Cell-Dyn 3200 System Reticulocyte Assay Service and Support Training Material

CELL-DYNâ 3200 System Reticulocyte Assay
Service and Support Training Material
Purpose:
This training material is intended to provide Area Service and Support Organizations
with information regarding the CELL-DYN 3200 Reticulocyte Assay. The assay is
similar to the Reticulocyte Mode used on the CELL-DYN 3500 and 3700 instruments.
The Reticulocyte reagent, Reticulocyte controls, and specimen requirements are the
same. A table containing similarities and differences has been developed to facilitate
understanding these differences between CELL-DYN analyzers.
NOTE: The CELL-DYN 3200 System Reticulocyte Assay Service and Support
Training Material is not intended to replace the reference documentation
available for the CELL-DYN 3200 System. This material should be used in
conjunction with the CELL-DYN 3200 System Operator’s Manual, which is the
guiding document for Service and Support training and customer training.
This document is not intended for customer distribution; however, it may be used as a
reference in creating customer training material. Refer to ISA 80-060 & 109-078 for
CELL-DYN 3200 System Customer Training Development Guide for Software Version
2.0.
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For Abbott Internal Use Only CELL-DYNâ 3200 Document Control No. 91124-101
User Must Verify Doc. is Current Provided by WWCS-SC November 2001
Table of contents
1. Introduction of the Reticulocyte Assay 3

a. Reticulocyte Overview 3
b. Reticulocyte Count 4
c. CELL-DYN 3200 Reticulocyte Assay 4
d. Specimen Requirements 4
e. Reagents 4
f. Control Material 5
2. Software 5
a. Turn On Reticulocyte Package 5
b. Patient Limit Sets 5
c. Unit Selection 5
d. QC files 5
e. Reticulocyte Data Log 5
3. Basic Operation 5
a. Running Reticulocyte Backgrounds 5
b. Running Controls 5
c. Running Patient Samples 5
d. Performance Specifications 6
e. Calibration/Standardization 6
f. Maintenance 6
4. Data review 7
a. Reticulocyte Parameters 7
b. Algorithms 8
c. Examples of Scatterplots 10
d. Reticulocyte Alerts 13
5. Troubleshooting 14
a. High Reticulocyte Backgrounds 14
b. Erratic Results 14
c. Excessive Alerts/Flagging 14
d. Out of Range Controls 15
e. Interfering Substances 15
6. Similarities and Differences of CELL-DYN Reticulocyte Methodology 16
7. References 18
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1. Introduction to the Reticulocyte Assay
a. Reticulocyte overview
• Reticulocytes are the precursor cells to the mature RBCs. They contain
ribosomal RNA and are the first stage after the exclusion of the nucleus [1].
• Reticulocytes remain in the bone marrow for two days before entering the
blood circulation. After a day in the peripheral blood they become mature
RBCs [6].
• On a Wright’s stained smear, polychromatophilic RBCs (Figure A) are
typically reticulocytes. Not all reticulocytes have this appearance on Wright's
stained smear.
• To identify reticulocytes under a microscope, special stains such as the
supravital stain New Methylene Blue precipitates RNA making the cells easily
identifiable (Figure B) [1].
Polychromatophilic
RBC
Figure A
Figure B
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b. Reticulocyte Count
• The reticulocyte count is used to assess the bone marrow’s erythropoietic
activity.
• There are several ways to enumerate reticulocytes. In the past the manual
method for counting reticulocytes was the only method. Now, with the
advancement of technology there are semi-automated and automated methods
for counting reticulocytes.
• Examples of Clinical Applications:
• Diagnostic applications for various anemias.
• Monitoring of a patient’s response to therapy.
• Bone Marrow response after a bone marrow transplant [6].
c. CELL-DYN 3200 Reticulocyte Assay

