Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay: 9140350A-October 2001

Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay
Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay
Overview
The CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay is an

immunology-based method for counting T-Lymphocytes and associated
subsets. The T-Cell Assay represents a valuable addition to the
CELL-DYN 4000 System because:
• this assay, which uses monoclonal antibodies, is accurate and
precise across a wide range of T-cell concentrations.
• it is rapid and fully automated.
This section contains all information needed for understanding and
running the T-Cell Assay. The organization of this section follows the
organization of the rest of the CELL-DYN 4000 System Operator’s
Manual.
CELL-DYN® 4000 Immuno T-Cell (CD3/4/8) Assay — CELL-DYN 4000 System Operator’s Manual Addendum 14-1
9140350A—October 2001
CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay
Overview Section 14
NOTES
14-2 CELL-DYN® 4000 Immuno T-Cell (CD3/4/8) Assay — CELL-DYN 4000 System Operator’s Manual Addendum
9140350A—October 2001
Section 14 Use or Function
Use or Function
Intended Use
The intended use of the CELL-DYN 4000 Immuno T-Cell (CD3/4/8)
Assay is to report the count of T-lymphocytes and associated T-cell
subsets in EDTA-anticoagulated human whole blood. The Assay is
intended for use on the CELL-DYN 4000 System, a fully automated
hematology analyzer. The Assay is intended for in vitro diagnostic use in
a clinical laboratory of a hospital or medical clinic, a reference laboratory,
or a research laboratory.
Specimen Collection
The specimen should be collected in a standard blood collection tube
containing EDTA.
CELL-DYN Immuno T-Cell (CD3/4/8) Reagents

NOTE: The CELL-DYN Immuno T-Cell (CD3/4/8) Reagents have been
formulated specifically for the CELL-DYN 4000 System in order to
provide optimal system performance. Use of reagents other than those
specified in this manual is not recommended as system performance can
be affected. Each CELL-DYN 4000 System is tested at the factory using
the specified reagents, and all performance claims are generated using
these reagents.
Two reagents are used in the T-Cell Assay. One of the reagents,
CELL-DYN CD3/4 T-Cell Reagent, is used to count T-helper/inducer
cells. The other reagent, CELL-DYN CD3/8 T-Cell Reagent, is used to
count T-suppressor/cytotoxic cells.
The key components of the CELL-DYN Immuno T-Cell (CD3/4/8)
Reagents are monoclonal antibodies (mAbs), designated anti-CD3,
anti-CD4, and anti-CD8. A fluorescent dye is bound to each mAb, with
fluorescein isothiocyanate (FITC) binding to anti-CD3 in both reagents.
Phycoerythrin (PE) is bound to anti-CD4 in CELL-DYN CD3/4 T-Cell
Reagent and to anti-CD8 in CELL-DYN CD3/8 T-Cell Reagent. The
mAbs in both reagents are freeze-dried (lyophilized) into a pellet and
packaged in a standard blood collection tube, as shown in the following
illustration. A stainless steel retainer keeps the pellet at the bottom of the
tube. The CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes have color
coded caps and are identified by bar code labels.
9140350A—October 2001
Use or Function Section 14
Close-up of Retainer
Top View
Retainer
Pellet containing
Monoclonal Antibodies
Figure 14.1: CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Tubes
Each box of reagent tubes contains two color-coded pouches, one pouch
containing 20 tubes of CELL-DYN CD3/4 T-Cell Reagent, and the other
containing 20 tubes of CELL-DYN CD3/8 T-Cell Reagent, as shown in
the following illustration.
Three packages of desiccant are included in each pouch. The indicator
granules in the desiccant packages turn from blue to lavender if the
contents of the pouch have been exposed to excessive moisture.
The color of the caps on the tubes allows the operator to differentiate the
reagents. The caps on tubes containing CELL-DYN CD3/4 T-Cell
Reagent are orange, while those on tubes containing CELL-DYN CD3/8
T-Cell Reagent are green.
Additional CELL-DYN Immuno T-Cell (CD3/4/8) Reagent bar code
labels are provided for use in labeling negative controls. For information
on running negative controls, refer to Subsection: Quality Control within
this section.
9140350A—October 2001
1 Box
Bar Code Labels for
Negative Control Tubes
Package Insert
2 Pouches per Box
Desiccant 20 CELL-DYN CD3/4 T-Cell Desiccant 20 CELL-DYN CD3/8 T-Cell

Reagent Tubes per Pouch Reagent Tubes per Pouch
Figure 14.2: Packaging for the CELL-DYN Immuno T-Cell (CD3/4/8) Reagents
9140350A—October 2001
Test Selection
The test selection for the T-Cell Assay is CBC+CD3/4/8, WBC Extended
Count.
The T-Cell Assay can be run in Autoloader mode only.
NOTE: When the T-Cell Assay is run, reticulocytes are not counted. To
obtain a reticulocyte count, rerun the specimen using test selection
CBC+RETC or CBC+RETC, Resistant RBC as appropriate.
Specimen Processing
NOTE: A CELL-DYN CD3/4 T-Cell Reagent tube and a CELL-DYN
Immuno CD3/8 T-Cell Reagent tube must be placed in the Autoloader
directly behind the specimen tube in this order and in the same rack.
CAUTION: If you are planning to run additional racks in the Autoloader

following the current rack, do not place tubes for a mAb test in the current
rack beginning with the specimen tube in the second tube position.
Specimen processing involves the following steps:

1. After first advancing the rack, mixing the contents of the tubes, and
advancing again, the CELL-DYN 4000 System aspirates 112.5 µL
from the specimen tube and begins to process the sample as a CBC
with WBC Extended Count.
2. The System backs up the rack two positions, advances one position,
mixes the tubes, and then advances one position.
3. The System aspirates 112.5 µL of blood from the specimen tube,
advances the rack one position, and deposits the blood into the
CELL-DYN CD3/4 T-Cell Reagent tube. An additional 112.5 µL of
CELL-DYN 4000 Diluent/Sheath Reagent is then deposited via the
Aspiration Probe into the CELL-DYN CD3/4 T-Cell Reagent tube
to suspend and dilute the mixture.
4. The System backs up the rack two positions and advances one
position.
5. The System aspirates 112.5 µL of blood from the specimen tube,
advances the rack two positions, and deposits the blood into the
CELL-DYN CD3/8 T-Cell Reagent tube. An additional 112.5 µL of
CELL-DYN 4000 Diluent/Sheath Reagent is then deposited via the
Aspiration Probe into the CELL-DYN CD3/8 T-Cell Reagent tube
to suspend and dilute the mixture.
6. The System moves the rack back three positions and forward one
position; the CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes
are then picked up by the Mix Head and gently rocked, with the Mix
Head sweeping through an arc of 36 degrees. During rack
movement and tube mixing, the blood and the CD3/4 and CD3/8
mAbs incubate at room temperature for approximately two minutes.
9140350A—October 2001
7. The Mix Head places the tubes back in the rack, and the rack is
advanced one position.
8. The System aspirates 150 µL of the mixture from the CELL-DYN
CD3/4 T-Cell Reagent tube and deposits 75 µL of it into the WBC
Dilution Cup. WBC Reagent is added to make a final dilution of
1:36.
9. The remaining 75 µL of the mixture is deposited into the RBC
Dilution Cup as waste.
10. The CD3/4 dilution is then transported to the Optical Flow Cell for
measurement and is injected into the flow cell via the same pathway
as is used for the WBC measurement.
11. After advancing the rack one position, the System aspirates 150 µL
of the mixture from the CELL-DYN CD3/8 T-Cell Reagent tube and
deposits 75 µL of it into the WBC Dilution Cup. WBC Reagent is
added to make a final dilution of 1:36.
12. The remaining 75 µL of the mixture is deposited into the RBC
Dilution Cup as waste.
13. The CD3/8 dilution is then transported to the Optical Flow Cell for
measurement and is injected into the flow cell via the same pathway
as is used for the WBC measurement.
9140350A—October 2001
NOTES
9140350A—October 2001
Section 14 Installation Procedures and Special Requirements
Installation Procedures and Special Requirements
Installation of Hardware for the T-Cell Assay

Before running the T-Cell Assay, you must have special hardware
installed on your CELL-DYN 4000 System. Abbott field personnel will
complete this installation at your request.
Installation of Software for the T-Cell Assay

