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LH750, Beckman Coulter; Operate

5C Control

5C Manual Aspirate

Activate Instrument in Standby

Body Fluids Controls

Body Fluids Patient

Change Report Type

Expected Results

Instrument Flags

Latron Primer/Control

Limitations

Materials

New Lot Latron Control

New Lot 5C

Patients Auto Aspirate

Patients Manual Aspirate

Quality Control

Reagent Update

Reagents

Result Derivation

Samples Pre-Dilute

Specimen

Transmit Results

I. Principle:

The Beckman/Coulter LH750 is an in vitro analytic instrument that provides complete blood cell counts (CBC), white cell differential counts, and Reticulocyte Counts on whole blood.
The Body Fluid Application provides analysis of cerebrospinal fluid, serous fluids (pleural, peritoneal, pericardial, peritoneal lavage, peritoneal dialysate and hyaluronidase treated synovial fluids). Only WBC and RBC results are reported on Body Fluids.

System Methodologies:

  1. WBC: Impedance counting. Pulse measured in the WBC aperture representing cells as 35fl or greater are classified as white blood cells.
  2. RBC: Impedance counting. Pulse measured in the RBC aperture representing cells as 36fl or greater are classified as red blood cells.
  3. Platelet Count: Impedance counting. Pulses measured in the RBC aperture representing cells from 2-20 fl are classified as platelets.
  4. HGB: The transmittance of light (525nm wavelength) through the lysed WBC solution in the hemoglobin cuvette is compared to the transmittance of the same light through a reagent blank. The system converts this ratio to a HGB value in g/dl using a calibration factor.
  5. MCV: The average volume of individual erythrocytes derived from the RBC histogram.
  6. RDW: The size distribution spread of the erythrocyte population derived from the RBC histogram.
  7. MPV: The average volume of individual platelets derived from the PLT histogram.
  8. Reticulocyte Count: The instrument makes 3 measurements (volume, conductivity and scatter) as each cell passes through the flow cell. The low frequency impedance measurement defines cell volume. The high frequency conductivity measurement indicates the internal conductivity. The light scatter measurement indicates the structure and shape.
  9. Absolute Reticulocyte: The absolute number of reticulocytes is calculated from RET% and RBC number.
  10. HCT, MCH, MCHC: Calculated parameters.
    HCT: The relative volume of packed erythrocytes to whole blood. HCT = RBC x MCV ÷ 10.
    MCH: The weight of HGB in the average erythrocyte. MCH = HGB ÷ RBC x 10
    MCHC: The average weight of HGB in a measured dilution. MCHC = HGB ÷ HCT x 100
  11. WBC Differential: The instrument makes 3 measurements (volume, conductivity and scatter) as each cell passes through the flow cell. The low frequency impedance measurement defines cell volume. The high frequency conductivity measurement indicates the internal conductivity. The light scatter measurement indicates the structure and shape. The AccuGate algorithm is applied to determine different cell populations.
  12. Absolute Neutrophils: Calculated parameter
    Total WBC x (%NE result ÷ 100) = absolute neutrophils.

    Note: Recalculate absolute neutrophils when a manual differential is reported.

II. Materials:

Racks

III. Reagents:

  1. Coulter LH Series 700 Diluent (Beckman/Coulter #8547194)
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 60 days after opening.
    3. Mixing instructions:
      1. Roll the cube 20 times.
      2. Let it set 24 hours to de-bubble.
  2. Coulter LH Series PAK Reagent Kit (Beckman/Coulter #8547195)
    contains Erythrolyse II and StabiLyse
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 60 days after opening.
  3. Coulter CBC Lyse S III diff Lytic Reagent (Beckman/Coulter #8546983)
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 60 days after opening.
  4. Coulter Clenz Cleaning Agent (Beckman/Coulter #8546931)
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 90 days after opening.
  5. Coulter LH Series Retic PAK Reagent Kit (Beckman/Coulter #8547196)
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 60 days after opening.
  6. Latron Primer (Beckman/Coulter #7546915)
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 30 days after opening.
  7. Latron Control (Beckman/Coulter #7546914)
    1. Store at 15 - 30°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 30 days after opening.
  8. Coulter 5C Cell Control (Beckman/Coulter #7547116)
    1. Store at 2 - 8°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 13 days after opening.
    3. Allow vials to equilibrate at room temperature for 15 minutes.
    4. Mix each vial using the 8x8x8 method.
      1. Hold the vials with the stoppers up and roll in hands 8 times.
      2. Hold the vials with the stoppers down and roll in hands 8 times.
      3. Gently invert the vials end to end 8 times.
    5. Visually check each vial after mixing to ensure cells are suspended. If cells are not suspended, then gently invert the controls 8 more times.
  9. Coulter Retic C Cell Control (Beckman/Coulter #7547125)
    1. Store at 2 - 8°C. Stable unopened until the expiration date printed on the label.
    2. Stable for 13 days after opening.
    3. Allow vials to equilibrate at room temperature for 15 minutes.
    4. Mix each vial using the 8x8x8 method.
      1. Hold the vials with the stoppers up and roll in hands 8 times.
      2. Hold the vials with the stoppers down and roll in hands 8 times.
      3. Gently invert the vials end to end 8 times.
    5. Visually check each vial after mixing to ensure cells are suspended. If cells are not suspended, then gently invert the controls 8 more times.
  10. Cell-Chex Auto Body Fluid Cell Count Control (Streck #200065)
    1. Store at 2 - 8°C. Stable unopened until the expiration date printed on the label.
    2. Controls are stable for 30 days after opening.
    3. Allow controls to warm to room temperature for 15 minutes before use.
    4. To mix:
      1. Hold the vial horizontally between the palms of the hands and roll the vial back and forth for 20-30 seconds.
      2. Mix by rapid inversion to ensure the cells are suspended.
      3. Gently invert the vials 8-10 times immediately before sampling.
      4. Vials stored for an extended period of time may require extra mixing.
  11. Hyaluronidase (Sigma #H3884)
    1. This reagent is salt-free with an activity of 750-3000 u/mg.
    2. Store at -20°C.

IV. Specimen:

  1. Samples should be mixed for 5 minutes before running. Refer to "Specimen Integrity: Hematology" in the Quality Assurance Manual.
    Note: Refrigerated specimens must be allowed to equilibrate to room temperature before mixing.