• Uses a New Methylene Blue based stain to stain RNA. The more RNA present
in the cell, the more stain is absorbed and the greater the light scatter.
• Available only in the Open mode.
• A semi-automated method that requires the sample to be prediluted in a
staining reagent and incubated for a period of time before being analyzed.
• Uses Light scatter information from the 0°, 10°, and 90° detectors to classify
the list mode events.
• Reticulocytes and mature RBCs have similar 10° scatter but reticulocytes
exhibit greater 90° scatter [1].
• Reticulocyte results reported are the Retic% and Retic absolute (only if an
RBC value was entered or retrieved from the Hematology data log).
d. Specimen Requirements
• K2EDTA, K3EDTA or Na2EDTA specimens can be used.
• Sample volume required to perform the assay is 20 µL.
• Specimens stored at room temperature can be processed up to 8 hours after
collection.
• If specimens are not analyzed within 8 hours they may be processed within 72
hours if stored at refrigerated temperature.
e. Reagents
• CELL-DYN Reticulocyte Reagent (List Number 03H40-01) uses the thiazine
dye New Methylene Blue N, a supravital dye that stains and precipitates RNA.
• Box contains 100 tubes, 3.7 mL of reagent in each 5 mL tube.
• Reagent must be stored away from light at room temperature.
Note: Reticulocyte Reagent (List Number 03H40-01) may be received in packaging
that does not specify use on CELL-DYN 3200 Systems, but are intended for
use on instruments that have been upgraded to software Version 2.0.
• The stain also stabilizes the RBCs and prevents them from lysing when in
contact with the WBC Lyse reagent as the sample is diluted further prior to
entering the optical flow cell.
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f. Control Material
• CELL-DYN Retic-RITE Controls (List number 03H41-01)
• Stabilized human red cells and reticulocyte surrogate.
• Available in two levels, Level I and II.
• 5 tubes of each level contained in the box.
• Must be stored at 2-8° C
• Each lot has a two month expiration date.
• 16-Day Open-Tube Stability.
• Quality Control checks should be performed at a minimum, every 8 hours
that reticulocytes samples are run according to the laboratory’s protocol.
Note: Reticulocyte Controls (List Number 03H41-01) may be received in packaging
that does not specify use on CELL-DYN 3200 Systems, but are intended for
use on instruments that have been upgraded to software Version 2.0.
2. Software
a. Turn on Reticulocyte Package: Reticulocyte package must be turned on in
order to process reticulocyte specimens.
• From the OPERATION SET UP menu, selecting the TURN ON RETIC
PKG soft key adjusts the instrument settings accordingly for reticulocyte
analysis.
b. Patient Limit Sets: Six (6) patient limit sets for Retic%, Retic Abs and RBC.
c. Unit Selection: A choice of five (5) types of units are available for reticulocytes
absolute. The default setting is USA units (K/µL).
d. QC Files: Six (6) QC files for Retic%.
• Each file can store up to 120 results.
• Westgard rules and Levey-Jennings chart.
e. Retic Data Log: Holds 2000 results and is accessible from both the Hematology
mode and Reticulocyte mode.
3. Basic Operation
This assay is available in the Open mode only.
a. Running Reticulocyte Backgrounds
• Reticulocyte Reagent tube suited for running Reticulocytes backgrounds can
be used for seven (7) consecutive days.
• Used to check the reagent and the fluidic pathway.
b. Running Controls
• Two levels of controls, CELL-DYN Retic-RITE I and II
• Dispense 20 µL of the well-mixed control material into the Reticulocyte
Reagent tube. Incubate for 15 minutes on a rotator or in a rack, after fully
inverting the stained specimens 5 times. The stained control specimens may
incubate for up to 30 minutes prior to processing on the system.
c. Running Patient Samples
• Dispense 20 µL of a well-mixed EDTA specimen into the Reticulocyte
Reagent tube. Incubate for 15 minutes on a rotator or in a rack, after fully
inverting the stained specimens 5 times. The stained reticulocyte specimens
may incubate for up to 2 hours prior to processing on the system.
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d. Performance specifications
• Background: [ 100 counts/count cycle
• Precision (CV%): Retic% [15% (specimen range 0.5-2.7%)
• Linearity Range: Retic% = 0.5-23%
• Carryover: Retic% [0.5% (specimen range 1.8%-20% with 30,000 listmode
events).
• Correlation: Retic% m 0.90
e. Calibration/Standardization
• No calibration required.
• Proper performance of the reticulocyte assay may require that the optics bench
be standardized with Opti-Cal. WOC gains and CVs should be verified using
7 µm latex beads.
• The WOC Autogain adjustment procedure will automatically copy the WOC
gains to the retic gains.
Note: Manual changes of the WOC gains require the operator to manually copy
the WOC gains to the retic gains.
• Gain entry is only available through the Hematology mode, Diagnostic menu.
f. Maintenance
• Extended Autoclean
• Must be performed weekly if routinely analyzing reticulocyte specimens.
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4. Data Review
a. Reticulocyte Parameters:
• Retic% = Retic count X 100

