MG-12087A Flow Cytometric Morphology Monograph.

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Advancements in Technology:
Flow Cytometric Morphology –
The Analytical Module. Chemistry
UniCel DxH 800 Coulter Cellular Analysis System Lab Automation
Information Systems
Molecular Diagnostics
Immunodiagnostics
Centrifugation
Disease Management
Hematology
Hemostasis
Flow Cytometry
Primary Care

MG-12087A
MG-12086A
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Flow Cytometric Morphology – Analytical Module

In Hematology, cellular analysis and the detection of cellular abnormalities
provides critical information to the physician. The UniCel DxH 800 Cellular
Analysis System has implemented the technology of Flow Cytometric
Morphology, providing the highest degree of resolution and sensitivity for
the science of cellular analysis. Crucial to Flow Cytometric Morphology is
sample preparation. The cytochemical reactions for flow cytometric analysis
are controlled by technological advancements within the UniCel DxH 800,
ensuring the accuracy and precision of each sample prepared.

The VCSn Module is the host analytical module within the UniCel DxH 800 that
functions to control sample preparation and delivery of prepared sample to the
flow cell for cellular analysis of the WBC differential, reticulocytes and NRBC.
The VCSn Module includes the following submodules: the Air Mix and
Temperature Control (AMTC), the Multi-transducer Module (MTM) and the
following assemblies: the VCSn panel, Flow-Cell Waste Assembly and
Distribution Valve Assembly.

Unique to the VCSn Module are the following patent-pending features:

• Temperature control for:

– All reagents used for sample preparation and delivery
– Reaction area, which includes the mix chambers
– Optical components and electronics
– Sample and sheath fluids
– Mixing chambers and focused air jet for sample mixing
• Flow cell design that supports additional light scatter measurements
• Distribution valve
• Solid state diluter design including solenoid valves and manifolds (no pinch
valves)
• Additional light scatter parameters for enhanced data acquisition for the WBC
differential, reticulocyte and NRBC analyses

The VCSn Module The VCSn Module prepares the WBC diff, reticulocyte and NRBC samples
Overview for flow cytometric analysis, generates the signals for all VCSn measurements
and handles the waste generated by the VCSn analyses. The integration of the
electronics, temperature control and hardware functionality for NRBC, WBC diff
and reticulocyte processes also resides within the VCSn Module. This includes
the reagent pumps, sample and sheath tanks, pressure regulators, sensors,
solenoid valves, sample distribution valve, flow cell waste chamber, the VCS
waste chamber, and the respective solenoid valves and manifolds.

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Sample Flow The whole blood sample used by the VCSn Module is delivered by the Sample
Aspiration Module. The Sample Aspiration probe delivers whole blood samples
to the temperature-controlled NRBC and WBC diff mix and Retic stain
chambers in the AMTC. The whole blood sample is delivered into the mix
chambers below the level of the reagents to ensure the blood sample is
combined with temperature controlled reagents (Figure 1).

Figure 1. Sample Delivery

Complete mixing of sample and reagents is accomplished using a temperature-

controlled focused jet of air regulated to four psi. (Figure 2)

Figure 2. Jet Air Mixing

Once sample preparation is complete, the prepared samples are directed via
the distribution valve from the mix chambers in the AMTC submodule to the
sample line. The sample line is then pressurized, sending the sample to the flow
cell for comprehensive flow cytometric cellular analysis.

A detailed description of the VCSn Module and the submodules is described on

the following pages. Figures 3 through 5 provide the dilution and analysis
details for all preparations controlled by the VCSn Module.

