Beckman Coulter AcT diff2 Series Analyzer Procedure Manual

Cowell Student Health Center Laboratory, 2006

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Policies and Procedures Approval Form Procedure: AcT diff 2 Series Analyzer Prepared By: _________________________________ Name

_____________________________ Title

Approved By: ________________________________________ Laboratory Director

Date Adopted: ______________________ Read By: Date

Date Discontinued: ___________________

Name/Title

Revisions: Review Date

Revision Date

Approved By

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Title

Personnel who actually perform moderately complex tests (e.g., the AcT diff2 Series Analyzer) are referred to as testing analysts by CLIA and must have at least a high school diploma. Testing personnel must have documented training and undergo periodic competency evaluations. In this laboratory, the following persons fulfill the role of: 

Laboratory director:________________________________



Clinical consultant: ________________________________



Technical consultant: ______________________________



Testing analyst(s): ________________________________ ________________________________

In this laboratory, the following personnel are authorized to operate the AcT diff2 Series Analyzer:

Employee Name

Employee Title

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Supervision Required? Y/N

Sup. Review to Report Results? Y/N

Principle The COULTER AcT diff2 Series Analyzer is a quantitative, automated hematology analyzer and leukocyte differential counter for In Vitro Diagnostic Use in clinical laboratories. AcT diff2 Series Analyzer parameters Parameter WBC - White Blood Cell or leukocyte count LY# Lymphocyte number LY% Lymphocyte percent (or ratio) MO# Mononuclear cell number MO% Mononuclear cell percent (or ratio) GR# Granulocyte number GR% Granulocyte percent (or ratio) RBC - Red Blood Cell or erythrocyte count Hgb - Hemoglobin concentration Hct - Hematocrit (relative volume of erythrocytes) MCV - Mean Corpuscular (erythrocyte) Volume MCH - Mean Corpuscular (erythrocyte) Hemoglobin MCHC - Mean Corpuscular (erythrocyte) Hemoglobin Concentration Plt - Platelet or thrombocyte count RDW - Red Cell (erythrocyte volume) Distribution Width MPV - Mean Platelet (thrombocyte) Volume

The purpose of the AcT diff2 Series Analyzer is to identify the normal human patient, with all normal system-generated parameters, and to flag or identify patient results that require additional studies. AcT diff2 analyzers are based on the Coulter method whereby cells are counted and sized by detecting and measuring changes in electrical resistance when a particle (such as a cell) in a conductive liquid passes through a small aperture. As each cell goes through the aperture, it impedes the current and causes a measurable pulse. The number of pulses signals the number of particles. The height of each pulse is proportional to the volume of that particle. While the number of pulses indicates particle count, the amplitude of the electrical pulse produced is proportional to the cell’s volume. The AcT diff2 analyzer has two operating modes: Open Vial Whole Blood and Closed Vial Whole Blood. Whole blood samples can be analyzed in either mode. Prediluted samples can be analyzed at the Open Vial Station.

Specimen Collection and Handling HANDLE BLOOD AS A POTENTIAL BIOHAZARD CAPABLE OF TRANSMITTING INFECTION. ALWAYS WEAR PROTECTIVE GLOVES AND LAB COAT WHEN PROCESSING SPECIMENS. Draw specimen in into a lavender-top Vacutainer tube containing K2EDTA. Thoroughly mix blood with EDTA anticoagulant. If hemolysis or small clots are observed, discard specimen.

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Mix venous blood sample at least 8 times by hand inversion. Gently turn capped sample upside down then back straight up. Alternatively, use a mechanical mixer for at least 5 minutes. Do not test samples that are incorrectly filled or that are clotted. If hemolysis or small clots are observed, discard specimen. Analyze venous blood samples within 24 hours of collection. Do not refrigerate samples for platelet and differential counts. If platelet or differential results are not required, store anticoagulated whole-blood specimens at 2 - 8 C. Warm samples to room temperature (16 35 C or 61 -95 F) before testing.

