Transcript
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Automated Hematology:
An Overview
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Session Outline• Principles of Automation• Initial Setup• Calibration• Quality Control• Flagging• Troubleshooting• Case Studies
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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
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ABX Pentra 60 C+
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Hematology Automation• Two General Principles
Electronic resistance ( impedance) Light scattering
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Cell counting• Coulter Principle
Dilution Vacuum and
pressure Electrical impedance Reagent systems
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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
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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
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TechnologiesABX
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Initial Setup
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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
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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
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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
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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.
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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.
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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
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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
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2. Check the waste container for: Sufficient capacity Proper connections
3. Perform daily startup4. In addition to verifying daily startup results, verify
acceptable: Reproducibility Carryover Control Results
Ensure the Instrument is Functioning Properly
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Quality Control
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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
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Quality Control Methods• Assayed or Unassayed stabilized material
(Commercial)• Previously analyzed patient samples
Easily obtained Cost effective Results and samples readily available
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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
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Quality Control• OUT OF CONTROL!!!
Repeat the assay ( One time occurrence ) Check for trends (from Levy Jennings) Check integrity of material Troubleshoot Verify instrumentation
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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.
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Remedial Action to Take When a Control is Outside its Expected Range
2. If any of the problems still exist, rerun the control; otherwise, proceed to the next step
3. Rerun the control to ensure the problem was not a statistical outlier.
4. Ensure the control material was not contaminated by running another vial or level of control.
Automated Hematology
Problem Solving – Troubleshooting
Specimen – Related OR Instrument
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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.
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• 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
Specimen-Related Problems
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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.
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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.
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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.
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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
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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.
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Correcting Reagent Problems
• You can correct most reagent problems by: changing the container of reagent priming the instrument with the new
reagent.
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Flagging• Condition flags
• Describes cell populationnormalabnormal
• WBC Suspect flags Blasts Imm Grans/Bands 1 Imm Grans/Bands 2 Variant lymphs Review Slide
Check Slide
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Check Slide
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
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Review ofHistograms
as aQualityControl
Test
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Histograms• RBC, PLT, and WBC
plotted on histogram• X-Axis
Cell size in femtoliters (fL)
• Y-Axis # of cells
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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
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RBC Histogram As A Quality Control Tool
ABN / INDICATOR PROBABLE CAUSE COMMENTLeft of curve does not touch baseline
Schistocytes and extremely small red cells
Review smear CBC and Platelet histogram
Bimodal peak Transfused cells, therapeutic response
Review Smear
Right portion of curve extended
Red cell autoagglutination
Review CBC & Smear
Left shift of curve Microcytes Review smear & CBC
Right shift of curve Macrocytes Review smear & CBC
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WBC Histogram As A Quality Control Tool
ABN / INDICATOR PROBABLE CAUSE COMMENTTrail extending downward at extreme left, or lymph peak not starting at baseline
NRBC, Plt clumping, unlysed RBC, cryoproteins, parasites
Review smear and correct WBC for NRBC
Peak to the left of lymph peak or widening of lymph peak towards left
NRBC Review smear & correct WBC for NRBC
Widening of lymph peak to right
Atypical lymphs, blasts, plasma cells, hairy cells, eosinophilia, basophilia
Review smear
Wider mono peak Monocytosis, plasma cells, eosinophilia, basophilia, blasts
Review smear
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WBC Histogram As A Quality Control Tool
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
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Platelet Histogram As A Quality Control Tool
ABN / INDICATOR PROBABLE CAUSE COMMENTPeak or spike at left end of histogram (2-8 fl)
Cytoplasmic fragments
Review smear
Spike towards right end of histogram
Schistocytes, microcytes, giant platelets
Review smear + CBC( MCV & RDW)( MPV & PDW)
Bimodal peak Cytoplasmic fragments
Review smear
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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:
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Interfering substances• Cold Agglutinins• Cryoglobulins• Lipemia• Platelet Clumps• RBC fragments• NRBCs
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Interferences Cont. All Parameters:
WBC:
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
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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
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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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