Abbott field personnel will ensure that the software for the T-Cell Assay
is fully functional when the CELL-DYN 4000 System is installed. If you
already own a CELL-DYN 4000 System, an Abbott Field Service
Representative will install the appropriate software revision.
System Customization for the T-Cell Assay

Most System customization is carried out using selections from the Setup
menu. Customization of control limits for the T-Cell Assay is carried out
separately, as described in Subsection: Quality Control within this
section.
The following table provides information on customizing the System for
use of the T-Cell Assay using the Setup menu. For additional information
regarding System customization, refer to the CELL-DYN 4000 System
Operator’s Manual, Section 2: Installation Procedures and Special
Requirements.
9140350A—October 2001
Installation Procedures and Special Requirements Section 14
Table 14.1: Customization for the T-Cell Assay Using the Setup Menu
Selection Action Reference
Parameter Sets> To display or print graphs related to the T-Cell CELL-DYN 4000 System
Assay on the Chartable Page, select from among Operator’s Manual, Section 2:
the following graphs: Installation Procedures and
• CD4T Viability∆: FL3 vs. FL2 for CD3/4 tube Special Requirements,
• CD8T Viability∆: FL3 vs. FL2 for CD3/8 tube Subsection: Customizing
• CD3/4 FL1-FL2: Scatterplot of FL1 vs. FL2 Parameter Sets
for CD3/4 tube
• CD3/8 FL1-FL2: Scatterplot of FL1 vs. FL2
for CD3/8 tube
• CD3/4 0° - 7°: Scatterplot of 0° vs. 7° for
CD3/4 tube
• CD3/8 0° - 7°: Scatterplot of 0° vs. 7° for
CD3/8 tube
• CD3/4 FL1: Histogram of FL1 for CD3/4 tube
Laboratory To display T-Cell parameters on the Laboratory CELL-DYN 4000 System

Worksheet… Worksheet Pageφ, select the 5-Part Differential Operator’s Manual, Section 2:
view from the Laboratory Worksheet Setup Installation Procedures and
window. Special Requirements,
To display or print graphs related to the T-Cell Subsection: Customizing the
Assay on the Laboratory Worksheet Page, select Laboratory Worksheet Page
from among the same graphs listed for the
Chartable Page.
Patient Limit To customize patient limit sets for the T-Cell CELL-DYN 4000 System
Sets> results, enter limits using the Patient Limit Set Operator’s Manual, Section 2:
window. Installation Procedures and
Special Requirements,
Subsection: Customizing
Patient Limit Sets
Data Log… To add T-Cell parameters to Data Sets for the CELL-DYN 4000 System
Data Log, select the desired parameters from the Operator’s Manual, Section 2:
Data Log Setup window. Installation Procedures and
Subsection: Customizing the
Data Log
∆ This graph is not used to calculate the parameters v4†, v8†, or v4/8†. For information on the calculation of these
parameters, refer to Subsection: Parameters and Report Units within this section.
† Clinical significance has not been established for this parameter. Therefore, it is not reportable in the U.S.A. It is provided
for laboratory use only.
φ The information on the Laboratory Worksheet Page is provided for laboratory use only.
9140350A—October 2001
Section 14 Installation Procedures and Special Requirements
Table 14.1: Customization for the T-Cell Assay Using the Setup Menu (Continued)
Selection Action Reference
Work List… To set Work List Standard Processing Conditions CELL-DYN 4000 System
to run the T-Cell Assay, choose CBC+CD3/4/8, Operator’s Manual, Section 2:
WBC Extended Count as the Test Selection. Installation Procedures and
NOTE: When setting Work List Standard Subsection: Customizing the
Processing Conditions for the T-Cell Assay Work List Standard Processing
(using the WORK LIST SETUP window), Conditions
always leave the number of replicates at the
default setting of one.
Paired To set paired difference criteria for the T-Cell CELL-DYN 4000 System
Difference… parameters, use the PAIRED DIFFERENCE Operator’s Manual, Section 2:
SETUP window. Installation Procedures and
NOTE: Paired Difference Analysis is not applied
Subsection: Customizing the
to the parameters v4†, v8†, or v4/8†.
Paired Difference Analysis
Program
Halt Behavior… No specific action is required, but the operator CELL-DYN 4000 System
should be aware that customization of halting Operator’s Manual, Section 2:
applies to violations of background and QC limits Installation Procedures and
for the T-Cell parameters. Special Requirements,
Subsection: Customizing Halt
Behavior
Units Format… The units format for T-Cell parameters is the CELL-DYN 4000 System
same as what has been selected for WBC. To Operator’s Manual, Section 2:
change the units format for the T-Cell Installation Procedures and
parameters, select the desired units format for Special Requirements,
WBC from the Units Format window. Subsection: Customizing the
Units Format
9140350A—October 2001
Installation Procedures and Special Requirements Section 14
NOTES
9140350A—October 2001
Section 14 Principles of Operation
Principles of Operation
Overview of Assay
Immunophenotyping of T-cell subsets can provide important information
for disease monitoring and can guide decisions about therapy. All T-cells
express the CD3 antigen. T-cells that express both the CD3 and CD4
antigens are T-helper/inducer cells, while those that express both the CD3
and CD8 antigens are T-suppressor/cytotoxic cells.
Technologies
The T-Cell Assay is based on technologies involving monoclonal
antibodies, fluorescence, and light scatter.
Monoclonal Antibodies
Monoclonal antibodies are produced by a cell formed when a cancer cell
(obtained from a continuously replicating cell line) is fused with a cell that
produces a single antibody. The resulting cell, called a hybridoma, has
two key characteristics: it replicates continuously and it produces the
desired antibody. The cell can be cultured so that it produces large
quantities of the single antibody.
Antibodies bind with extremely high specificity to a single antigen.
Typically, the antibody-producing cell used to generate a monoclonal
antibody is selected on the basis of its binding to an antigen of interest.
The antibody-producing cells used to generate the monoclonal antibodies
for the T-Cell Assay were selected on the basis of their binding to the
following antigens:
• for T-cells: CD3 receptor
• for T-helper/inducer cells: CD4 receptor
• for T-suppressor/cytotoxic cells: CD8 receptor
Monoclonal antibodies are named based on their CD (Cluster
Designation) number. The mAbs used for the T-Cell Assay are called
Anti-Human CD3, CD4, and CD8.
The antibodies used in this assay bind to certain antigens only as follows:
• Anti-CD3 antibody reacts with the CD3 antigen, which is expressed
on T-cells. T-cells make up 61 to 85% of peripheral blood
lymphocytes.1
on T-helper/inducer cells and also on monocytes (in low density).
T-helper/inducer cells make up approximately 28 to 58% of
peripheral blood lymphocytes.1
9140350A—October 2001
Principles of Operation Section 14

on T-suppressor/cytotoxic cells, and also on a subset of Natural
Killer (NK) cells. T-suppressor/cytotoxic cells make up 19 to 48%
of peripheral blood lymphocytes.1
Fluorescence
Measurements using monoclonal antibodies are possible because
fluorescent dyes can be covalently bonded to the antibodies. The dyes
used in the T-Cell Assay are fluorescein isothiocyanate (FITC) and
phycoerythrin (PE). When excited at 488 nm, the two dyes exhibit
fluorescence peaks centered at the following wavelengths:
• FITC: 530 nm
• PE: 580 nm
The fluorescence of FITC is centered near the 530-nm peak wavelength
emitted by the dye used in the reticulocyte measurement on the
CELL-DYN 4000 System. This commonality makes it feasible for the
T-Cell Assay to time-share the FL1 optical subsystem with the
reticulocyte measurement. The FL2 optical subsystem is used to measure
PE fluorescence.
The FL3 optical subsystem is used to assess the viability of lymphocytes.
Lymphocytes with FL3 fluorescence measurements below a certain level
are considered viable.
Light Scatter
The T-Cell Assay also uses light scatter technology. The 0° light loss and
7° light scatter measurements are used by the algorithms in conjunction
with the fluorescence measurements, as described in the following
subsection.
Algorithms
Channel data for the 0° light loss, 7° light scatter, and FL3 measurements
are used in hardware thresholding. Data that meet the requirements are
then stored in the T-Cell Assay List Mode Data Table. Storage of data in
List Mode Data Tables is discussed in the CELL-DYN 4000 System
Operator’s Manual, Section 3: Principles of Operation, Subsection: List
Mode Data Storage.
The algorithms use the 0° light loss, 7° light scatter, FL1, FL2, and FL3
channel data to identify and count T-Cells, T-helper/inducer, and
T-suppressor/cytotoxic cells among the other cells and cell fragments in
the specimen. FL3 data from the CBC with Extended WBC Count run are
used to quantify the viability of lymphocytes in order to allow
computation of the concentration of viable CD4+ and CD8+ T-cells.
9140350A—October 2001
Parameters and Report Units

