- Cambridge Latin Course Unit 1 Stage 8

Beckman Coulter
AcT diff2
Series Analyzer
Procedure Manual
Cowell Student Health Center Laboratory, 2006
1
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
2
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
3
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.
4
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.
5
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).
6
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.
8
Criteria for reflex to slide review
Parameter
WBC, RBC, HGB, PLT
WBC
PLT
MCV
MCHC
Range
Exceeds linearity
<4.0 or >15,000
<100,000 or >500,000
<75 or >105 fl
> 36
RDW
No diff or incomplete diff
Neut #
Neut %
Bands
Lymph#
Mono#
-----
>22%
+++++
<1.0 or >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
<50 or >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.
9
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
10
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
<3,000 ; >15,000
<8g/dL >20g/dl
<25 >60
<100,000 >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.
11
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
12
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.
13
Appendix A