    CBC/CBC Diff:

    Automatic Aspiration Mode

    Manual Aspiration Mode

    Prediluted Mode
  2. Body Fluid Application provides analysis of cerebrospinal fluid, serous fluids (pleural, peritoneal, pericardial, peritoneal lavage, peritoneal dialysate) and hyaluronidase treated synovial fluids. Refer to “Body Fluids” section
    1. Cerebrospinal fluids should be processed within 2 hours of collection when stored at room temperature.
    2. Serous fluid should be processed within 8 hours of collection when stored at room temperature.
    3. Synovial fluids:
      1. Should be processed within 8 hours of collection when stored at room temperature.
      2. Pretreat synovial fluid with hyaluronidase before running on the instrument. Transfer 1 ml of synovial fluid to a small test tube that contains 5 mg hyaluronidase. Mix for 5 minutes.
  3. Reticulocyte Counts are not to be run on non-human species. The LH750 Reticulocyte Count is not validated for non-human species.

V. Reagent Update:

  1. Record the new reagent information:
    1. Click on the System Setup icon. (Yellow diamond, located in the lower left corner of the screen.)
    2. Select QA.
    3. Select the Reagent Tab.
    4. Click on Reagent Setup.
    5. Use the handheld barcode reader to scan the 2 barcodes on the new reagent container. (The barcodes may be scanned in any order.)
    6. Verify that all the fields of the “Reagent Information Preview” box have populated correctly.
    7. Click the Save button (green checkmark) to complete the importing of the new reagent data.
  2. Open the new reagent container. Change the expiration date on the container to reflect that data that was imported.
  3. Unscrew the pickup tube assembly off the old reagent container.
  4. Lift the assembly straight up and out. Be careful not to touch part of the assembly or let it touch any lab surfaces.
  5. Transfer the pickup tube assembly to the new reagent container and secure it.
    Note: Retic reagent is in use at UCL-Finley only, but UCL-Mercy DBQ needs to keep an active retic barcode in the Reagent Setup Screen. At Mercy the retic barcode is taped to a small bottle of diluent. When the barcode expires, call Finley to send another barcode with a later outdate to scan into the LH750 at Mercy DBQ.
  6. Prime the reagent that was replaced using the function feature.
  7. Run two 5C cell controls before assaying any patient samples. (See section VIII.)

VI. Running Latron Primer and Control:

  1. Select Function 57 on the Analytical Unit. Press F57 and then Enter, on the Numeric Keypad.
  2. Press the Clear Aperture button. This identifies the sample as the Latron Primer.
  3. Mix the Primer bottle by gentle inversion. Be careful not to introduce any bubbles into the bottle.
  4. Aspirate the Latron Primer in the manual aspiration mode.
  5. Review the Latron Primer results on the QA screen.
    1. Select “QA” on the Command Center to display the Quality Assurance application.
    2. Select “QC” to display the control windows.
    3. Select the Latron Control. The control results table, statistics and graphs appear on the window. Use the scroll bars on the window to view other parameter results and graphs.
  6. The results for the Primer must be ≤ 500. The control trial will be highlighted in red if it is unacceptable. If the Latron Primer results are unacceptable, repeat the Primer once. If repeat is still unacceptable the flowcell may need to be purged, or the Primer may have excessive bubbles.
  7. Function 57 will be displayed on the Analytical Unit. Press Enter.
  8. Mix the Latron Control bottle by gentle inversion. Be careful not to introduce any bubbles into the bottle.
  9. Aspirate the Latron Control in the manual aspiration mode.
  10. Review the Latron Control results on the QC screen. The control trial will be highlighted in red if results are unacceptable. If the Latron Control results are unacceptable, repeat the control once. If the repeat is unacceptable, Purge the Flowcell and then repeat the Control. “PURGE the Flowcell” is Function 13.
  11. Press the STOP button on the Analytical Unit and the instrument will go into the Ready mode.

VII. Activating the Instrument in Standby:

  1. If the diluter is idle for about 1 hour, the pneumatic subsystem turns off. The analyzer screen will go blank and the display screen will read “Compressor Off”.
  2. To activate the pneumatic subsystem, press PRIME APERT button on the analyzer.
  3. Wait for the analyzer to display READY and then proceed with patient or control sampling.

VIII. Running 5C Cell Controls & Retic Controls (Automatic Aspiration Mode):

  1. Allow the controls to reach room temperature and mix according to handling instructions in Reagents section. It will take approximately 15 minutes for controls to reach room temperature.
  2. Place control vials in a rack with the barcode labels facing up.
  3. Place the rack in the autosampler with the barcode labels up and caps toward the operator. The instrument will automatically start cycling.
  4. Review the control results in the QA file.
    1. Select “QA” on the Command Center to display the Quality Assurance application.
    2. Select “QC” to display the control files.
    3. Select the specific control for which you want to review results. The control results table, statistics and graphs appear on the window. Use the scroll bars on the window to view other parameter results and graphs.
  5. The Control Trial will be highlighted in red if it is unacceptable. Individual constituents will be flagged with “L” or “H” indicating which constituent(s) was unacceptable.
  6. If controls are unacceptable, refer to section XXIII.8. (Quality Control, Definitive language for handling “control out” situations).

IX. Running 5C Cell Controls & Retic Controls (Manual Aspiration Mode):

Note: Under most circumstances, 5C and Retic-C Controls should be run using the automatic aspiration mode.

  1. Allow the controls to reach room temperature and mix according to handling instructions in Reagents section. It will take approximately 15 minutes for controls to reach room temperature.
  2. Press “ID” button and then the “Prime Aperture” button on the analyzer.
  3. Enter the control barcode ID (including preceding zeroes) using the numeric keypad on the analyzer and then press ENTER.
  4. Unscrew the cap from the control vial and immerse the tip of the manual aspiration probe into the control vial. Aspiration is activated automatically.
  5. When a beep sounds, remove the control vial from the manual aspiration probe. The probe retracts, rinses and dries.
  6. Review the control results in the QA file:
    1. Select “QA” on the Command Center to display the Quality Assurance application.
    2. Select “QC” to display the control files.
    3. Select the specific control desired for review. The control results table, statistics and graphs display. Use the scroll bar to review other parameter results and graphs.
  7. The Control Trial will be highlighted in red if it is unacceptable. Individual constituents are flagged “L” or “H” indicating which one is unacceptable.
  8. If controls are unacceptable, refer to section XXIII.8. (Quality Control, Definitive language for handling “control out” situations).