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~582 μL WB/Elyse +
210 – 250 μL Stabilyse
Start Acquisition
1:23 dilution
Stabilyse temp at ~24°C
~34 μL WB +
~548 μL Erythrolyse
1:16 dilution
Ar Mix and Elyse temp ~24°C

Figure 3. WBC Diff Cycle Specifications

~34 μL WB + ~200 μL 4 μL of aspirated WB/Stain solution + 2000 μL Retic Lyse

~35 μL WB/Stain Start Acquisition
Stain 1:8 dilution aspirated 1:500 dilution

~34 μL WB + ~200 μL 4 μL of aspirated WB/Stain solution + 2000 μL Retic Lyse

Stain 1:8 dilution ~35 μL WB/Stain aspirated 1:500 dilution Start Acquisition

Figure 4. Retic Cycle Specifications

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422 μL WB/diluent + – 155 μL Cell Lyse

1:25 dilution Start Acquisition
Reaction @ ~24°C

22 μL WB + – 400 μL Diluent
1:18 dilution
Ar Mix temp ~24°C
Blood in diluent for 0.96 – 1.04 sec

Figure 5. NRBC Cycle Specifications

Note: Reaction temperatures are system controlled depending on laboratory

temperature.

The VCSn Module hosts the VCSn panel, the flow cell waste assembly, the
Submodules and distribution valve, the air mix temperature control module (AMTC) and the
Assemblies Multi-transducer Module (MTM). These submodules and assemblies are
described below.

• The VCSn panel provides vacuum,

pressure and electrical support and
the distribution of these functions
throughout the VCSn module. The
panel includes reagent pumps
exclusively designed and
manufactured for Beckman Coulter,
solenoid valves, sheath and sample
pressure regulators, pressure sensors
and chambers, and electronic circuitry
and manifolds (Figure 6).

Figure 6. VCSn Panel

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• The flow-cell waste assembly collects

and removes the waste from the AMTC
module, the flow-cell waste chamber
and the distribution valve and directs it
to the VCS waste chamber for
disposal. The VCS waste chamber
is a new design that includes level
sensing to prevent overflow (Figure 7).

• The distribution valve assembly is a

two-piece ceramic valve that precisely
rotates in a clockwise rotation at 40°
intervals. The valve assembly includes Figure 7. VCS Waste Assembly
an optical sensor and a nine-
position code wheel that verifies
the position of the valve with
each 40° interval. This design
improves the accuracy of port
alignment. The distribution valve
directs the prepared sample to
the sample line and directs
cleaner to the mix chambers and
finally to waste (Figure 8).

Figure 8. Distribution Valve

Hardware Modules • The Air Mix Temperature
Control (AMTC) Module
receives and controls the sample preparation reactions for the WBC diff,
NRBC and reticulocytes. The reagents, NRBC, Diff, Retic and Stain Mix
chambers and the air for mixing are all temperature controlled for consistent
results. The AMTC includes one Peltier Module, one heater and three
sensors. The function of this module is to store heat or cold energy from the
thermoelectric device (Peltier), transfer heat or cold to the reagents and mix
chambers for a temperature controlled sample preparation environment
(Figure 9).

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Stain
Chamber
Retic
Chamber

Diff Chamber
Heat Exchange Block
with Insulation
NRBC
Peltier Heater Chamber
Assembly

Figure 9. Air Mix Temperature Control Module

• The major components and functions within the AMTC include:

– The heat exchange block is the core of the temperature control system.
The Peltier provides the heat/cold energy within the heat exchange block
using sensors mounted on the block that monitor the heat exchange block
and the ambient temperatures. This information is provided to the control
board that signals the Peltier to heat or cool.
– Air mixing is accomplished by using a precisely positioned air jet tube that
generates three-dimensional flow patterns within the mix chambers. The
temperature-controlled, three-dimensional flow ensures a homogenous
blood and reagent mix within the chambers. The air mix assembly has only
one moving part, a design that dramatically enhances reliability, reduces
vibration and greatly improves reaction control within the mix chambers.
There are no mixing motors.
– The Mix chambers are specially designed chambers that facilitate the
receipt and thorough mixing of a precise volume of delivered blood sample
and reagents for WBC diff, NRBC and reticulocyte analyses. The WBC diff,
NRBC and reticulocyte mix chambers are located in the heat exchange
block to provide a temperature-controlled environment for sample
preparation. The fourth mix chamber used for reticulocyte stain and blood
preparation is configured with an external heater and thermal control.