The results generated by the T-Cell Assay are shown in the following
table.
Table 14.2: Parameters and report units for the T-Cell Assay
Parameter Description Units (USA)
CD3T Concentration of T-cells (CD3+ lymphocytes); computed as the average 10e3/µL

of the CD3+ lymphocyte concentrations from the CELL-DYN CD3/4 and
CD3/8 T-Cell Reagent tubes
%CD3 Percentage of lymphocytes that are T-cells (CD3+ lymphocytes); %

computed as the average of the CD3+ lymphocyte percentages from the
CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes
CD4T Concentration of T-helper/inducer cells (CD3+CD4+ lymphocytes) 10e3/µL
%CD4 Percentage of lymphocytes that are T-helper/inducer cells %

(CD3+CD4+ lymphocytes)
CD8T Concentration of T-suppressor/cytotoxic cells (CD3+CD8+ lymphocytes) 10e3/µL
%CD8 Percentage of lymphocytes that are T-suppressor/cytotoxic cells %

(CD3+CD8+ lymphocytes)
4/8 Ratio of T-helper/inducer cell (CD3+CD4+ lymphocyte) concentration to

T -suppressor/cytotoxic cell (CD3+CD8+ lymphocyte) concentration
v4† Concentration of T-helper/inducer cells (CD3+CD4+ lymphocytes) that are 10e3/µL

viable (computed as CD4T multiplied by the viable lymphocyte fraction, as
calculated from FL3 data obtained during the CBC with Extended WBC
Count run)
v8† Concentration of T-suppressor/cytotoxic cells (CD3+CD8+ lymphocytes) 10e3/µL

that are viable (computed as CD8T multiplied by the viable lymphocyte
fraction, as calculated from FL3 data obtained during the CBC with
Extended WBC Count run)
v4/8† Ratio of v4†, viable T-helper/inducer cell (viable CD3+CD4+ lymphocyte)

concentration to v8†, viable T -s uppressor/cytotoxic cell (viable
CD3+CD8+ lymphocyte) concentration
9140350A—October 2001
Flagging
Suspect Parameter Flags
Suspect Parameter Flags require review and validation of results before
affected results can be reported. The following two tables show the
System Faults and Data Faults specific to multi-tube assays. The System
generates Suspect Parameter Flags for these faults. Following these tables
are listed additional Suspect Parameter flagging conditions that invalidate
results from the T-Cell Assay, causing results to be marked with an
asterisk [*] or left blank [ ].
NOTE: When a Suspect Parameter Flag occurs, the operator can
determine which fault(s) caused the flag by checking the invalid data
messages. These messages are displayed in the INVALID DATA window,
which automatically appears when a record is initially displayed on the
Chartable Page and the Laboratory Worksheet Page. If the INVALID
DATA window is closed, open it by selecting the *Invalid Data… button
on the Laboratory Worksheet Page.
For more information on Suspect Parameter flagging, refer to the

CELL-DYN 4000 System Operator’s Manual, Section 3: Principles of
Operation, Subsection: Suspect Parameter Flags.
System Faults also generate System Initiated Messages (SIMs) in most
cases. SIMs for the T-Cell Assay are described in Subsection:
Troubleshooting and Diagnostics within this section. Data Faults that are
specific to the T-Cell Assay do not lead to SIMs.
For general information on SIMs, refer to the CELL-DYN 4000 System
Operator’s Manual, Section 10: Troubleshooting and Diagnostics,
Subsection: Responding to System Initiated Messages.
9140350A—October 2001
Table 14.3: System Faults
Numerical Results Marked with [*]

Invalid Data Message Explanation of Fault Condition
or Left Blank [ ]
[Missing Reagent Tube, A mAb bar code label was not All absolute and % T-Cell results, 4/8,
Assay Not Performed] detected when expected. v4†, v8†, v4/8†, 4mn1†◊, 8mn1†◊,
4mn2†◊, 8mn2†◊
[Incorrect Reagent Tube, An unexpected mAb bar code label All absolute and % T-Cell results, 4/8,
Assay Not Performed] was detected. v4†, v8†, v4/8†, 4mn1†◊, 8mn1†◊,
4mn2†◊, 8mn2†◊
NOTE: This message
applies to mAb tubes for
immunology-based tests
other than the T-Cell Assay
(e.g., the CELL-DYN 4000
ImmunoPlt™ (CD61)
Assay).
[Insufficient Reagent to There is insufficient reagent for a All absolute and % T-Cell results, 4/8,
Complete Multi-tube multi-tube assay (such as the T-Cell v4†, v8†, v4/8†, 4mn1†◊, 8mn1†◊,
Assay] Assay). 4mn2†◊, 8mn2†◊
[CD3/4/8 Count Rate/ Two possible causes: All absolute and % T-Cell results, 4/8,
Mismatch Violation] v4†, v8†, v4/8†, 4mn1†◊, 8mn1†◊,
• There is inadequate agreement 4mn2†◊, 8mn2†◊
between the hardware counts
from the specimen tube and the
CELL-DYN CD3/4 T-Cell
Reagent tube or between the
hardware counts from the
specimen tube and the
Reagent tube.
• There is an anomaly in the

hardware count rate from the
specimen tube, the CELL-DYN
CD3/4 T-Cell Reagent tube, or
the CELL-DYN CD3/8 T-Cell
Reagent tube.
◊ This parameter is available for display in QC files only. See description on page 14-46.
NOTE: In the first three of these System Faults, all of the affected
numerical results display as a blank [ ].
NOTE: The CD3/4/8 Count Rate/Mismatch Error fault does not lead to
a SIM. There is no halt in Autoloader operation.
9140350A—October 2001
NOTE: Under the following additional System Fault conditions, the

T-Cell-related results (all absolute and % T-Cell results, 4/8, v4†, v8†,
v4/8†, 4mn1†, 8mn1†, 4mn2†, 8mn2†) are marked with an asterisk [*] or
left blank [ ]:
• [Autoloader Failure]
• [Clot Detected During Aspiration]
• [Laser Current Too High]
• [Pneumatics Failure, 40 psi]
• [Pneumatics Failure, 15 in. Hg Vacuum]
• [Sample Aspiration Pump Failed To Home]
• [Analyzer Lost Communication with Pneumatic Unit]
(No Calculation/Display Flag, all results left blank)
• [Aspiration Probe Failed to Return to Upper Position]
(No Calculation/Display Flag, all results left blank)
• [Short Sample] (from the specimen tube)
• [Residual Fluid Detected in WBC Dilution Cup]
• [WBC Dilution Cup Temperature Out of Range]
• [WBC Dilution Cup Motor Voltage Out of Range]
• [Hemoglobin Reagent Syringe Failed to Home]
• [Diluent/Sheath Reagent Syringe Failed to Home]
• [WBC Reagent Syringes Failed to Home]
9140350A—October 2001
Table 14.4: Data Faults
Numerical Results Marked with [*] or

Invalid Data Message Explanation of Fault Condition
Left Blank [ ]
[Too Many Events Too many events fell outside Absolute and % T-Cell results, 4/8, v4†,
Outside Limits During predetermined limits during the v8†, v4/8†, 4mn1†, 8mn1†, 4mn2†, 8mn2†
CD3/4/8 Assay] CD3/4/8 Assay.
[Outside Reportable The result fell outside the linear One or more of the following: CD3T,
Range] range limits for one or more of the CD4T, CD8T, calculated parameters that
following: WBC, CD3T, CD4T, are based on one or more of these
CD8T. The linear range limits for measured parameters.
the T-Cell Assay are given in
Subsection: Linearity within this
section.
NOTE: An asterisk may appear on

a result indicating a linear range
violation even though the result is
at a limit. This is due to a rounding
error of a true value that is outside
the limit and the asterisk should
be considered valid.
NOTE: Under the following additional Data Fault conditions, all