X. Running Patient Samples/Automatic Aspiration Mode:

  1. Load samples into the rack with the barcode label facing up.
  2. Place the rack securely into the loading bay on the right side of the instrument. The instrument will automatically start cycling.
  3. Review patient results. The instrument has been programmed with “Review by Exception”.
    1. Samples that pass through these filters will be “Autovalidated” and sent to CLICS Instrument Review Release.
    2. Samples that did not pass through the review criteria will be in the “Review List” folder.
  4. To review patient results, select the “Patient Result” icon on the Command Center.
  5. Click on “Patient File Folder” icon located at the top of the screen. The patient results are placed in folders based on status and Run Type.
  6. Select the “Review List” folder. The patient results that did not pass through the review criteria will be in this folder. Results are listed with the most recent at the top.
  7. Highlight the patient results to be reviewed and then select the “Results and Graphics” icon on the right side of the screen. Patient results will be displayed. The CBC Data including histograms, and the Diff Data including cytograms, are available by selecting the appropriate tabs on the patient results screen.
  8. Refer to “Peripheral Blood Smear (PBS) Review, Technologist/Pathologist” for appropriate review steps.
  9. If the barcode label was not read, the sample ID will be displayed as “------“. To enter the Sample ID, select the “Edit” button on the right of the screen. Enter the correct Sample ID and press the Tab key. The edited field will be flagged with an “E”. Click on the “Save Edit” icon.
  10. Editing of patient results should be done on the CLICS Instrument Review Release screen.
  11. To release results to CLICS, select the “Validate” icon on the top of the screen.
  12. Close out the patient result screen to return to the “Review List” folder.

XI. Running Patient Samples/Manual Aspiration Mode:

Note: Run all Vet specimens in the manual mode after checking specimen for clots.

  1. If the sample has a barcode label:
    Place the cursor in the barcode field at the Workstation Command Center. Scan the sample barcode using the handheld barcode reader. Press the “ID” button and then the “.” button on the analyzer, to transfer the Sample ID to the Analytical Unit. The barcode number will appear on the Analytical Unit screen. Press the ENTER button on the Analytical Unit to accept the barcode number, or STOP to reject the barcode ID.
  2. If you are entering the Sample ID manually:
    Place the cursor in the barcode field at the Workstation Command Center, and type in the Sample ID to be used and then press the Tab key. Press the “ID” button and then the “.” button on the analyzer, to transfer the Sample ID to the Analytical Unit. The barcode number will appear on the Analytical Unit screen. Press the ENTER button on the Analytical Unit to accept the barcode number, or STOP to reject the barcode ID.
  3. If you fail to send the sample ID to the instrument within 60 seconds of data entry in the Barcode ID field, the sample ID entered is cleared. This minimizes the risk of sample misidentification.
  4. Remove the stopper from the specimen tube.
  5. Immerse the aspirator tip in the tube. The manual aspiration process activates automatically.
  6. When you hear a beep, remove the tube from the aspirator tip. The probe retracts, rinses and dries.
  7. After the instrument cycles the sample, review the sample results on the Workstation. Refer to steps listed in the above section for sample results review.

XII. Running Samples/Prediluted Mode:

  1. Dispense diluent into a test tube if necessary using Function F04. Press F04 on the numeric keypad and place a tube under the Manual aspiration probe. Press ENTER and the diluent will be dispensed. Then press STOP.
  2. Select the Prediluted Mode. A window will be displayed asking for the dilution factor. If a citrate tube is being sampled due to platelet clumps the dilution factor to enter is 1.1. If a 1:2 dilution is being run because a sample has results above the linear range, the dilution factor to enter is 2.0. The instrument will automatically correct the results for the dilution factor entered.
  3. If the sample has a barcode label:
    Place the cursor in the barcode field at the Workstation Command Center. Scan the sample barcode using the handheld barcode reader. Press the “ID” button and then the “.” button on the analyzer, to transfer the Sample ID to the Analytical Unit. The barcode number will appear on the Analytical Unit screen. Press the ENTER button on the Analytical Unit to accept the barcode number, or STOP to reject the barcode ID.
  4. If you are entering the Sample ID manually:
    Place the cursor in the barcode field at the Workstation Command Center, and type in the Sample ID to be used and then press the Tab key. Press the “ID” button and then the “.” button on the analyzer, to transfer the Sample ID to the Analytical Unit. The barcode number will appear on the Analytical Unit screen. Press the ENTER button on the Analytical Unit to accept the barcode number, or STOP to reject the barcode ID.
  5. If you fail to send the sample ID to the instrument within 60 seconds of data entry in the Barcode ID field, the sample ID entered is cleared. This minimizes the risk of sample misidentification.
  6. Remove the stopper from the specimen tube.
  7. Immerse the aspirator tip in the tube. The manual aspiration process activates automatically.
  8. When you hear a beep, remove the tube from the aspirator tip. The probe automatically retracts, rinses and dries.
  9. After the instrument cycles the sample, review the sample results on the Workstation. Refer to steps listed above for sample results review.

XIII. Running Body Fluid Cell Count Controls (Manual Aspiration Mode):

Note: Body fluid controls are not barcoded and do not have a QC file on the LH, they must be run in the manual aspiration mode.