Equipment and Materials Equipment Performance Parameters The AcT diff2 Analyzer operates at ambient temperature (16 - 35 C or 61 - 95 F) at humidity no higher than 85% without condensation Materials, Reagents COULTER diff AcT Tainer reagent pack or diff AcT Pak™, both of which contain diluent (Reagent 1) and lytic reagent (Reagent 2). The diff AcT Tainer reagent pack also contains AcT Rinse™ Shutdown Diluent (Reagent 3). Reagent 1 is an isotonic electrolyte solution that dilutes whole blood samples, stabilizes cell membranes for accurate counting and sizing, conducts aperture current, rinses instrument components between analyses, and prevents duplicative cell counts by using the sweep-flow process. Reagent 2 is a lytic reagent that lyses red blood cells for white blood cell count and hemoglobin measurement. Caution: Eye irritant. Avoid contact with skin and eyes. Avoid breathing gas. Contact with acid liberates poisonous gas. Reagent 3, AcT Rinse Shutdown Diluent, prevents protein buildup that occurs in and around the apertures. Caution: Avoid eye and skin contact. Do not ingest. Reagent Preparation No reagent preparation is required. Appropriate safety precautions for handling reagents are contained on the respective Material Safety Data Sheets located in the laboratory’s MSDS/ HAZARDS binder. Reagent Storage Store reagents at ambient room temperature (2 -25 C). Keep containers closed. Discard reagents at the expiration date. Replace reagents when the screen prompt appears or when the reagent container is empty. Reagent Tracking When opening new reagents, log on the Reagent Log indicating date opened, lot number, and expiration date. COULTER 4C PLUS cell control: abnormal low, normal and abnormal high. COULTER S-CAL calibrator.

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Control/Calibrator Preparation Handle controls and calibrators using universal precautions. Controls and calibrator contain stabilized human erythrocytes and no test method can offer complete assurance that Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) or other infectious agents are absent. Take appropriate safety measures to avoid contact from aerosols when removing the cap/stopper assembly. Control/Calibrator Storage Store at 2 -8 C (35 -46 F). Sealed control and calibrator vials are stable until the expiration date. Opened calibrator vials are stable for 1 hour. Discard expired control/calibration materials. Before use, inspect controls/calibrators for indications of instability or deterioration. Gross hemolysis (darkly colored supernatant) is indicative of product deterioration. Do not use deteriorated product. Waste Container Be sure the waste container is in a safe place and is properly connected. Do not overfill the waste container. When the "waste full" icon appears, replace the waste container with an empty one.

Quality Control Procedures QC Frequency Test 3 levels of QC samples once per day of testing using Coulter 4C PLUS cell controls. QC Procedure Using COULTER 4C PLUS Cell Control 1. Be sure the 4C PLUS cell control information and values have been correctly entered from the TABLE OF EXPECTED RESULTS listed on the package insert. For information on how to enter the values, see "Entering 4C PLUS Cell Control Information" in chapter 2 of the AcT diff2 Operator’s Guide. 2. Ensure that 4C PLUS cell control is not past its expiration date and that it is at the correct storage temperature. 3. Allow refrigerated controls/calibrators to reach room temperature before use. Mix by rolling slowly between the palms of the hands 8 times then invert and roll 8 times. Gently invert the tube 8 times. Inspect the tube contents to determine if all cells have been uniformly distributed. Repeat above steps if tube contents have not been uniformly distributed Inspect the tube contents to ensure that all cells are uniformly distributed; if not, repeat this step. 4. At the Main screen, touch the QA icon. 5. At the QA screen, touch the 4C PLUS Run icon. 6. Select the correct control level: L Low N Normal H High The square darkens next to your selection. Make sure that the level of control you are testing matches the one selected (L, N or H).

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7. Invert the tube once or twice prior to cycling. IMPORTANT: Risk of misleading results if Cap Pierce Station door is opened before the sample analysis is completed. Do not open the door. The door will open automatically.