T-Cell-related results (all absolute and % T-Cell results, 4/8, v4†, v8†,
v4/8†, 4mn1†, 8mn1†, 4mn2†, and 8mn2†) are marked with an asterisk
[*] or left blank [ ]:
• [Known Interference with WBC Results, likely due to PLT

Clumping]
• [Suspected Interference with WBC Results, likely due to PLT
Clumping]
• [Number of WBC Events Out-of-Bounds Greater than Preset
Limit]
• [Unidentified Fluorescent Population Greater than Preset
Limit]
• [Resistant RBC Interference with WBC Results]
• [Optical Interference]
Under the following additional Data Fault condition, all % T-Cell
results are marked with an asterisk or left blank:
• [Significant Number of Cells Removed from WBC
Differential Analysis] and the LYM parameter is marked with an
asterisk
9140350A—October 2001
NOTES
9140350A—October 2001
Section 14 Specifications
Specifications
Volume and Throughput Specifications

• Aspiration Volume: 337.5 µL (for CBC + CD3/4/8,
WBC Extended Count
test selection)
• Minimum Specimen Volume: 1.0 mL
• Minimum Cycle Time: 7 minutes (Ready to Ready)
Performance Specifications
T-Cell Background Counts
Table 14.5: T-Cell Background Counts
Parameter SI USA
CD3T ≤0.02 x 109 cells/L ≤0.02 x 103 cells/µL
(For information on running background counts for the T-Cell Assay,

refer to Subsection: Running T-Cell Background Count within this
section.)
Precision
Precision was determined by running 10 EDTA-anticoagulated whole
blood specimens (≤4 hours since collection) in duplicate using the T-Cell
Assay. The original specimens were concentrated or diluted as needed to
obtain the desired test specimens. Results were subjected to Paired
Difference Analysis to calculate the Coefficient of Variation (CV).
9140350A—October 2001
Specifications Section 14
Table 14.6: Precision
Specimen Range
Parameter Limits
SI USA
CD3T 0.1-3.5 x 109 cells/L 0.1-3.5 x 103 cells/µL CV ≤4.0%
Linearity
Specimens for the linearity study were obtained by first enriching with
WBCs a specimen with a normal hemogram and then serially diluting it.
The dilutions yielded T-cell counts across the ranges shown.
Table 14.7: Linearity
Specimen Range
Parameter Limits♦
SI USA
CD3T 0.025-4.4 x 109 cells/L 0.025-4.4 x 103 cells/µL ± 0.02 x 103/µL or ≤8%
♦ The number to be used is the greater of the two at a given concentration.
Correlation
The correlation of the T-cell concentrations from the CELL-DYN 4000
System to corresponding concentrations from the FACScanTM flow
cytometer is expressed by the correlation coefficient (r).
Table 14.8: Correlation
Specimen Range
Parameter r
SI USA
CD3T 0.025-3.5 x 109 cells/L 0.025-3.5 x 103 cells/µL ≥ 0.95
9140350B—December 2002
Section 14 Specifications
Carryover
Carryover was tested as follows:
1. A CBC+CD3/4/8, WBC Extended Count test was run on a fresh
specimen without Suspect Parameter or Suspect Population flags.
2. A T-Cell Background Count was run. (For instructions on running
T-Cell Background Count, refer to Subsection: Running T-Cell
Background Count within this section.)
3. For each of the T-Cell parameters (CD3T, CD4T, CD8T), carryover
was computed as the ratio of the background count obtained in
Step 2 (T-Cell Background Count) to the corresponding T-Cell
count obtained from the specimen in Step 1, multiplied by 100.
T-Cell Background Count

Carryover = × 100
T-Cell Count
Carryover specification for CD3T, CD4T, and CD8T: ≤1%
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Specifications

Tube Dimensions
13 x 75 mm tube, with cap
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Stability Period

Table 14.9: CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Stability Period
Condition Stability Period
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Within expiration date provided on the pouch label
Pouch Stability (unopened)
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent 60 days at room temperature

Pouch Stability (opened, then resealed)
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent tube 8 hours at room temperature

Stability (after removal from pouch)
9140350A—October 2001
Specifications Section 14
NOTES
9140350A—October 2001
Section 14 Operating Instructions
Operating Instructions
Preparing to Run the T-Cell Assay

CAUTION: Before the T-Cell Assay is run, special hardware must be
installed on your CELL-DYN 4000 System. If the T-Cell Assay is run
without the special hardware, data from the assay must not be reported.
CAUTION: Once blood and Diluent/Sheath Reagent are added to the

CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes, the dilutions are
usable for one test only; the used reagent tubes must then be discarded.
For this reason, be sure to check for used reagent tubes whenever racks
are removed from and later replaced in the Autoloader.
NOTE: If you are planning to run additional racks in the Autoloader

following the current rack, do not place tubes for a mAb test in the current
rack beginning with the specimen tube in the second tube position.
NOTE: Before running the T-Cell Assay on a specimen, it is suggested

that a CBC, WBC Extended Count test be run. If the WBC result is
flagged as suspect and invalidated, as indicated by an asterisk [*] or, in
some cases, a blank [ ], do not run the T-Cell Assay on the specimen. If
the LYM result is flagged as invalid, but the WBC result is not, the
percentage T-Cell results will be invalidated, but the absolute T-Cell
results will be reportable.
NOTE: To open a new pouch of CELL-DYN Immuno T-Cell (CD3/4/8)

Reagent, cut the pouch above the resealing seam. When removing tubes
from the pouch, make sure the indicator granules in the desiccant
packages are blue. If the indicator granules have turned lavender, this
indicates exposure to excessive moisture; do not use tubes from the
pouch. Alteration in the appearance of the reagent pellet, such as a change
in color (pale pink to red), or shrinking of the pellet (so that it can pass
through the stainless steel retainer when the tube is inverted), indicates
instability and deterioration. In such cases, the reagent tube should be
discarded.
The T-Cell Assay can be run in Autoloader mode only. As shown in the
following figure, the specimen tube is placed first, followed by the
CELL-DYN CD3/4 T-Cell Reagent tube and then the CELL-DYN
CD3/8 T-Cell Reagent tube. This series of tubes must be in the same rack.
9140350A—October 2001
Operating Instructions Section 14
Specimen Tube
CELL-DYN CD3/4 T-Cell Reagent Tube
Specimen Tube CELL-DYN CD3/4 T-Cell
Reagent Tube
Reagent Tube
Figure 14.3: Placement of tubes for the T-Cell Assay
Running T-Cell Background Count

A T-Cell Background Count should be run whenever the lot changes for
the CELL-DYN Immuno T-Cell (CD3/4/8) Reagents, Diluent/Sheath
Reagent, WBC Reagent — Part A, or WBC Reagent — Part B. The T-Cell
Background Count is the T-Cell background effect attributable to both the
instrument and the CELL-DYN Immuno T-Cell (CD3/4/8) Reagents.
The specification for the T-Cell Background Count is as follows:
Table 14.10: T-Cell Background Counts
Parameter SI USA
CD3T ≤0.02 x 109/L ≤0.02 x 103/µL
CD4T ≤0.02 x 109/L ≤0.02 x 103/µL
CD8T ≤0.02 x 109/L ≤0.02 x 103/µL
9140350A—October 2001
Preparing to Run T-Cell Background Count

Before running a T-Cell Background Count, perform the following steps:
1. Run a CBC Background Count and verify that the System meets
background specifications. For information on specifications for
background counts, refer to the CELL-DYN 4000 System
Operator’s Manual, Section 4: Performance Characteristics and
Specifications, Subsection: Background Counts.
2. Make sure the System is functioning properly by running
hematology controls. For information on running hematology
controls, refer to the CELL-DYN 4000 System Operator’s Manual,
Section 11: Quality Control, Subsection: Procedures for
Monitoring with Controls.
3. For each T-Cell Background Count, obtain an empty blood
collection tube without anticoagulant, place the tube in the
Autoloader rack, and place behind it the CELL-DYN CD3/4 and
CD3/8 T-Cell Reagent tubes. See the following figure.
Empty Specimen Tube