  1. Allow the controls to reach room temperature and mix according to handling instructions in Reagents section. It will take approximately 15 minutes for controls to reach room temperature.
  2. Perform a Diluent Blank when entering the Body Fluid (CBC) mode. The WBC must be ≤ 0.2 and the RBC must be ≤ 0.01. If the results of the Diluent Blank are unacceptable, run another Diluent Blank. To dispense LH Diluent form the Manual Open Vial aspiration probe, press F04 on the analyzer and place a test tube under the aspiration probe. Press ENTER and the diluent will dispense. Refer to “Running Patient Samples/Manual Mode Aspiration” for instructions on running sample in the manual mode.
  3. Place the cursor in the barcode field at the Workstation Command Center. Manually enter the control lot # in the field and hit TAB on the keyboard.
  4. Enable the Body Fluids mode by clicking on the box next to the Body Fluids (CBC) application.
  5. Press “ID” and then the “.” button on the analyzer. When the analyzer status is READY, press ENTER.
  6. Unscrew the cap from the control vial and immerse the tip of the manual aspiration probe into the control vial. Aspiration is activated automatically.
  7. When a beep sounds, remove the control vial from the manual aspiration probe. The probe retracts, rinses and dries.
  8. Review the Control results:
    1. Select Patient Results on the Command Center.
    2. Click on “All” under completed.
    3. Select the lot # of the control and review the results.
    4. Document the control result on the LH 750 Body Fluid Cell Count QC Log.

XIV. Running Body Fluids Patient Samples:

  1. Ensure that the specimen has been properly collected and sufficient volume is available. Synovial fluids must be treated with hyaluronidase prior to running on the instrument. Refer to Specimen section of this procedure.
  2. Enable the Body Fluids mode on the workstation Command Center by clicking on the box next to the Body Fluids application. Body fluid analysis defaults to the CBC mode.
  3. Perform a Diluent Blank when entering the Body Fluid mode. (If QC was run just prior to the patient sample and the blank was already performed and passed, another blank is not necessary.) The WBC must be ≤ 0.2 and the RBC must be ≤ 0.01. If the results of the Diluent Blank are unacceptable, run another Diluent Blank. To dispense LH Diluent from the Manual Open Vial aspiration probe, press F04 on the analyzer and place a test tube under the aspiration probe. Press ENTER and the diluent will be dispensed. Refer to “Running Patient Samples/Manual Mode Aspiration” for instructions on running samples in the Manual Mode.
  4. Enable the Body Fluids mode by clicking on the box next to the Body Fluids application. Body fluid analysis defaults to the CBC mode.
  5. Mix the sample and aspirate in the Manual Mode. Refer to “Running Patient Samples/Manual Mode Aspiration” for instructions on running samples in the Manual Mode.
  6. Review sample results. If the WBC is <0.20 x 103 cells/µl, or if the RBC is <0.010 x 106cells/µl, do not report LH750 results. An “R” Flag on a Body Fluid result indicates that the result is below the reportable range. The cell count must be performed on a hemacytometer.
  7. Convert LH750 results to cells/mm3.
    WBC: Move the decimal three positions to the right, or multiply the result by 1000.
    RBC: Move the decimal six positions to the right, or multiply the result by 1,000,000.
  8. Report the WBC and RBC in cells/mm3.
  9. Body Fluid Survey:
    1. Run the body fluid survey specimens according to survey instructions.
    2. The body fluid survey lists reporting units as:
      WBC x 109/L
      RBC x 1012/L
      These units are comparable to the units used on the LH750:
      WBC x 103/µl
      RBC x 106/µl.
      Report results obtained on the LH750. DO NOT convert results for the survey to cells/mm3.
  10. To run another Body Fluid, enable the Body Fluid mode again. The selection applies to only one sample at a time.
  11. Run all body fluid controls and patient samples consecutively. Do not run any venous blood samples between controls or patient body fluid samples.

XV. Importing New Control Lot Number for Beckman/Coulter 5C Control:

  1. Select the Setup Icon on the Command Center.
  2. Select the QA icon.
  3. Select the Control Tab.
  4. Select the “Set up New Control Folder” icon.
  5. Select BCI- 5C-All Levels.
  6. Select the “Set Up New Lot” icon.
  7. Follow the directions on the pop-up window. Check Auto Stop and Auto Transmit.
  8. Select OK.
  9. Select the Close icon.
  10. Select OK to close the System Setup/QA window.
  11. After scanning the assay sheets, ensure that the correct features are still selected or enabled.
    1. Double-click on the yellow diamond icon.
    2. The System Setup window is displayed; double-click on the QA-Quality Assurance icon.
    3. The System Setup (Quality Assurance) window is displayed; click on the Control tab.
    4. All controls are displayed: At the middle of the screen, review “Auto Xmit” and “Auto Stop” columns.
      1. MHC LH750: There should be an “x” (enabled) in the Normal, Abnormal I and Abnormal II “Auto Stop” and “Auto Xmit” boxes and “x” in both the CControl and MControl “Auto Xmit” boxes. All other boxes should be blank/grey.
      2. Finley LH750: There should be and “x” enabled in the Normal, Abormal I, Abnormal II and Retic. Level I, Retic. Level 2 and Retic. Level 3 “Auto Stop” and “Auto X-mit” boxes and “x” in both the CControl and MControl “Auto X-mit” boxes. All other boxes should be blank/grey.
    5. If “x”s are not in the above configuration and need to be edited, click on the left side of the line to be edited (should backlight in black) and then click on the “pencil” icon.
    6. The Control Folder Setup window is displayed.
    7. At the top center of the window is an Automatic Data Handler box containing “Auto Transmit” and “Auto Stop” boxes.
    8. Click a check mark into the desired box (or de-select).
    9. Click on the green check mark to close out and Save the selection.
      Note: This must be done every time a new lot number of QC data is imported via the 2D BCR
      .
  12. When switching to a new lot number of the same control, a 3 day overlap is performed to determine the new target means to be used. The laboratory staff uses the following protocol to review the data.
    1. A 3 day overlap must be performed when switching to a new lot of control.
      1. The new lot number of controls will be imported according to the above instructions.
      2. A 3 day overlap is performed. The new lot number of control and the current lot number of control are run together. The data for each control will be stored in their respective files per control code.
      3. Following the 3 day overlap, the results obtained from the new lot number control are printed and reviewed. Refer to step 12 for printing overlap data.
      4. The printed control data will include calculated mean, SD and CV values for all constituents. A Mean Plot chart will also show where each constituent mean fits within the 2SD limits. If the mean values fall within the expected 2SD ranges, as noted on the Mean Plot chart, then the control target values for each parameter will be edited to the mean value obtained from the control overlap.
      5. If any of the control values fall outside of the expected 2SD limits, the site supervisor must be notified before proceeding.
    2. To apply Lab Limits to the control file and calculate the mean from the overlap data:
      1. Select the specific control for which you want to adjust limits. The control results table, statistics and graphs appear n the window.
      2. Select “Mean=>Lab Target” to replace the assigned values on this window with the values that currently appear as the mean values. The Workstation also replaces the expected ranges. The Workstation now displays the Lab Target and Lab Limit values.
  13. Printing Controls Prior To Deletion:
    Following a successful control overlap, print a hard copy of the old lot# of control prior to deleting from the control dictionary.
    1. Select the QA icon on the Command Center.
    2. Select the QC icon.
    3. Highlight the control lot number to be printed and press the Printer Icon.
    4. Select “Selected Lot” in the List Runs section. Select “Thumbnail” in the Levey-Jennings Graph section and then select “OK”.
  14. Deleting A Control From The Control Dictionary:
    1. To delete the old control lot numbers, select the QA icon on the Command Center.
    2. Select the QC icon.
    3. Highlight the control lot number to be deleted and click on the Delete icon.
    4. Select “ All data including set-up tables” and then select “OK”.