8. Place the well-mixed sample in the tube holder at the Cap Pierce Station and close the door. 9. When the tube holder door opens, remove the vial and return it to the refrigerator. 10. Results appear on the screen. Unless non-numeric results occur for one or more parameters, the control results are automatically stored:  If Autoprint is off you can manually print results by touching the Print icon.  To manually reject these results, touch the Trash icon.  If results are not within the expected range, rerun the control starting at step 6. If results are still out of range, see the Special Procedures and Troubleshooting Section of the AcT diff2 Operator's Guide. 11. Repeat steps 6 through 10 for each required control level. 12. If the results are within the expected range, you are finished running controls. If you do all of the above steps and the results still do not meet your performance expectations, call your Beckman Coulter Representative. 13. First try re-running the control with the same vial of control. 14. If QC is still not in range, open a new vial of QC material and re-run. 15. Refer to troubleshooting procedures in the Coulter Operator’s Guide section 6.8. Do not report patient test results until control values are acceptable.

Patient testing Precautions  When you operate the AcT diff2 instrument, be sure all covers and doors are closed.  If the probe is loose or bent, do not run the instrument. Call your Beckman Coulter Representative.  Do not place hands in the area of the peristaltic pumps.

Sample Analysis - Closed Vial Whole Blood Mode 1. At the Main screen, select Closed Vial Whole Blood mode. 2. At the Main screen, touch the Sample Results Screen icon. NOTE: If the door is inadvertently closed after it has opened automatically, or if it is closed at a screen where samples are not run, you can open the door by touching the Main Menu icon and then the Sample Results icon.

3. Touch the Patient Range icon until the desired range (1, 2 or 3) appears. 4. Verify that the sample ID is correct. If autosequencing is on, the 9-digit sample ID number automatically increments by 1. If autosequencing is off, manually enter the sample ID and touch the Save icon. Be careful not to duplicate an existing sample ID number that may have been used previously. Unique sample accession numbers will be automatically assigned to specimens by the Orchard Harvest LIS. 5. Mix the sample on the mechanical rocker prior to sampling. 7

6. Be sure you are in the Closed Vial Whole Blood mode. 7. Place the well-mixed sample in the tube holder at the Cap Pierce Station and close the door. 8. When the tube holder door opens, remove the tube. 9. Sample results are automatically saved by the instrument, and the results appear on the screen. 10. Print the results:  If Autoprint is on, the results print automatically.  If Autoprint is off, touch the Print icon.  If Autosequence is on, the instrument is ready to run the next sample.  If Autosequence is off, you must manually enter an ID number before the probe descends for the next sample.  If flags appear, see the Special Procedures and Troubleshooting Section of the AcT diff 2 Operator's Manual.

Sample Analysis - Open Vial Whole Blood Mode 1. At the Main screen, select Open Vial Whole Blood mode. 2. At the Main screen, touch the Sample Results Screen icon. 3. Touch the Patient Range icon until the desired range (1, 2 or 3) appears. 4. Verify that the sample ID is correct. If autosequencing is on, the 9-digit sample ID number automatically increments by 1. If autosequencing is off, manually enter the sample ID and touch the Save icon. Be careful not to duplicate an existing sample ID number that may have been used previously. 5. Mix the sample thoroughly on the mechanical rocker. 6. Be sure you are in the Open Vial Whole Blood mode. 7. Present the well-mixed sample to the probe so that the tip is well into the tube, and press the aspirate switch. 8. When you hear the beep, remove the sample, and put the cap back on the tube. 9. The analyzer displays the sample results on the screen and automatically saves them. 10. Print the results:  If Autoprint is on, the results print automatically.  If Autoprint is off, touch the Print icon.  If Autosequence is on, the instrument is ready to run the next sample.  If Autosequence is off, you must manually enter an ID number before the probe descends for the next sample.  If flags appear, see the Special Procedures and Troubleshooting Section of the AcT diff2 Operator’s Manual.

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Criteria for reflex to slide review Parameter WBC, RBC, HGB, PLT WBC PLT MCV MCHC

Range Exceeds linearity 15,000 500,000 105 fl > 36

RDW No diff or incomplete diff Neut # Neut % Bands Lymph# Mono# -----

>22%

+++++

7.0 >85% >5% on slide rev. > 5.0 >1.5 Total voteout

MCV +++++

Results over range for Plt, WBC, RBC, HGB, HCT GRAN, LYM 130

XXXXX

Aperture alert

…..

Incomplete calculation

Action Dilute and re-run Slide review Slide review Slide review Check for cold agglutinin by placing in 37C incubator for intervals of 15 min; check for lipemia, hemolysis.