Reagent Tube
Figure 14.4: Placement of Tubes for a T-Cell Assay Background Count
9140350A—October 2001
Performing T-Cell Background Count

Perform the T-Cell Background Count as follows:
1. Select the Work List button on the Control Panel.
2. In the WORK LIST window, select the Create Entry… button.
3. From the Specimen Type> menu in the WORK LIST ENTRY
window, select Background; then, select CD3/4/8 from the
Sub Type> menu.
4. Enter the rack and tube position number (e.g., 5901 for rack #59, 1st
tube position) for the empty tube in the Rack/Tube Number (RRTT)
field.
NOTE: Make sure there is no bar code label on the empty
specimen tube.
5. Select the Save Entry button.
6. Select the OK button after a message window is displayed saying to
ensure correct placement of tubes.
7. Select the Close button.
8. Select the Run Loader button.
Running Patient Specimens

Procedural Guidelines
• A negative and a positive control for the T-Cell Assay should be run
at least once during every 8 hours of operation when running the
T-Cell Assay, before reporting any T-Cell results. For information
on running controls, refer to Subsection: Quality Control within this
section.
• For information on collection tubes recommended for use on the
Autoloader, refer to the CELL-DYN 4000 System Operator’s
Manual, Section 7: Operational Precautions and Limitations,
Subsection: Specimen Preparation, Storage, and Handling.
• The number of replicates ordered using the Work List (set via the
WORK LIST SETUP or WORK LIST ENTRY window) must
always be one.
• The recommended procedure for running the T-Cell Assay on
patient specimens depends on the number of patient specimens
being run:
– To run the T-Cell Assay on multiple patient specimens, use the
WORK LIST SETUP window to set the Test Selection for
Work List Standard Processing Conditions to CBC+CD3/4/8,
WBC Extended Count.
9140350A—October 2001
– To run the T-Cell Assay on a small number of patient

specimens, use the WORK LIST ENTRY window to select
CBC+CD3/4/8, WBC Extended Count as the Test Selection.
Table 14.11: Running the T-Cell Assay
Task Steps
Run the T-Cell Assay on 1. From the Setup> menu, select Work List…
Multiple Patient 2. From the Specimen Type> menu in the WORK LIST SETUP window, select
Specimens Patient.
3. From the Test Selection> menu, select CBC+CD3/4/8, WBC Extended
Count.
4. Select the OK button.
5. Place specimen tubes and CELL-DYN CD3/4 and CD3/8 T-Cell Reagent
tubes in the racks, placing the reagent tubes in the next tube positions in the
rack directly behind each specimen tube in the correct order.
Run the T-Cell Assay on 1. Select the Work List button on the Control Panel.
a Small Number of 2. In the WORK LIST window, select the Create Entry… button.
Patient Specimens 3. From the Specimen Type> menu in the WORK LIST ENTRY window, select
Patient.
Count.
5. To identify the specimen tube by Specimen ID, select the Match Specimen ID
radio button; then, enter the Specimen ID.
6. To identify the specimen tube by rack and tube position number, select the
Match RRTT radio button; then, enter the rack and tube position numbers
(e.g., 5901 for rack #59, 1st tube position) for the specimen tube in the Rack/
Tube Number (RRTT) field.
NOTE: Make sure there is no bar code label on the specimen tube.
8. Select the OK button if a message window is displayed saying to ensure
correct placement of tubes.
9. For each additional specimen to be run using the T-Cell Assay, repeat the
previous steps beginning with step 2.
11. Place specimen tubes and CELL-DYN CD3/4 and CD3/8 T-Cell Reagent
tubes in the racks, placing the reagent tubes in the next tube positions in the
rack directly behind each specimen tube in the correct order.
9140350A—October 2001
Data Review of Single Records

Chartable Page
Numerical Results
When CBC+CD3/4/8, WBC Extended Count is the test selection, the test
selection (CBC+CD3/4/8,C) is indicated in the upper right corner of the
Chartable Page.
The following parameters are displayed:
• CD3T (This parameter is computed as the average of the
CD3+ concentrations of lymphocytes from the
CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes.)
• %CD3 (This parameter is computed as the average of the
CD3+ percentages of lymphocytes from the
• CD4T
• %CD4
• CD8T
• %CD8
• 4/8
9140350A—October 2001
Graphs
The following graphs display data obtained from the T-Cell Assay:
• CD4T Viability∆: FL3 vs. FL2 for CD3/4 tube
• CD8T Viability∆: FL3 vs. FL2 for CD3/8 tube
• CD3/4 FL1-FL2: Scatterplot of FL1 vs. FL2 for CD3/4 tube
• CD3/8 FL1-FL2: Scatterplot of FL1 vs. FL2 for CD3/8 tube
• CD3/4 0°-7°: Scatterplot of 0° vs. 7° for CD3/4 tube
• CD3/8 0°-7°: Scatterplot of 0° vs. 7° for CD3/8 tube
These graphs can be selected for any of the 16 parameter sets for display
and printing on the Chartable Page. (For information on how to set up
parameter sets, refer to the CELL-DYN 4000 System Operator’s
Manual, Section 2: Installation Procedures and Special Requirements,
Subsection: Customizing Parameter Sets.) In the Single Record View
graphs can also be selected for display (but not printing) using the icon at
the upper-left corner of each graph. To include a graph on a printed report
of the Chartable Page, a parameter set with the graph must be selected.
∆ This graph is not used to calculate the parameters v4†, v8†, or v4/8†. For information on the calculation of these parameters,
refer to Subsection: Parameters and Report Units within this section.
9140350A—October 2001
Figure 14.5: Chartable Page
9140350A—October 2001
Laboratory Worksheet Page
Numerical Results
When CBC+CD3/4/8, WBC Extended Count is the test selection, the test
selection (CBC+CD3/4/8,C) is indicated in the upper right corner of the
Laboratory Worksheet Page printed report.
The following parameters are displayed:
• CD3T (This parameter is computed as the average of the
CD3+ concentrations of lymphocytes from the CELL-DYN CD3/4
and CD3/8 T-Cell Reagent tubes.)
• %CD3 (This parameter is computed as the average of the
CD3+ percentages of lymphocytes from the
• CD4T
• %CD4
• CD8T
• %CD8
• 4/8
• v4†
• v8†
• v4/8†
† Clinical significance has not been established for this parameter. Therefore, it is not
reportable in the U.S.A. It is provided for laboratory use only.
Graphs
The T-cell-specific graphs can be selected for display and printing on the
Laboratory Worksheet Page via the setup of the Laboratory Worksheet
Page. When viewing the Laboratory Worksheet Page, graphs that are not
displayed can also be selected for display (but not printing) using the icon
at the upper-left corner of each graph. For information on selecting graphs
for display and printing on the Laboratory Worksheet Page, refer to the
CELL-DYN 4000 System Operator’s Manual, Section 2: Installation
Procedures and Special Requirements, Subsection: Customizing the
Laboratory Worksheet Page.
9140350A—October 2001
Figure 14.6: Laboratory Worksheet
Graphs Page
The Graphs Page allows the operator to view up to 10 graphs, including
those not displayed on the Laboratory Worksheet Page. If the graphs
specific to the T-Cell Assay are not displayed, they may be selected for
viewing.
Data Review of Multiple Records

The Log View allows viewing of multiple records containing numerical
results. For information on changing and selecting Data Sets, refer to the
CELL-DYN 4000 System Operator’s Manual, Section 2: Installation
Procedures and Special Requirements, Subsection: Customizing the
Data Log.
9140350A—October 2001
Section 14 Calibration Procedures
Calibration Procedures
The System does not require a separate calibration procedure to run the
T-Cell Assay. Calibration of the WBC parameter constitutes calibration
for the T-Cell Assay.
9140350A—October 2001
Calibration Procedures Section 14
NOTES
9140350A—October 2001
Section 14 Operational Precautions and Limitations
Operational Precautions and Limitations
Requirement for Special Hardware before Running the T-Cell Assay

Before the T-Cell Assay is run, special hardware must be installed on your
CELL-DYN 4000 System. If the T-Cell Assay is run without the special
hardware, data from the assay must not be reported.
Maximum Suggested Elapsed Time after Collection of Venous Whole