XVI. Importing New Control Lot Number for Latron Control:

Note: Latron Primer does not require importing new ranges for new lot numbers. Acceptable range is always <500.

  1. Select the Setup Icon on the Command Center.
  2. Select the QA icon.
  3. Select the Control Tab.
  4. Select the “Set up New Control Folder” icon.
  5. Select BCI- Latron.
  6. Select the “Set Up New Lot” icon.
  7. Enter the information listed on the package insert, starting with the lot number. Use the tab key to advance to the next field.
  8. Select OK.
  9. Select the Close icon.
  10. Select OK to close the System Setup/QA window.
  11. Printing Controls Prior To Deletion:
    Print a hard copy of the old lot# of control prior to deleting from the control dictionary.
    1. Select the QA icon on the Command Center.
    2. Select the QC icon.
    3. Highlight the control lot number to be printed and press the Printer Icon.
    4. Select “Selected Lot” in the List Runs section. Select “Thumbnail” in the Levey-Jennings Graph section. Select OK.
  12. Deleting A Control From The Control Dictionary:
    1. To delete the old control lot numbers, select the QA icon on the Command Center.
    2. Select the QC icon.
    3. Highlight the control lot number to be deleted and click on the Delete icon.
    4. Select “ All data including set-up tables” and then select “OK”.

XVII. Changing Report Type:

In the event that CLICS or the Hospital/Clinic computer system is down, the report type can be changed so the Suspect and Definitive Messages do not print.

  1. Select the Setup icon on the Command Center.
  2. Select the General Settings icon.
  3. Select Reporting Options.
  4. Select Chartable Report as the Printout Option.
  5. Deselect Laboratory Report.
  6. Click on OK.

XVIII. Transmit Results to LIS:

  1. Select the Patient Results icon.
  2. Select the appropriate patient results folder.
  3. Click on the “Host Transmission” icon on the top left corner of the patient results screen.
  4. Click the green check mark icon to transmit, or the red X to cancel.

XIX. Limitations:

  1. White Blood Cells (WBC): Certain unusual RBC abnormalities that resist lysing, NRBC, fragmented WBC, agglutinated WBC, any unlysed particles greater than 35fl, very large or aggregated platelets, specimens containing fibrin, cell fragments, or other debris such as in pediatric and oncology specimens.
  2. Red Blood Cells (RBC): Very high WBC count, high concentration of very large platelets, agglutinated RBC, RBC smaller than 36fl, specimens containing fibrin, cell fragments, or other debris such as in pediatric and oncology specimens.
  3. Hemoglobin (HGB): Very high WBC count, severe lipemia, heparin, certain unusual RBC abnormalities that resist lysing, or anything that increases the turbidity of the sample such as elevated levels of triglycerides.
  4. Mean Corpuscular Volume (MCV): Very high WBC count, high concentration of very large platelets, agglutinated RBC, RBC fragments that fall below the 36fl threshold, or rigid RBC.
  5. RBC Distribution Width (RDW): Very high WBC count, high concentration of very large or clumped platelets, agglutinated RBC, RBC below the 36fl threshold, two distinct populations of RBC, or rigid RBC.
  6. Platelet (PLT): Very small red blood cells near the upper threshold, cell fragments, Giant or clumped platelets, platelet fragments, or cellular debris near the lower platelet threshold, electronic noise.
  7. Mean Platelet Volume (MPV): Known factors that interfere with the platelet count and shape of the histogram or known effects of EDTA.
  8. Hematocrit (HCT): Known factors that interfere with the parameters used for computation: RBC and MCV.
  9. Mean Corpuscular Hemoglobin (MCH): Known factors that interfere with the parameters used for computation: HGB and RBC.
  10. Mean Corpuscular Hemoglobin Concentration (MCHC): Known factors that interfere with the parameters used for computation: HGB, RBC and MCV
  11. Diff Parameters: Known factors that affect the WBC count as listed above or high triglycerides that affect lysing. Hypogranular or agranular granulocytes, lyse resistant red cells, very small or multi-population lymphocytes, precipitated elevated proteins.
  12. Samples with cold agglutinins may falsely lower the red blood cell count. Refer to “Cold Agglutinin Artifact in Hematology Analysis (CBC), Prevention of”.
  13. Very high glucose levels (≥ 800 mg/dl) may falsely increase MCV. This results in falsely high hematocrit, and falsely low MCHC.ref 8