Slide review Manual differential Slide review Slide rev. for bands Manual differential Slide review Slide review Thoroughly mix and repeat specimen, zap apertures if persists Check bath shield; make a dilution with normal saline Use spun crit or slide rev to verify Check sample for clots; if persists, repeat with known sample; if persists, zap apertures. Address voteout (above)

If a patient has a positive mononucleosis test and the CBC for that patient yields results consistent with infectious mononucleosis (i.e. inverted differential), then the slide review need not be performed. A comment will be inserted into the results which states that a slide review is not indicated due to positive mono test. If the CBC results are not consistent with infectious mononucleosis (i.e. increased neutrophils) then a slide review should be performed.

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Reference Ranges Coulter’s Normal Range Study derived the following reference ranges (below) for the AcT diff2 Series Analyzer. You may use these default ranges, or establish your own. If you use Coulter’s ranges, write “same” in the last column of the following chart. Parameter

Units

WBC RBC HGB HCT MCV MCH MCHC PLT RDW MPV LY LY MO MO GR GR

x103 cells/ uL x106 cells/ uL g/dL ratio fL pg g/dL x103 cells/ uL % FL % # % # % #

Our Reference Ranges 3.8-10.8 4.2-5.8 13.2-17.1 38.5-50.0 80.0-100.0 27.0-33.0 32.0-36.0 140-400 11-15.0 7.5-11.5 20-45 0.85-3.90 0-12 0.2-0.95 40-83 1.52-8.55

Reportable Ranges The operating range listed below is the range of results over which the AcT diff2 Series AnalyzerSeries instruments display, print and transmit results. The linear (reportable) range is also listed below. Linearity limits apply only to directly measured parameters. The AcT diff2 Series Analyzer flags values between the linear range and the operating range. Parameter

Operating Range

WBC RBC Hgb MCV Plt LY% MO% GR% LY# MO# GR#

0.0 - 150 0.00 - 8.00 00.0 - 30.0 50.0 - 130.0 000 - 3000 0 - 100 0 - 100 0 - 100 0 - 99.9 0 - 99.9 0 - 99.9

Linearity Limit/ Reportable Range 0-99.9 0-7.0 0-25.0 0-999.0

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Units x 103 cells/ x 106 cells/ g/dL fL x 103 cells/ % % % 3 x 10 cells/ x 103 cells/ x 103 cells/

L L

L

L L L

The AcT diff2 Series Analyzer system confirms parameter results prior to reporting. After the computer corrects for coincidence, it compares the three counts each for WBC, RBC, Plt. If the unit finds disagreement among all count periods or does not meet other internal criteria, the instrument displays a total voteout. Note: In rare instances, a transient or partial aperture blockage may not be detected by any of these methods. Therefore, verify flagged results for accuracy and review any result that exceeds your patient reference ranges.

Reporting Results Results are reported to the ordering physician through the LIS interface. Critically abnormal results are reported verbally to the clinician and documented on the patient report.

Panic/Alert Value Procedures Parameter WBC Hgb Hct Plt

Critical/Panic/ Alert Values 15,000 20g/dl 60 600,000

All critical values are to be reported directly to the clinical staff. The verbal report is documented in the patient report. Information in the verbal report should be read back by the clinician. Documentation of the value should include the time and date of the report and the names of the technologist reporting the result and of the clinician receiving the results.

Procedures for Using Alternative Methods If the AcT diff2 Series Analyzer is unavailable for use, specimens will be sent to Quest Laboratories for analysis. Instrument malfunction or persistently out of range QC would be instances where the Analyzer was unavailable Limitations of the Procedure K2EDTA is the recommended anticoagulant. K3EDTA and Na2EDTA are also acceptable. Use of other anticoagulants can yield misleading results.

Interfering Substances These can also yield misleading results for the parameters listed below: WBC: Certain unusual RBC abnormalities that resist lysing, nucleated RBCs, fragmented WBCs, any unlysed particles greater than 35 fL, very large or aggregated platelets as when anticoagulated with oxalate or heparin. RBC: Very high WBC count, high concentration of very large platelets, agglutinated RBCs and RBCs smaller than 36 fL.