Blood
36 hours at room temperature (Results for the CD3T, CD4T, and CD8T
parameters within 10% of results
obtained on a fresh, normal specimen run
up to four hours after collection)
NOTE: This time and storage condition applies only to T-Cell Assay
results. For corresponding information for CBC results, refer to the
CELL-DYN 4000 System Operator’s Manual, Section 7: Operational
Precautions and Limitations. For corresponding information for CD61
Assay results, refer to the CELL-DYN 4000 System Operator’s Manual,
Section 13: CELL-DYN 4000 ImmunoPlt™ (CD61) Assay.
Reagent Storage and Handling

If the indicator granules in the desiccant packages have turned from blue
to lavender, this indicates exposure to excessive moisture; the tubes in the
pouch must not be used.
The CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes must not be
used if they are exposed to ambient light for more than 8 hours.
Once blood and Diluent/Sheath Reagent are added to the CELL-DYN
CD3/4 and CD3/8 T-Cell Reagent tubes, the dilutions are usable for one
test only; the used reagent tubes must then be discarded. For this reason,
be sure to check for used reagent tubes whenever racks are removed from
and later replaced in the Autoloader.
Collection Tube Handling

Specimen tubes run in Autoloader mode should not be punctured more
than five times. The T-Cell Assay involves three punctures of the
specimen tube, so the collection tube cap should be changed before
rerunning the T-Cell Assay on a specimen.
9140350A—October 2001
Operational Precautions and Limitations Section 14
Interfering Substances and Conditions

The following interfering substances and conditions can affect the T-Cell
results:
• Lyse-resistant RBCs
• PLT clumps
• Human anti-mouse antibodies
• Lymphocyte auto-fluorescence
• NRBCs
• Immunosuppressive therapy (e.g., OKT3, ATGAM)
• Blast cells
• Leukemia∆
NOTE: Any substance or condition that can interfere with the System’s
principles of operation should be considered an interfering substance or
condition. For example, an infused fluorescent dye that fluoresces at a
wavelength used by the System can affect the associated parameter(s).
∆ The T-Cell Assay is not intended for screening samples for the presence of leukemic cells or for use in phenotyping samples
from leukemic patients.
9140350A—October 2001
Section 14 Troubleshooting and Diagnostics
Troubleshooting and Diagnostics
Observable Problems
For information concerning Autoloader-related problems and other
observable problems, refer to the CELL-DYN 4000 System Operator’s
Manual, Section 10: Troubleshooting and Diagnostics, Subsection:
Troubleshooting Observable Problems.
System Initiated Messages (SIMs)

NOTE: When the T-Cell Background Count limits are exceeded, [SIM
0099: Background Count Limit Exceeded] is displayed. This SIM is
documented in the CELL-DYN 4000 System Operator’s Manual,
Section 10: Troubleshooting and Diagnostics, Subsection: Responding
to System Initiated Messages.
9140350A—October 2001
Troubleshooting and Diagnostics Section 14
0260 Missing Reagent Tube, Assay Not Performed
NOTE: After reading the corrective actions, select the OK or Save button
to remove the Message window.
NOTE: There is no halt; the Autoloader advances to the next specimen
tube position. The System processes the specimen tube as a CBC, WBC
Extended Count, and displays 0.00* or a blank [ ] for T-Cell numerical
results.
NOTE: Because the specimen tube was aspirated, but the CELL-DYN
CD3/4 and CD3/8 T-Cell Reagent tubes were not, the System detects a
count mismatch, triggering the [CD3/4/8 Count Rate/Mismatch Violation]
Suspect Parameter Flag.
Probable Cause(s) Corrective Action(s)
CELL-DYN CD3/4 or CD3/8 Place the reagent tubes in the next tube positions in
T-Cell Reagent tube was not placed the rack, directly after the specimen tube.
in the correct tube position in the
rack following the specimen tube.
The Autoloader Mixing Assembly Clean the Autoloader Mixing Assembly (as directed
mishandled one or both of the in the CELL-DYN 4000 System Operator’s Manual,
CELL-DYN CD3/4 or CD3/8 T-Cell Section 9: Service and Maintenance).
Reagent tubes.
The Autoloader rack did not move Clean the Autoloader racks and tray (as directed in
correctly. the CELL-DYN 4000 System Operator’s Manual,
Section 9: Service and Maintenance).
The bar code label on one or both of Rerun the test using one or both new reagent tubes.
the CELL-DYN CD3/4 or CD3/8
T-Cell Reagent tubes is scuffed, dirty,
or missing and was not read
successfully.
9140350A—October 2001
0261 Unexpected Reagent Tube, Assay Not Performed
NOTE: There is no halt; the Autoloader advances to the next tube
position.
A mAb reagent tube was placed at Remove the mAb reagent tube from the rack or order
this tube position, but a multi-tube a multi-tube test.
test was not ordered.
0262 Incorrect Reagent Tube, Assay Not Performed
NOTE: This message applies to reagent tubes for immunology-based tests

for the CELL-DYN 4000 System other than the T-Cell Assay (e.g., the
CELL-DYN 4000 ImmunoPltTM (CD61) Assay).
NOTE: There is no halt; the Autoloader advances to the next specimen
tube position. The System processes the specimen tube as a CBC, WBC
Extended Count and displays 0.00* or a blank [ ] for T-Cell numerical
results.
NOTE: Because the specimen tube was aspirated, but the mAb reagent
tube was not, the System detects a count mismatch, triggering the
[CD3/4/8 Count Rate/Mismatch Violation] Suspect Parameter Flag.
An incorrect mAb reagent tube was Place a correct mAb reagent tube at this tube position
placed at this tube position. and rerun the requested test for this specimen.
9140350A—October 2001
0642 Insufficient Reagent to Complete Multi-tube Assay
NOTE: After reading the corrective actions, select the Clear or Save
button to remove the Message window.
NOTE: This SIM halts sample processing in Autoloader mode. The
System processes the specimen tube as a CBC, WBC Extended Count and
displays 0.00* or a blank [ ] for T-Cell numerical results.
NOTE: Because the specimen tube was aspirated, but the CELL-DYN
CD3/4 or CD3/8 T-Cell Reagent tube was not, the System detects a count
mismatch, triggering the [CD3/4/8 Count Rate/Mismatch Violation]
Suspect Parameter Flag.
The Diluent/Sheath and/or WBC Replace the appropriate reagent container(s) (as
Reagent — Part A and/or — Part B is directed in the CELL-DYN 4000 System Operator’s
low in the cubitainer. Manual, Section 9: Service and Maintenance).
Select the Clear Fault button.
NOTE: Run a background count in addition to the
background count already run and ensure that the
results are within specifications before running
specimens.
The tubing from the Diluent/Sheath Uncrimp or reconnect the tubing from the
and/or WBC Reagent — Part A and/ appropriate reagent container(s) to the Analyzer.
or — Part B container to Analyzer Select the Clear Fault button.
has been crimped or has been
disconnected.
The tubing from the Diluent/Sheath Contact your Hematology Customer Support Center.
and/or WBC Reagent — Part A and/ NOTE: The Analyzer will remain in Not Ready
or — Part B container to the status until repairs are completed.
Analyzer is damaged.
9140350A—October 2001
0642 Insufficient Reagent to Complete Multi-tube Assay (Continued)
A pinch valve is sticking. Select the Save button to remove the message.
Review the diagrams in the CELL-DYN 4000
System Operator’s Manual, Section 10:
Troubleshooting and Diagnostics, Subsection:
Troubleshooting Data-Related Problems that
indicate the pinch valves that control the flow of
reagent.
Clean the indicated pinch valves (as directed in the
CELL-DYN 4000 System Operator’s Manual,
Section 9: Service and Maintenance).
Clear the fault in the MESSAGES window (as
directed in the CELL-DYN 4000 System Operator’s
Manual, Section 5: Operating Instructions).
Perform your laboratory’s QC procedures.
The vacuum is too low or is See corrective actions given in SIM 0830.
obstructed. (If this is the case, [SIM Check the reservoir’s vacuum supply tubing and
0830: 15 in. Hg Vacuum Too associated pinch valves.
Low] would also be displayed.)
Select the Clear Fault button.
An electronic and/or mechanical Contact your Hematology Customer Support Center.
failure has occurred. NOTE: The Analyzer will remain in Not Ready
status until repairs are completed.
9140350A—October 2001
Data-Related Problems
T-Cell Background Count Outside Specification
For information on running a T-Cell Background Count, refer to Subsection:

Running T-Cell Background Count within this section.
The Analyzer flow system is dirty. Run CBC Backgrounds Counts followed, as needed,
by Autoclean.
The Diluent/Sheath Reagent, Install a new cubitainer of Diluent/Sheath Reagent.
WBC Reagent — Part A, or WBC
Reagent — Part B is contaminated.
One or both of the CELL-DYN Rerun the specimen with one or more new reagent
Immuno T-Cell (CD3/4/8) Reagents tubes.
is contaminated. If the problem occurs repeatedly, contact your
Hematology Customer Support Center.
Imprecise T-Cell Results
The instrument requires Confirm that routine maintenance is up to date.

maintenance.
9140350A—October 2001
Section 14 Quality Control
Quality Control
Patient controls for the T-Cell Assay (specimens from an adult with
hemogram and WBC differential values in the normal range, without
Suspect Parameter or Suspect Population flags) should be run at least
once during every 8 hours of operation when running this assay, before
reporting any T-Cell results. Patient controls for the T-Cell Assay should
also be run whenever the lot changes for the CELL-DYN Immuno T-Cell
(CD3/4/8) Reagents, Diluent/Sheath Reagent, or WBC Reagent — Part A
or — Part B.
CAUTION: Do not use commercial control products for quality control
of the T-Cell Assay.
Patient control specimens are run as both negative and positive controls.
A negative control is run by placing the patient control in the Autoloader
and placing behind it two new blood collection tubes without
anticoagulant, labeled with appropriate CELL-DYN Immuno T-Cell
(CD3/4/8) Reagent bar code labels. A positive control is run by placing
the patient control in the Autoloader and placing behind it, in proper order,
CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes.
The purpose of the negative control is to confirm that there is no
significant contribution to the T-Cell counts from the instrument.
The purpose of the positive control is to confirm that when cells with a
given marker are present, the System, including instrument and reagents,
is capable of properly identifying them.2
Parameters Used in Quality Control

NOTE: Before running T-Cell Assay Controls, make sure the System is
functioning properly by running hematology controls. For information on
running hematology controls, refer to the CELL-DYN 4000 System
Operator’s Manual, Section 11: Quality Control, Subsection:
Procedures for Monitoring with Controls.
For the negative control, CD3T, CD4T, and CD8T must be within the
following limits:
• CD3T: 0-10 cells/µL
9140350A—October 2001
Quality Control Section 14
For the positive control, CD3T, CD4T, and CD8T must be within limits to
be determined by the laboratory. The following ranges3 may be
appropriate for use as limits for the positive control:
In addition, for the positive control, the mean channel parameters for the
fluorescence signals for certain cell populations must be within preset
limits, as follows:
NOTE: All four parameters must be within preset limits.
• 4mn1† – This parameter is the mean channel of the CD3+ cells in the
CELL-DYN CD3/4 T-Cell Reagent tube detected by the FL1
subsystem. The control limits for this parameter are 175-205.
• 8mn1† – This parameter is the mean channel of the CD3+ cells in the
CELL-DYN CD3/8 T-Cell Reagent tube detected by the FL1
• 4mn2† – This parameter is the mean channel of the CD3+CD4+ cells
in the CELL-DYN CD3/4 T-Cell Reagent tube detected by the FL2
• 8mn2† – This parameter is the mean channel of the CD3+CD8+ cells
in the CELL-DYN CD3/8 T-Cell Reagent tube detected by the FL2
NOTE: These parameters are available for display in QC Files only.
For information on entering limits for control results, refer to the

CELL-DYN 4000 System Operator’s Manual, Section 11: Quality
Control, Subsection: Entering Limit Sets in a QC File.
Running Whole Blood Controls for the T-Cell Assay

Procedural Guidelines
Controls are run as QC specimen type, directly into a QC File.
NOTE: If T-Cell results are marked with a Suspect Parameter Flag [*]
following a negative control run, investigate the cause of the flag by
checking the INVALID DATA window on the Laboratory Worksheet
Page. If the only message listed in the window is [Too Many Events
Outside Limits During CD3/4/8 Assay], disregard this Suspect
Parameter Flag and accept the control results if results are within preset
limits.
9140350A—October 2001
There are two possible ways to run controls for the T-Cell Assay,
depending on how the control tube is identified.
• If the control tube is identified with a Q Label showing the QC File
number, then the test can be selected using the QC FILE SETUP
window.
• If the control tube is not identified with a Q Label, then it can be
identified either by the Specimen ID or by rack and tube position
numbers in the Work List and the test can be selected using the
WORK LIST ENTRY window.
Table 14.12: Running Whole Blood Control for the T-Cell Assay if Control Tube Is Identified with a Q Label
Task Steps
Set Up a QC File 1. Select the QC> button on the Control Panel.

2. Select the desired QC File from the displayed menu.
NOTE: QC Files with CBC+CD3/4/8, WBC Extended Count as the
default test selection (entered using the QC FILE SETUP window) will
not be accessible from the NEXT OPEN TUBE SETUP window (as
indicated by parentheses).
3. From the QC FILE window, select the Setup… button.
4. Because you will be running whole blood controls as patient controls, check
that the For Patient Control radio button in the QC FILE SETUP window is
selected and, if it is not, select it.
Count.
NOTE: If this file was last used in Open Tube mode, then the Test
Selection menu item for the T-Cell Assay will be shown as unavailable
by parentheses. In this case, take the following steps:
a. Select the Run Open Tube… button to open the NEXT OPEN TUBE
SETUP window.
b. Change the Specimen Type to Patient.
c. Select the Close button to close the NEXT OPEN TUBE SETUP window.
d. To verify that all test selections are now available in the QC FILE SETUP
window for the desired QC File, select the Test Selection> menu. All menu
items should now be available.
6. Select the OK button in the QC FILE SETUP window.
Run Controls 1. Label the negative and positive controls with Q labels to match the appropriate
QC Files.
2. For the negative control, label two empty specimen tubes with the appropriate
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent bar code labels provided in the
T-Cell Assay kit.
3. Place the negative control in the rack, and place directly behind it, in the same
rack, and in the correct order, the two empty specimen tubes labeled with the
appropriate CELL-DYN Immuno T-Cell (CD3/4/8) Reagent bar code labels.
4. Place the positive control in the rack, and place directly behind it in the same
rack the CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes in the correct
order.
9140350A—October 2001
Table 14.13: Running Whole Blood Control for the T-Cell Assay if Control Tube Is Not Identified with a Q Label
Task Steps
Set Up a QC File Refer to the previous table.
Create a Work List Entry 1. Select the Work List button on the Control Panel.
2. In the WORK LIST window, select the Create Entry… button.
3. From the Specimen Type> menu in the WORK LIST ENTRY window, select
QC.
4. From the Sub Type> menu, select the desired QC File.
Count.
6. To identify the patient control specimen tube by Specimen ID, select the Match
Specimen ID radio button. Ensure that the referenced QC File has a Specimen
ID associated with it. If the referenced QC File does not have a Specimen ID
associated with it:
a. Open the QC File.
b. Set it up as a patient control.
c. Specify the Replicate ID to match the Specimen ID.
7. To identify the patient control specimen tube by rack and tube position number,
select the Match RRTT radio button and enter the rack and tube position
numbers (e.g., 5901 for rack #59, 1st tube position) for the patient control
specimen tube.
NOTE: Ensure that there is no bar code label on the specimen tube.
9. Select the OK button after a message window is displayed saying to ensure
correct placement of tubes.
Run Controls 1. For the negative control, label two empty specimen tubes with the appropriate
CELL-DYN Immuno T-Cell (CD3/4/8) Reagent bar code labels provided in the
T-Cell Assay kit.
2. Place the negative control in the rack, and place directly behind it, in the same
rack, and in the correct order, the two empty specimen tubes labeled with the
appropriate CELL-DYN Immuno T-Cell (CD3/4/8) Reagent bar code labels.
3. Place the positive control in the rack, and place directly behind it in the same
rack the CELL-DYN CD3/4 and CD3/8 T-Cell Reagent tubes in the correct order.
Reviewing Control Results Using Levey-Jennings Plots