XX. Instrument Flags:

  1. “V” indicates one of the three counts voted out.
    Action:
    The count was calculated using 2 counts. Report result. If this becomes a chronic problem: Clean the aperture using “Zap Aperture Function 09”.
  2. ”-----“ Indicates Total Voteout (LH500)
    Action:
    Check the sample for clots. If clots are seen, collect a new sample. If no clots are seen, perform the aperture cleaning procedure and then rerun the sample.
  3. “:::::” The instrument detected a clog in the flow cell, PC1, PC2 or FC will be displayed on the y-axis of the dataplot. This could also be due to resistant RBC. If the latter is the case the flag will be limited to one patient and a manual differential will be required.
    Action:
    If the flag is limited to one patient, check the patient sample for clots. If clots are found, a new sample must be collected. If flag occurs on multiple samples, there is a clog in the flowcell. In either case, clean the flowcell.
    1. “Purge Flowcell”, “Function 13” first. If this does not correct the problem perform “Function 44”. This will usually clear out the plug. If it does not, perform “Function 45” and then “Function 46” for the real tough clots. Be sure to fill out an IPR.
  4. “+++++” Result exceeds the instrument’s operating range. Refer to “section XXI. Results Derivation”.
  5. “.....” Incomplete computation. This could occur on calculated parameters because of a voteout (-----) or over range results(+++++) for the parameters used in the calculation. Refer to “section XXI. Results Derivation”.
  6. “P” indicates partial aspiration. This occurs in the “Auto” mode when the blood detectors sense a possible clot, or a HGB of approximately ≤ 5 gm/dl.
    Note: If chronic “P” flags occur, contact an Instrument Specialist. The manual mode does not detect partial aspiration. Do not run samples in manual mode in an attempt to clear up the flag. Doing so may mask a problem.
    1. Check the sample for clots.
      1. If no clots are seen, rerun the sample. Refer to “Specimen Integrity, Hematology” protocol.
      2. If clots are seen, collect a new sample.
    2. If HGB is in the 5-6 gm/dl range, the blood detectors may be triggering a “P” flag because of the low HGB.
      1. Release instrument results to CLICS.
      2. Delta Check GO () – release the results.
      3. No Delta Check or Delta STOP (): check the sample for clots.
        1. If no clots are seen, release the results. Refer to “Specimen Integrity, Hematology” protocol.
        2. If clots are seen, collect a new sample.
  7. “R” indicates review. This flag is not used as a criterion for review in this procedure. It may display in conjunction with other flags that are defined.

XXI. Results Derivation:

The linear ranges for the LH 750 are:
Note: Results above the upper linear limit must be diluted with LH Diluent and rerun in the Prediluted Mode. The dilution factor must be entered so the instrument can correct the results for the dilution. If the WBC must be diluted, refer to “Hemoglobin Correction for High WBC” procedure for corrections needed for HGB, HCT, MCH and MCHC.

    1. WBC (103/µl): 0.00 - 300.00 (reported as thou/µl)
      If the WBC is ≥ 300.0 dilute the sample 1:3 with LH Diluent and reassay. (Refer to section XII. “
      Running a Prediluted Sample”.) Perform a manual differential.3
    2. RBC (106/µl): 0.00 - 8.00 (reported as mil/µl)
      If the RBC is ≥ 8.00, which is commonly seen with veterinary samples, dilute the sample 1:2 with LH Diluent and reassay in the Prediluted mode.
    3. HGB (g/dl): 0.0 - 25.0
    4. PLT (103/µl): 0 - 3000 (reported as thou/µl)
    5. Retic (109/l): 0 – 750 (reported as thou/µl)
      Note: LH750 uses 109/l. CLICS reports as thou/µl units. These units are equivalent.
      1. If a retic count is 0.00% and the absolute retic count is ….., repeat the patient sample. If the results are unchanged, verify that the retic channel is operating properly by running a retic control.
      2. If the retic control result is acceptable enter the Canned Comment “Retic count below threshold.” in the CLICS Report Comment field.
  1. If a glucose ≥ 800 mg/dl occurs on a patient, enter a Canned Comment “MCV may be falsely elevated due to very high glucose, resulting falsely elevated hematocrit, and falsely low MCHC.”ref 7
  2. Body Fluids: If the WBC is <0.20 x 103 cells/µl, or if the RBC is <0.010 x 106cells/µl, do not report LH750 results. An “R” Flag on a Body Fluid result indicates that the result is below the reportable range. The cell count must be performed on a hemacytometer.
  3. In the event that a critical value is encountered on a test that is not ordered, the following steps are performed: (Example: A critical WBC is obtained on a sample that has only a HGB/HCT ordered.)
    1. Verify the critical value according to “Critical Value Chart & Protocol”.
    2. Call the nursing unit or doctor’s office and inform them that a critical value (high or low) has been obtained on the patient sample on a test that was not ordered. Report the result following the “Critical Value Chart & Protocol”.
    3. Request that an order be placed for the test with the critical value so that the critical value result can be officially entered in CLICS.
    4. Manually data enter the actual critical value result when/if the order is received.
  4. CLICS will automatically insert the Canned Comment “Reference intervals interpreted by veterinarian.” on all veterinary samples performed on the LH750.

XXII. Expected Result(s) and/or Critical Values:

WBC: ref 5

sex

age range

expected range

critical low

critical high

M & F

0-1 yr

6.0-17.5 x thou/µl

1.0

50.0

M & F

1-2 yr

6.0-17.0 x thou/µl

1.0

50.0

M & F

2-4 yr

5.5-15.5 x thou/µl

1.0

50.0

M & F

4-6 yr

5.0-14.5 x thou/µl

1.0

50.0

M & F

6-8 yr

4.5-13.5 x thou/µl

1.0

50.0

M & F

8-10 yr

4.5-13.5 x thou/µl

1.0

50.0

M & F

10-16 yr

4.5-13.0 x thou/µl

1.0

50.0

M & F

≥ 16 yr

4.5-11.0 x thou/µl

1.0

50.0

RBC: ref 5

sex

age range

expected range

critical low

critical high

M & F

0-15 days

3.9-5.9 x mil/µl

   

M & F

15 days-1mo

3.3-5.3 x mil/µl

   

M & F

1-4 month

3.5-5.1 x mil/µl

   

M & F

4-6 month

3.9-5.5 x mil /µl

   

M & F

6-9 month

4.0-5.3 x mil/µl

   

M & F

9 month-1yr

4.1-5.3 x mil/µl

   

M & F

1-3 yr

3.8-4.8 x mil/µl

   

M & F

3-6 yr

3.7-4.9 x mil/µl

   

M & F

6-9 yr

3.8-4.9 x mil/µl

   

M & F

9-12 yr

3.9-5.1 x mil/µl

   

M

12-15 yr

4.1-5.2 x mil/µl

   

F

12-15 yr

3.8-5.0 x mil/µl

   

M

15-18 yr

4.2-5.6 x mil/µl

   

F

15-18 yr

3.9-5.1 x mil/µl

   

M

18-45 yr

4.3-5.7 x mil/µl

   