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– VCSn block Peltier receives input from the heat exchange block sensors
and directs the heating and cooling of the heat exchange block to maintain
a controlled sample preparation temperature.
• The Multi-transducer Module (Figure 10) functional components include:
– The MTM optical system includes a 5 mW diode red laser, beam shaping
optics and associated opto-mechanical components. The benefits of the
new design include:
• Custom-designed laser, proprietary to Beckman Coulter
• Ultra-low-noise laser operation for increased signal-to-noise ratio
• Enhanced laser power stability
• Newly designed opto-mechanical components to precisely position the
focused light within the sensing zone of the flow cell.
• Temperature-controlled hardware and sample analysis

Figure 10. Multi-transducer Module

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• The MTM Flow cell (Figure 11)

– The flow cell provides hydrodynamic focusing, placing the cells in the
sensing zone, one cell at a time.
– The design allows for the simultaneous measurement of Volume (DC),
Conductivity (RF), and five angles of Light Scatter (Sn) for each cell
analyzed.
– There are a total of 29 data measurements associated with each cell
analyzed.
• The MTM optical sensors measure Median Angle Light Scatter (MALS) and
Low Angle Light Scatter (LALs).
– MALS sensor detects Low Median Angle Light Scatter (LMALS), Upper
Median Angle Light Scatter (UMALS) and MALS.
– LAS sensor detects Axial Light Loss (ALL) and Low Angle Light Scatter
(LALS).

Figure 11. Multi-transducer Flow Cell

• Thermal control has been implemented to ensure that the MTM maintains a
stable temperature regardless of environmental variances. This ensures
mechanical and optical stability of the MTM, increasing the integrity of the
system alignment. The MTM Peltier controls the MTM hardware temperature at
approximately 24° C (75° F).

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• Sample and sheath diluent temperature control brings prepared sample

solutions and sheath fluid to a desired temperature range for consistency. The
MTM sample line Peltier controls the temperature of these solutions with the
ultimate goal to eliminate any thermal influences to the sheath / sample stream
that may affect sample results.
• The MTM covers provide individual protection for the optics, flow cell and
VCSn preamp. The dustproof covers prevent the analytical electronics from
electrical and magnetic interference as well as any environmental
disturbances. All optical covers include a safety interlock.
• The MTM electronics assembly has the responsibility to support the following
instrument functions:
– Provide stable DC and RF stimulus utilizing a newly designed temperature
compensation feedback circuitry
– Convert the response of the cell from the DC and RF stimulus to electronic
pulses that are captured by the advanced DxH 800 circuitry.
– Convert the response of the cell to the optical stimulus from the laser to
electronic pulses that are captured by the DxH 800 circuitry
– Monitor flow characteristics within the flow cell to detect electrical and/or
optical stability of the sample or sheath streams.

Conclusion Since the release of VCS technology in the 1980s, Coulter Corp. and Beckman
Coulter have utilized the most advanced technology for the analysis of the WBC
differential and reticulocytes. The UniCel DxH 800 expands the parameter
capabilities of VCS technology to include NRBC. Advanced hardware and
algorithm technology elevates the comprehensive analysis of the WBC
differential, reticulocytes and NRBC to a new level. The VCSn Module within the
UniCel DxH 800 ensures consistent sample results through uniform sample
preparation. Temperature control of the reagents and the cytochemical reactions
in the newly designed mixing chambers alleviates external environmental
variables for more reportable results. The design includes new system hardware
elements; unique flow cell design, the distribution valve and MTM opto-
mechanicals and electronics, which were engineered to provide the most
consistent, reliable and accurate sample measurements for use by the UniCel
DxH 800 advanced algorithms. The hardware innovations within the VCSn
Module, in conjunction with the advanced algorithm applications, provide
the foundation for the science of Flow Cytometric Morphology unique to the
UniCel DxH 800.

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