The T-Cell parameters and fluorescence mean channel parameters† can be
reviewed in historical context using Levey-Jennings plots. For
information on Levey-Jennings plots, refer to the CELL-DYN 4000
System Operator’s Manual, Section 11: Quality Control, Subsection:
Levey-Jennings Graphs.
† Clinical significance has not been established for these parameters. Therefore, they are not reportable in the U.S.A.
They are provided for laboratory use only.
9140350A—October 2001
Troubleshooting Control Results

If the results for the negative or positive control are outside preset limits,
these failures must be investigated.
Troubleshooting a Negative Control Failure

To troubleshoot a failure of the negative control:
1. Run a different blood specimen as the negative control.
2. If the negative control fails again, run a CBC Background Count and
verify that the System meets background specifications. For
information on specifications for background counts, refer to the
CELL-DYN 4000 System Operator’s Manual, Section 4:
Performance Characteristics and Specifications, Subsection:
Background Counts.
If the results for WBC Background Count are outside specifications,
troubleshoot for optical background counts outside specifications.
For the procedure on troubleshooting optical background counts
outside specifications, refer to the CELL-DYN 4000 System
Operator’s Manual, Section 10: Troubleshooting and Diagnostics,
Subsection: Troubleshooting Optical Data-Related Problems.
3. If the results for WBC Background Count are within specifications,

run a T-Cell Background Count.
For the procedure on running a T-Cell Background Count, refer to
Subsection: Running T-Cell Background Count within this
section.
For specifications for T-Cell Background Counts, refer to
Subsection: Performance Specifications within this section.
If the results for T-Cell Background Count are outside
specifications, troubleshoot for this problem. For the procedure,
refer to Subsection: Data-Related Problems within this section.
4. If problems persist, call your Hematology Customer Support
Center.
Troubleshooting a Positive Control Failure

To troubleshoot a failure of the positive control:
1. Run a different blood specimen as the positive control.
2. If the positive control fails again, run the control with new
CELL-DYN CD3/4 and/or CD3/8 T-Cell Reagent tubes.
3. If problems persist, call your Hematology Customer Support
Center.
9140350A—October 2001
On-Line Monitoring
NOTE: Data from the T-Cell Assay are not used in Moving Averages or
Delta Check Analysis.
9140350A—October 2001
Section 14 Parts and Accessories
Parts and Accessories
CELL-DYN Immuno T-Cell (CD3/4/8) Reagents are available as an

optional accessory. The orderable item (List Number 07H22-01) is a box
containing two pouches, one pouch containing 20 CELL-DYN CD3/4
T-Cell Reagent tubes and the other pouch containing 20 CELL-DYN
CD3/8 T-Cell Reagent tubes, and extra CELL-DYN Immuno T-Cell
(CD3/4/8) Reagent bar code labels.
9140350A—October 2001
Parts and Accessories Section 14
NOTES
9140350A—October 2001
Section 14 Bar Codes
Bar Codes
The System identifies the CELL-DYN CD3/4 and CD3/8 T-Cell Reagent
tubes using a Code 128 bar code label.
9140350A—October 2001
Bar Codes Section 14
NOTES
9140350A—October 2001
Section 14 Trademark
Trademark
FACScanTM is a trademark of Becton Dickinson and Company.
9140350A—October 2001
Trademark Section 14
NOTES
9140350A—October 2001
Section 14 References
References
1. Reichert T, DeBruyere M, Deneys V, Totterman T, Lydyard P,

Yuksel F, Chapel H, Jewell D, Van Hove L, Linden J, Buchner L.
Lymphocyte Subset Reference Range in Adult Caucasians. Clinical
Immunology and Immunopathology. 1991; 60: 190-208.
2. National Committee for Clinical Laboratory Standards. Clinical
Applications of Flow Cytometry: Quality Assurance and
Immunophenotyping of Lymphocytes; Approved Guidelines.
NCCLS Document H42-A (ISBN 1-56238-364-7) NCCLS, 940
West Valley Road, Suite 1400, Wayne, PA 19087-1898, 1998.
3. Rose NR, Friedman H, Fahey JL. Manual of Clinical Laboratory
Immunology - Third Edition, 1986.
9140350A—October 2001
References Section 14
NOTES
9140350A—October 2001

Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay: 9140350A-October 2001

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay: 9140350A-October 2001

Uploaded by

Copyright:

Available Formats

Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay

Section 14 CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay

The CELL-DYN 4000 Immuno T-Cell (CD3/4/8) Assay is an

CELL-DYN Immuno T-Cell (CD3/4/8) Reagents

Figure 14.1: CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Tubes

2 Pouches per Box

Desiccant 20 CELL-DYN CD3/4 T-Cell Desiccant 20 CELL-DYN CD3/8 T-Cell

CAUTION: If you are planning to run additional racks in the Autoloader

Specimen processing involves the following steps:

Installation Procedures and Special Requirements

Installation of Hardware for the T-Cell Assay

Installation of Software for the T-Cell Assay

System Customization for the T-Cell Assay

Selection Action Reference

Laboratory To display T-Cell parameters on the Laboratory CELL-DYN 4000 System

Selection Action Reference

• Anti-CD8 antibody reacts with the CD8 antigen, which is expressed

Parameters and Report Units

Parameter Description Units (USA)

CD3T Concentration of T-cells (CD3+ lymphocytes); computed as the average 10e3/µL

%CD3 Percentage of lymphocytes that are T-cells (CD3+ lymphocytes); %

CD4T Concentration of T-helper/inducer cells (CD3+CD4+ lymphocytes) 10e3/µL

%CD4 Percentage of lymphocytes that are T-helper/inducer cells %

CD8T Concentration of T-suppressor/cytotoxic cells (CD3+CD8+ lymphocytes) 10e3/µL

%CD8 Percentage of lymphocytes that are T-suppressor/cytotoxic cells %

4/8 Ratio of T-helper/inducer cell (CD3+CD4+ lymphocyte) concentration to

v4† Concentration of T-helper/inducer cells (CD3+CD4+ lymphocytes) that are 10e3/µL

v8† Concentration of T-suppressor/cytotoxic cells (CD3+CD8+ lymphocytes) 10e3/µL

v4/8† Ratio of v4†, viable T-helper/inducer cell (viable CD3+CD4+ lymphocyte)

For more information on Suspect Parameter flagging, refer to the

Table 14.3: System Faults

Numerical Results Marked with [*]

• There is an anomaly in the

NOTE: Under the following additional System Fault conditions, the

• [Residual Fluid Detected in WBC Dilution Cup]

• [WBC Dilution Cup Temperature Out of Range]

• [WBC Dilution Cup Motor Voltage Out of Range]

• [Hemoglobin Reagent Syringe Failed to Home]

• [Diluent/Sheath Reagent Syringe Failed to Home]

• [WBC Reagent Syringes Failed to Home]

Table 14.4: Data Faults

Numerical Results Marked with [*] or

NOTE: An asterisk may appear on

NOTE: Under the following additional Data Fault conditions, all

• [Known Interference with WBC Results, likely due to PLT

Volume and Throughput Specifications

CD3T ≤0.02 x 109 cells/L ≤0.02 x 103 cells/µL

CD4T ≤0.02 x 109 cells/L ≤0.02 x 103 cells/µL

CD8T ≤0.02 x 109 cells/L ≤0.02 x 103 cells/µL

(For information on running background counts for the T-Cell Assay,

Table 14.6: Precision

CD3T 0.1-3.5 x 109 cells/L 0.1-3.5 x 103 cells/µL CV ≤4.0%

CD4T 0.1-2.0 x 109 cells/L 0.1-2.0 x 103 cells/µL CV ≤5.0%

CD8T 0.1-2.0 x 109 cells/L 0.1-2.0 x 103 cells/µL CV ≤6.0%

♦ The number to be used is the greater of the two at a given concentration.

CD3T 0.025-3.5 x 109 cells/L 0.025-3.5 x 103 cells/µL ≥ 0.95

CD4T 0.025-2.0 x 109 cells/L 0.025-2.0 x 103 cells/µL ≥ 0.95

CD8T 0.025-2.0 x 109 cells/L 0.025-2.0 x 103 cells/µL ≥ 0.90

T-Cell Background Count

CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Specifications

CELL-DYN Immuno T-Cell (CD3/4/8) Reagent Stability Period

Condition Stability Period

CELL-DYN Immuno T-Cell (CD3/4/8) Reagent 60 days at room temperature