RBC + Retic count
Note The RBC value used in this calculation is extracted from list mode events
of the reticulocyte analysis.
• RBC: The value is either entered by the operator or retrieved from the
Hematology Data log if the patient specimen was run less than eight (8) hours
prior to the reticulocyte analysis.
• Retic Abs = RBC X Retic%

(Calculated only if the RBC value is entered by the operator or retrieved from
the Hematology Data log.)
CELL-DYN 3200 Graphics Printout

(90°/0° Scatterplot/Histogram and 90°/10° Scatterplot/Histogram)
Note: The 90°/0° scatterplot appearance is different from the CELL-DYN 3500
and 3700. This difference is due to the internal 0° gain change
implemented in this software.
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b. Algorithms
The analyzer collects 30,000 list mode events. Each event is measured and
classified using the 0°, 10°, and 90° light scatter. The algorithm uses a three-
dimensional analysis using radial histograms from the 0°/10°/90° cube and the
90° light scatter to identify the list mode events.
• Typically, events such as WBCs and NRBCs saturate the 0°, 10° and 90°
detectors (fall into channel 255) and are removed from analysis.
• On the 0°°/10°° scatterplot (not viewable to the operator, but provided here as
an example) a fixed threshold is set. Events below this threshold (displayed
as yellow) such as noise, platelets, large platelets, platelet aggregates and
degenerative RBCs are removed from analysis. Once events are identified,
they retain their classification regardless of where they appear on other
scatterplots.
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• On the 90°°/0°° and 90°°/10°° scatterplots, a 60° arc is drawn from a fixed point
and within these areas radial histograms are created. From the radial
histograms and 90° light scatter, thresholds are determined between the RBCs
(red) and reticulocytes (blue).
Retics 60º
Retics
60°
RBC RBCS
• On the 90°°/10°° scatterplot, another radial histogram is created that determines

the threshold that separate RBCs from coincidence. Events that fall to the
right of the threshold are identified as coincidence, WBCs, or NRBCs (WBCs
and NRBCs are displayed in white).
WBCs/NRBCs
Retics
60°
Coincidence
RBCS
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c. Examples of scatterplots
Color of cell populations on scatterplots

Red RBCs
Blue/Light Blue Reticulocytes
Yellow Noise, Platelets, Degenerative RBCs
White (Black on printout) WBC/NRBC
• Normal Sample
Noise, platelets,
degenerative RBCs
Reticulocytes
RBC
The noise, platelet, degenerative RBC population are minimal on this type of sample. The
RBC population is well defined and clearly separated from the reticulocyte population.
Notice that the 90° scatter of the reticulocyte population is greater than the RBC
population. The WBC/NRBC populations typically saturate the detectors and are
removed. If they are present on the scatterplot, their 90° and 10° light scatter clearly
separate them from the other cell populations.
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• Control Results
Level I Retic-RITE control
Level II Retic-RITE control
Note: Control material commonly display retic flags such as Fragile RBCs and
Excessive RBC Loss (ERL). These flags can be disregarded when running
control material. Ensure the control values are within the assay range.
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• Bad Reticulocyte Reagent
Notice the noise/platelet population in the bottom left corner of the 90°/0° and 90°/10°
scatterplots. It is more apparent than in a normal sample. A portion of the RBC
population is included into the noise/platelet population triggering the ERL flag.
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d. Reticulocyte Alerts
Reticulocyte Alerts
Instrument Alerts with Suppressed Reticulocyte Results
Alerts Software Criteria Probable Cause Corrective Action
Flow Error § Increase in average count § Air bubbles § Run a background count to cycle
rate during the reticulocyte § Hardware malfunction air through the system.
count cycle § Rerun the reticulocyte specimen.
§ If alert still occurs, refer to
Chapter 10, Troubleshooting, of
the Operator’s Manual.
Too Few Events § Fewer than 3000 events § Inadequate whole blood sample § Prepare and run another dilution.
counted mixing § If the flag persists, verify the
§ Cold Agglutinin reticulocyte results by an
alternative method.
>>>> § The reticulocyte § The value exceeds the reportable § Verify reticulocyte results by an
(Chevrons) percentage exceeds the linear range. alternate method.
reportable linear range.
Data Invalidating Alerts
Alerts Software Criteria Probable Cause Corrective Action
Fragile RBC’s § The average count rate § Air bubbles § Run a background count to cycle
decreases during the § Staining a fragile RBC specimen air through the system. Rerun the
reticulocyte count cycle. too long in the reticulocyte reagent reticulocyte specimen.
§ Fragile RBCs § Prepare another dilution and run
after 15 minutes of incubation.
§ If flag persists, verify reticulocyte
results by an alternate method.
Excessive RBC Loss § The percentage of list § Specimen staining time too long in § Prepare another dilution and run
(ERL) mode events below the the reticulocyte reagent after 15 minutes of incubation.
0°/10° threshold exceeds § Rapid degeneration of RBCs. § Rerun the specimen.
4.0% or § High concentration of platelets, § If the flag persists, verify the
§ The mode of the histogram platelet aggregates or other reticulocyte results by an
created in the 0°/10° interfering substances. alternative method
scatterplot is <channel 20 § Microcytic RBCs
§ Improper instrument settings.
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5. Troubleshooting
a. High Reticulocyte background counts