F

18-45 yr

3.8-5.1 x mil/µl

   

M

45-65 yr

4.2-5.6 x mil/µl

   

F

45-65 yr

3.8-5.3 x mil/µl

   

M

≥ 65 yr

3.8-5.8 x mil/µl

   

F

≥ 65 yr

3.8-5.2 x mil/µl

   

HGB: ref 5

sex

age range

expected range

critical low

critical high

M & F

0-15 days

13.4-19.8 g/dl

12.5

22.0

M & F

15 days-1month

10.7-17.1 g/dl

8.0

17.0

M & F

1-2 month

9.4-13.0 g/dl

8.0

17.0

M & F

2-4 month

10.3-14.1 g/dl

8.0

17.0

M & F

4-6 month

11.1-14.1 g/dl

8.0

17.0

M & F

6month-5 yr

11.0-14.0 g/dl

8.0

17.0

M & F

5-9 yr

11.5-14.5 g/dl

8.0

17.0

M & F

9-12 yr

12.0-15.0 g/dl

8.0

17.0

M

12-15 yr

12.0-16.0 g/dl

7.0

20.0

F

12-15 yr

11.5-15.0 g/dl

7.0

20.0

M

15-18 yr

11.7-16.6

7.0

20.0

F

15-18 yr

11.7-15.3

7.0

20.0

M

18-45 yr

13.2-17.3

7.0

20.0

F

18-45 yr

11.7-15.5

7.0

20.0

M

45-65 yr

13.1-17.2

7.0

20.0

F

45-65 yr

11.7-16.0

7.0

20.0

M

≥ 65 yr

12.6-17.4

7.0

20.0

F

≥ 65 yr

11.7-16.1

7.0

20.0

HCT: ref 5

sex

age range

expected range

critical low

critical high

M & F

0-15 days

41-65 %

   

M & F

15 days-1month

33-55%

   

M & F

1-2 month

28-42%

   

M & F

2-4 month

32-44%

   

M & F

4-6 month

31-41%

   

M & F

6-9 month

32-40%

   

M & F

9 month-1 year

33-41%

   

M & F

1-3 year

32-40%

   

M & F

3-6 year

32-42%

   

M & F

6-9 year

33-41%

   

M & F

9-12 year

34-43%

   

M

12-15 year

35-45%

   

F

12-15 year

34-44%

   

M

15-18 year

37-48%

   

F

15-18 year

34-44%

   

M

18-45 year

39-49%

   

F

18-45 year

35-45%

   

M

45-65 year

39-50%

   

F

45-65 year

35-47%

   

M

≥ 65 year

37-51%

   

F

≥ 65 year

35-47%

   

Absolute Reticulocyte Count: ref 5

sex

age range

expected range

critical low

critical high

M & F

0-14 days

239-404 x thou/µl

   

M & F

14 days-12 yr

46-154 x thou/µl

   

M & F

≥ 12 yrs

59-146 x thou/µl

   

The rest:

Analyte

Expected Range

Critical Low

Critical High

MCV: ref 5

80-100 fl

   

MCH: ref 5

26-34 pg

   

MCHC: ref 5

32-37 g/dl

   

RDW: ref 1

12.1 - 15.2%

   

Platelet Count: ref 6

150-400 x thou/µl

<30

>1,000

Platelet oncology:

 

<10

 

MPV: ref 1

7.5 - 11.2 fl

   

Neutrophils: ref 1

42-74%

   

Lymphocytes: ref 1

18-44%

   

Monocytes: ref 1

5-13%

   

Eosinophils: ref 1

0-8%

   

Basophils: ref 1

0-1%

   

Absolute Neutrophils

 

<0.5

 