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Hgb: Very high WBC count, severe lipemia, certain unusual RBC abnormalities that resist lysing, anything that increases the turbidity of the sample such as elevated levels of triglycerides. MCV: Very high WBC count, high concentration of very large platelets, agglutinated RBCs, RBC fragments that fall below the 36-fL threshold, rigid RBCs. Plt: Very small red blood cells near the upper threshold, cell fragments, clumped platelets as with oxalate or heparin, platelet fragments or cellular debris near the lower platelet threshold. Hct: Known factors that interfere with the parameters used for its computation, RBC and MCV. MCH: Known factors that interfere with the parameters used for its computation, Hgb and RBC. MCHC: Known factors that interfere with the parameters used for its computation, Hgb, RBC and MCV. LY,MO,GR: Known factors that affect the WBC count as listed above, such as high triglycerides, that can affect lysing. Precautions System integrity might be compromised and operational failure might occur if: The equipment is used in a manner other than specified. Operate the instrument as instructed in the AcT diff2 Product Manuals. Software that is not authorized by Coulter is introduced into your computer. Only operate your system's computer with the software card authorized by Coulter. Observe the copyright statement on the card. If there is a power failure or brownout, turn the instrument off. When the power returns, turn the instrument back on. It automatically reboots. If you are processing a sample when you turn the instrument off, you lose the sample's results. You must rerun the sample when you turn the instrument back on.

References Use the Operator's Guide for: Getting started and running the instrument day-to-day Reviewing unusual results (how to read a result report and what flags mean) Performing special procedures such as cleaning, replacing, or adjusting a component of the instrument Troubleshooting problems with your instrument. Use the Reference Manual for: What the instrument does and methods it uses Instrument specifications and requirements How to interface your analyzer to your laboratory's host computer How to safely use the instrument. Use the Installation and Training Guide for: Initially setting up the instrument and printer

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Powering up the instrument Customizing the software. Use the Operating Summary for: Running your instrument using a quick reference set of procedures Verifying screen icon definitions.

COULTER AcT diff2, AcT Pak, AcT Tainer, AcT Rinse, 4C PLUS and S-CAL are trademarks of Coulter International Corp.

References 1. Clinical Laboratory Improvement Amendments of 1988; Final Rule, 42 CFR Part 493 et al, changes through Jan. 24, 2003. Available at http://www.phppo.gov/clia/regs/toc.asp. 2. Henry JB, ed. Todd Sanford’s Clinical Diagnosis and Management by Laboratory Methods. Philadelphia: W.B. Saunders; 2001. 3. Tietz NW. Fundamentals of Clinical Chemistry. Philadelphia: W.B. Saunders; 2001. 4. The State Operations Manual, Appendix C: Survey Procedures and Interpretative Guidelines for Laboratories and Laboratory Services. National Technical Information Services. Available at http://www.cms.hhs.gov/clia/appendix.asp?. 5. Clinical Laboratory Standards Institute; Procedures for the Handling and Processing of Blood Specimens; H18-A3 (2004). Available at http://www.nccls.org. 6. Clinical Laboratory Standards Institute; Protection of Laboratory Workers from Occupationally Acquired Infections; M29-A3 (2005). Available at http://www.nccls.org. 7. Clinical Laboratory Standards Institute; Clinical Laboratory Technical Procedure Manuals; GP2-A4 (2002). Available at http://www.nccls.org. 8. Brochure #2 – Verification of Performance Specifications. Available at http://www.cms.hhs.gov/clia/. 9. Brochure #3 – Calibration and Calibration Verification. Available at http://www.cms.hhs.gov/clia/. 10. Brochure #4 – Equivalent Quality Control Procedures. Available at http://www.cms.hhs.gov/clia/. 11. List of Proficiency Testing (PT) Providers. Available at http://www.cms.hhs.gov/clia/ptlist.pdf. 12. 20 Hour Continuing Medical Education (CME) Courses for Laboratory Directors of Moderate Complexity Laboratories. Available at http://www.cms.hhs.gov/clia/cmecourses.pdf.

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Appendix A