Results Misleading results can occur if the specimen was not properly collected, stored, or transported. The following situations can also yield misleading results for the parameters listed:

  46. Interfering substances • Cold Agglutinins • Cryoglobulins • Lipemia • Platelet Clumps • RBC fragments • NRBCs

  47. Clotted specimen. Inspect every specimen for clots Unusual RBC abnormalities that resist lysing malarial parasites giant platelets, platelet clumps, NRBCs fragmented white cells, agglutinated white cells, lyse-resistant red cells, cryoglobulin, some extremely elevate proteins, unlysed particles greater than 35 fL in size Interferences Cont. • All Parameters: • WBC:

  48. Interferences • RBC: Very high WBC count, high concentration of very large platelets, auto-agglutination • Hgb: Very high WBC count, severe lipemia, heparin, certain unusual RBC abnormalities that resist lysing • MCV: Very high WBC count, high concentration of very large platelets, auto-agglutination • RDW: Very high WBC count, high concentration of very large platelets, auto-agglutination • Plt: Giant platelets, platelet clumps, white cell fragments, electronic noise, very small red cells, red cell fragments • Hct: Known references related to RBC and MCV

  49. Interferences • MCH: Known interferences related to Hgb and RBC • MCHC: Known interferences related to Hgb, RBC, and MCV Troubleshooting Flagged Results: • Refer to the Instrument Manual to troubleshoot codes, flags, and messages displayed with patient results

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