• Reagent
• Repeat the background count.
• Run new reticulocyte reagent tube.
• Run with new lot of reticulocyte reagent.
• Check storage conditions of reagent tubes.
• Ensure the laboratory’s room temperature is within specifications
(15-30ºC).
• Hardware
• Run a blank cycle without a reticulocyte reagent tube to determine
whether the problem is reagent or hardware.
• Check background in hematology mode, if elevated troubleshoot
accordingly.
• Perform Autoclean.
• Clean shear valve.
b. Erratic results
• Reagents
• Prepare another dilution and run after 15 minutes incubation.
• Prepare another dilution with a new lot of reticulocyte reagent and run
after 15 minutes incubation.
(15-30ºC).
• Hardware
• Check reticulocyte background.
• Check reticulocyte gain setting vs. hematology gain setting and ensure that
they are the same.
• Check WBC Lyse and Optical injection syringe for bubbles and proper
movement.
• Check Optical transfer pump tubing.
• Confirm WBC mixing chamber is draining.
• Confirm 7 µm latex bead CV and recovery (Hematology mode).
• Confirm Opti-Cal recovery (Hematology mode).
c. Excessive alerts/flagging
• Reagents
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(15-30ºC).
• Hardware
they are the same.
d. Out of range controls

• Reagents:
• Check storage of reagent tubes.
(15-30ºC).
• Controls
• Rerun with new tube of control.
• Check other level of control.
• Hardware
they are the same.
e. Interfering substances [7]