XXIII. Quality Control:

  1. Routine Controls:
    1. Beckman/Coulter 5C™ Hematology Controls are assayed every 8 hours alternating controls. Two levels of control are run following morning start up. One level is then run on other shifts. Two levels of controls are also run after replacing reagents. All commercial controls are run in the Auto Mode.
    2. In lieu of using commercial control material for the Open Vial Sample Mode, patient whole blood is used as control material. The same patient whole blood sample must be assayed in both the Auto Mode and the Open Vial Mode every 8 hours. An EDTA whole blood patient sample, with results in normal range, is obtained from available samples.4
      1. Run an EDTA sample in the Auto Mode using barcode ID MCONTROL. Print results.
      2. Run the same sample immediately in the Open Vial Sample mode using barcode ID MCONTROL. (It must be the next sample run.) Print results.
      3. Compare the results of both trials. The results from the Auto and Open Vial modes must match within stated limits. Attach the Auto and Open Vial reports together and sign them indicating results were reviewed and were acceptable.
      4. If results do not match within the stated limits rerun the sample in both modes. If results are still not within the stated limits contact the Site Supervisor.
      5. Forward the reports to the Instrument Specialist Office at the end of the day.
    3. City Control samples are run once per day. Results are entered into the “City Control Data Acq Logs”. Refer to the “Hematology City Control: Preparation, Application and Management” protocol.
    4. The Latron Primer and Control are run once a day, typically following Startup.
    5. Streck Cell-Chex Auto Body Fluid Cell Count Control, Level 2 and Level 3, are run once each shift that a body fluid cell count is performed. The LH systems recover very close to the group specific means on the package insert of this control product. The targets are necessarily low – miscellaneous fluids. So low that any significant deviation from the means would recover values exceeding the manufacturer’s posted ranges. These ranges are more than adequate for the clinical application. (See MVR 20080910 for representative data.)
  2. Criteria for accepting or rejecting a patient run is based on the “Quality Control; General” protocol in the Quality Assurance Manual.
  3. Analytical Range: (For Instrument Specialist use only.)
    1. WBC (103/µl): 0.00 - 400.00
    2. RBC (106/µl): 0.00 - 8.00
    3. HGB (g/dl): 0.0 - 25.0
    4. PLT (103/µl): 0 - 3000
    5. Retic (109/l): 0 - 750
  4. Calibrator: Beckman/Coulter S-CAL Calibrator.
  5. Calibration Frequency: Calibration is performed every 6 months. Calibration may be performed after major maintenance and/or replacement of critical parts that may influence test performance.
  6. Calibration Verification: Calibration verification is performed every 6 months and whenever there is major preventative maintenance or replacement of critical parts that may influence test performance.
  7. Control Ranges: Manufacturer target mean values are initially imported to the LH750 when a new lot number of control is being overlapped. These mean values are then evaluated and edited, if necessary, following a 3 day control overlap.
    Lab action limits for hematology controls are based on locally established system performance from no fewer than 60 consecutive trials over at least one and a half months after initial baseline studies and set at ± 3 SD for all reported analytes unless specifically stated otherwise in our written operational procedure.
    Action limits are set in the LH750 by entering the “set range” into the Lab limits fields for each analyte on each control screen.
    Target means for each analyte on fresh lots of control material will be set based upon a three trial overlap with active (current) control prior to pressing the fresh lots into service. Action limits will be fixed against these calculated means. If the initial, calculated mean from the overlap varies more than 5% from its package insert mean, contact the office of the Technical Director for instructions before releasing the control parameter for routine Q.C.
  8. Definitive language for handling “control out” situations: When a control result is outside the posted limits, first, check the Incidence Log for evidence of imprecision in that constituent.
    1. If the Incidence Log indicates that there is evidence of imprecision, stop testing and report to a Supervisor.
    2. If there is no evidence of imprecision, repeat the control once. If the control result is acceptable, proceed with patient testing. Make an Incidence Log entry including the control results and the posted limits.
    3. If the control result is not acceptable and the control result errs in the other direction, stop testing. This indicates imprecision. There are a few maintenance steps to perform to correct the problem. Check to see if there are any flags associated with the control results. This may help to determine what maintenance may be appropriate to perform first. Proceed with the following:
      1. WBC, RBC, PLT, MCV, etc.:
        1. Perform aperture zap, Function: F09.
        2. Clean outside of Blood Sampling Valve (BSV).
      2. Latron Control or differential:
        1. Purge the flowcell, Function: F13.
        2. Flowcell unclog 1, 2 or 3, Function: F44, F45, F46
          1.   After maintenance is done, retest controls. If control results are acceptable, proceed with testing.
          2.   If controls are not acceptable, call an Instrument Specialist.
          3.   In either event, make an entry in the Incidence Log.
    4. If the control result errors in the same direction, report results to Supervisor. This indicates a drift or shift in either the control material or the analyzer. Review the Incidence Log for evidence that the control constituent has been running off its target in the same direction. At that point the supervisor assumes that the error is real and determines whether or not the error has a clinical impact.
    5. If the error does not have a clinical impact, proceed with testing and report to the office of the Technical Director. The data will be reviewed to determine if the mean should be reset.
    6. If the error does have clinical impact stop testing for that constituent. There are a few maintenance steps to perform to correct the problem. Proceed with the following:
      1. WBC, RBC, PLT, MCV, etc.:
        1. Perform aperture zap, Function: F09
        2. Clean outside of BSV.
      2. Latron Control or differential:
        1. Purge the flowcell, Function: F13
        2. Flowcell unclogs 1, 2 or 3, Function: F44, F45, F46.
    7. After maintenance is done, retest controls. If control results are acceptable, proceed with testing.
    8. If controls are not acceptable, call an Instrument Specialist.
    9. In either event, make an entry in the Incidence Log.

Analyte Mode Match Limits

      Analyte

      Limits*

      WBC

      0.4

      RBC

      0.11

      HGB

      0.4

      MCV

      1.5

      RDW

      0.5

      MPV

      0.5

      Platelet Count

      23

      % Retic Count

      0.3

      *Limited by the forced significant digit.

XXIV. Reference:

  1. LH750 Operate Instructions. Beckman/Coulter.
  2. Cell-Chex Auto Body Fluid Cell Count Control package insert. Streck. Omaha, NE. 01-2006.
  3. Urgent Product Corrective Action for Coulter LH750 Hematology Analyzers. Beckman Coulter. 2/7/2007.
  4. Q.C. for Hematology Analyzers with Two Sample Modes. The Joint Commission. 5/23/2005.
  5. Wu, Alan H.B., Tietz Clinical Guide to Laboratory Tests. 4th Ed.
  6. http://www.nlm.nih.gov/medlineplus/ency/article/003647.htm
  7. Steve Raymond, Dubuque Pathology Associates.
  8. Morse, E.E., G. Kalache, W. Germino, R. Stoddard; Annals of Clincal and Laboratory Science, Vol 11, Issue 2, pp 184-187 “Increased Electronic Mean Corpuscular Volume Induced By Marked Hyperglycemia”.

 

  1. January 2006 L. McGovern
  2. July 2006 L. McGovern (Revised: III.10.; XII.7-10.; XX.1.D., 8.; XXI.2.)
  3. September 2006 L. McGovern (Revised: IV.3., XVIII.6.)
  4. February 2007 L. McGovern (Revised: XVIII.1.A.)
  5. September 2007 L. McGovern (Revised: XX.1.B.; XXI.4.)
  6. October 2007 L. McGovern (Revised: XX.1.B.d.)
  7. October 2007 L. McGovern (Revised: XX.5.)
  8. June 2008 L. McGovern (Revised: III.11. added)
  9. September 2008 S. Raymond/L. McGovern (Revised: XX.1.E.)
  10. August 2009 L. McGovern (Revised: IV.; XVIII.)
  11. September 2009 L. McGovern (Revised: XVIII.E.a-b. added)
  12. November 2009 L. McGovern (Revised: XX.1.A.,B., 6.)
  13. July 2010 L. Reif, S. Dunn, L. McGovern (Revised: V.1.,3.,5.note; VII.; VIII.6.; IX.; XIII.; XIV.3.; XV.7., 11.A.c., 12.D., XVIII.3-4.; XX.; XXI.1.A.,B., XXII.; XXIII.8.C.a.-b, F.a-b.; XXIV.5.; align ref rngs w/ ref materials)
  14. December 2010 L. McGovern, P. Ellerbeck (Revised: XXII.plt ct; XXIV.6)

 

Comprehensive Review:

Instrument Specialist:

Technical Director:

 

Interim Review:
December 2011 D. Stence/L. McGovern/G. Miller (Revised: V.2., 6.; XI.Note; XIV.11.; XV.11.added; XIX.13.; XXI.2.; XXIV.7-8.)


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