Known or potential interferents
Cellular Elements Cellular Inclusions Miscellaneous
• Platelet clumps • Howell-Jolly bodies • Abnormal red cells
• Basophilic Stippling • Heinz bodies • Paraproteins
• Giant Platelets • Pappenheimer bodies • Cold agglutinins
• Leukocytes and • Parasites (malaria, • Platelet/erythrocyte
leukocyte fragments babesia) coincidence
• Nucleated erythrocytes • Hemolysis
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6. Similarities and Differences of CELL-DYN Reticulocyte Methodology
Similarities and Differences of CELL-DYN Reticulocyte Methodology
Analyzer CELL-DYN CELL-DYN CELL-DYN CELL-DYN
3200 3500 3700 4000
Method Light Scatter Light Scatter Light Scatter Fluorescence and Light
Semi-automated Semi-automated Semi-automated Scatter
Automated
Detectors 0°,10°,90° 0°,10°,90° 0°,10°,90° 7°, FL1
List Mode events 30,000 30,000 30,000 50,000
Retic Parameters Retic %, Retic Abs Retic%, Retic Abs Retic%, Retic Abs, IRF Retic%, Retic Abs, IRF
Specimen Type EDTA EDTA EDTA EDTA
Specimen Volume 20 µL 20 µL 20 µL 112.5 µL (225 µl of
RBC/PLT dilution)1
Incubation time 15 min-2 hrs 15 min-2 hrs 15 min-2 hrs N/A
Number of Patient
6 4 4 8
limit sets
Reticulocyte Stain New Methylene Blue New Methylene Blue New Methylene Blue Proprietary dye
(List Number)* (03H40-01) (03H40-01) (03H40-01) (01H76-01)
Reticulocyte Reagent Room temperature in the Room temperature in the Room temperature in the Refrigerated before use
storage dark dark dark
Reticulocyte Reagent Labeled expiration date Labeled expiration date Labeled expiration date Expires 30 days after
stability opening or if unopened
according to labeled
expiration date,
whichever comes first
Controls CELL-DYN Retic-RITE CELL-DYN Retic-RITE CELL-DYN Retic-RITE CELL-DYN Reticulated
(List Number)* Control, 2 levels Control, 2 levels Control, 2 levels Cell Control, 2 levels
(03H41-01) (03H41-01) (03H41-01) (01H72-01)
Control storage Refrigerated (2-8°C) Refrigerated (2-8°C) Refrigerated (2-8°C) Refrigerated (2-8°C)
Control Lot expiration Each lot has a 2 month Each lot has a 2 month Each lot has a 2 month Each lot has a 2 month
expiration date expiration date expiration date expiration date
* CELL-DYN Reticulocyte Reagent and Retic-RITE Control may be received in packaging that does not specifiy use on the CELL-DYN 3200 but are intended
for use on instruments that have been upgraded to software Version 2.0.
16 of 18
Similarities and Differences of CELL-DYN Reticulocyte Methodology
Analyzer CELL-DYN CELL-DYN CELL-DYN CELL-DYN
3200 3500 3700 4000
Control Stability 16-Day Open-Tube 16-Day Open-Tube 16-Day Open-Tube 7-Day Open-Tube
stability, or if unopened stability, or if unopened stability, or if unopened stability, or if unopened
until the labeled until the labeled until the labeled until the labeled
expiration date, expiration date, expiration date, expiration date,
whichever comes first whichever comes first whichever comes first whichever comes first
Control incubation
15-30 minutes 15-30 minutes 15-30 minutes N/A
time
Number of QC Files 6 6 6 25
Maintenance Weekly Extended Weekly Extended Weekly Extended No specific maintenance
Autoclean Autoclean Autoclean
Precision (CV) [ 15% [ 15% [ 15% [ 15%
Linearity (Range) Retic%= 0.5-23% Retic%= 0-30% Retic%= 0-30% Retc = 0.0–1500.0 x 10 3
/µL
Correlation m 0.90 m 0.90 m 0.90 m 0.95
Coefficient
Correlation performed CELL-DYN 4000 Becton Dickinson® CELL-DYN 4000 Becton Dickinson®
to: FACScan FACScan
Background [ 100 counts [ 100 counts [ 100 counts [ 5.00 x 103/µL
Carryover [ 0.5% [ 0.5% [ 0.5% [ 1%
Reticulocyte Fragile RBCs, ERL Fragile RBCs, ERL, Fragile RBCs, ERL, IR
Alerts/Flags ENC ENC
Are the ERL and/or
ENC alerts dependent
on the hematology No Yes2 Yes2 N/A
results taken from the
Data log?
1. Reticulocytes are run in conjunction with the CBC analysis. The sample volume is the nominal volume required for the combinations of tests.
2. RBC values manually entered will not trigger the ERL and/or ENC alerts (ENC flag applies to CELL-DYN 3500 and 3700 Systems only).
17 of 18
7. References
Photomicrographs:
Figure A: School of Medicine, University of Washington, HuBio 552 hematology
course, morphology plates, 1999; polychromatophilic or shift cell.
Figure B: Aleman, M .Department of Cell Biology, Vanderbilt University Medical
Center. Histology lessons, Blood outline, Erythrocytes, 1995; reticulocytes
Literature:
1. Abbott Laboratories, Abbott Park, IL 60064. CELL-DYN System 3200
Operator’s Manual, October 2001; 14:1-79.
Operator’s Manual, June 1999; Chapter 4.
Operator’s Manual, November 2000; Chapter 4.
Operator’s Manual, September 1999; Section 4
5. McKenzie, S. Textbook of Hematology, 1996; 665.
6. Abbott Laboratories, Abbott Park, IL 60064. CELL-DYN System 3500R System
Training Guide, September 1997; 10:5.
7. NCCLS, Wayne, PA 19087. Methods for Reticulocyte Counting (Flow Cytometry
and Supravital Dyes); Approved Guideline, NCCLS document H44-A, October
1997; Vol. 17, No. 15:12.
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CELL-DYNâ 3200 System Reticulocyte Assay

Service and Support Training Material

1. Introduction of the Reticulocyte Assay 3

6. Similarities and Differences of CELL-DYN Reticulocyte Methodology 16

c. CELL-DYN 3200 Reticulocyte Assay

• Retic% = Retic count X 100

• Retic Abs = RBC X Retic%

CELL-DYN 3200 Graphics Printout

• On the 90°°/10°° scatterplot, another radial histogram is created that determines

Color of cell populations on scatterplots

Level I Retic-RITE control

Level II Retic-RITE control

a. High Reticulocyte background counts

d. Out of range controls

e. Interfering substances [7]

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