manufacturer :       Becton Dickinson

model :                   FACScan

annotation :           Dokumente engl.

FACScan is Becton Dickinson Immunocytometry Systems" benchtop flow cytometer designed for applications ranging from routine clinical to advanced research. lt analyzes cells as they pass one at a time through a focused laser beam. FACScan can measure up to five parameters—forward light scatter (FSC), side light scatter (SSC), and three tluorescence parameters (FL1, FL2, FL3). And FACScan can measure the pulse height, area, and width of each tluorescence parameter.

Because FACScan requires no optical alignment, daily setup can be perfolined quickly and consistently using FACSComp setup software.

Principle of Operation

FACScan analyzes cells as they travel in a moving fluid stream past a fixed laser beam. As a cell passes in front of the laser, several measurements are made based an the physical characteristics of the cell. These characteristics, which pertain to how the cell scatters the laser light and emits fluorescence, provide us with information about the cell" s size, intemal complexity, and relative fluorescence intensity. This information is collected and transmitted to the computer.

Intended Use

The FACScan flow cytometer is designed for a wide range of applications. FACScan is for in vitro diagnostic use for enumeration of leucocyte (non-blast) subsets or reticulocytes. In addition, FACScan can be used for the following research applications: DNA cell-cycle analysis, platelet studies, three-color immunofluorescence applications, chromosome classification, or intracellular ionized calcium measurements. For a brief description of some of the various applications, refer to Chapter 5, Applications.

Requirements

Your system includes CELLQuest software for acquisition and analysis and FACSComp software for daily setup and quality control. Other application­specific software programs are also available. The operating supplies are minimal and can be purchased separately through BDIS,

Hardware Requirements

A FACScan flow cytometer can be operated by any Macintosh computer system purchased through BDIS. Other platforms may also be supported for off-line data analysis; contact your Becton Dickinson Sales Representative for detailed information.

Software Requirements

• Macintosh system software, version 7.1 or later
• CELLQuest software, version 1.0 or later (for acquisition and analysis)
• FACSComp software, version 1.0 or later (for instrument setup)
• FACSConvert" software, version 1.0 or later (if CONSORT -generated data will be analyzed)

Supplies Required

• 12 x 75-mm polystyrene sample test tubes (Falcon®)
• Haema-Line® 2 sheath fluid (Serono-Baker Diagnostics)
• CaliBRITE" quality control beads (for use with FACSComp software)
• spare sheath and waste reservoirs (for cleaning procedures)
• chlorine bleach (for instrument cleaning)

Clorox and other brand-name bleaches are preferred because they are filtered to remove particles and are a known concentration of 5% sodium hypochlorite.

Optional Products

Retic-COUNT" Reticulocyte Enumeration software (for reticulocyte applications)

SimulSET" software (for automated acquisition and analysis of two-color immunophenotyping applications)

Instrument Overview

Take a few minutes to study Figures 1-2 through 1-5 and their descriptions. This

will familiarize you with die various instrument components and help you understand the instructions in this user's guide.

FACScan Instrument

FACScan (Figure 1-2) is a five-detector flow cytometer, which consists of optical, electronic, and fluidic systems, as well as a built-in, air-cooled, argon-ion laser.
Samples are introduced through a stainless steel injection tube equipped with an outer droplet containment sleeve. The sleeve works in conjunction with a vacuum pump to eliminate droplet formation of sheath fluid as it backflows from the injection tube. Fluid controls allow you to select die fluidics mode and sample flow rate.

The fluidics compartment provides easy access to the removable sheath and waste reservoirs as well as die sheath fluid Filter. A vent valve toggle switch allows you to quickly depressurize die sheath reservoir should you need to refill it during a run.

Figure 1-2 FACScan instrument

control panel

All instrument controls except the fluid control dial and sample flow rate switch are located on this panel. For a detailed description, see Section 1.3.2, FACScan Control Panel.

power switch

This switch turns the FACScan on and off

optics door

The door shielding die compartment that houses the flow cell assembly. Open this door to view the flow cell (Figure 1-3) while draining and filling. The laser light is blocked by a shutter bar when the door is open.

flow cell —A quartz cuvette where die sample suspension is injected into a moving stream of sheath fluid. The flow cell is the area where die laser beam intercepts die fluid stream.

fluid control panel

This panel contains the fluid control dial and the sample flow rate switch.

fluid control dial —This dial allows you to select among five fluidics modes (see Figure 1-4).

DRAIN stops the flow of sheath fluid and reverses the pressure to force fluid out of die flow cell and into die waste reservoir. Follow DRAIN with FILL to refill die flow cell.

FILL fills die flow cell with sheath fluid at a controlled rate to prevent bubble formation or entrapment.

RUN aspirates sample from die sample tube and establishes a constant pressure differential between the sample and the sheath fluid as they flow through the flow cell.

STANDBY restricts fluid flow and reduces laser power to conserve laser life.

  NOTE: When a sample is removed from the sample injection port (SIP), the instrument switches to an automatic STANDBY status to conserve sheath fluid.

BACKFLUSH reverses the flow of sheath to flush fluid out of the sample injection tube to help remove clogs. Follow BACKFLUSH with DRAIN, then FILL to refill the flow cell.

sample flow rate s-vvitch —This switch allows you to select between two sample flow rates (see Figure 1-4).

LO delivers 12 µL ±3 µL/min of sample through the flow cell.

HI delivers 60 µL ±7 µL/min of sample through the flow cell.

sample injection port (SIP)

The area where the sample tube is installed on the instrument. lt includes the sample injection tube and the tube support arm.

sample injection tube —This stainless steel tube carries cells from die sample test tube to die flow cell. The sample injection tube is covered with an outer sleeve that serves as part of a droplet containment system.

tube support arm —This arm supports die sample tube. lt also functions to activate die droplet containment system vacuum. The vacuum is on when die arm is positioned to the right and off when the arm is centered.

fluidics compartment

The lower-left panel on die instrument opens for easy access to the fluid reservoirs and sheath filter. For more details on die contents of this compartment, see Section 2.1, Before You Begin.

sheath reservoir— This 4-L container holds enough sheath fluid for approximately 3 hours of ran time. lt is equipped with a fluid level detector that indicates, via the software, a near-empty condition.

waste reservoir —This 4-L container collects the fluid waste after it flows from the flow cell. A fluid level detector indicates, via die software, a near­full condition.

sheath filter —This 0.22-Inn filter cleans the sheath fluid before it enters die flow cell. This minirnizes die possibility that debris from sheath fluid will be acquired with your samples.

vent valve toggle switch —This toggle switch relieves die sheath reservoir of air pressure to allow removal when refilling. When the switch is in die up position, the sheath reservoir is pressurized; in the down position, pressure is released.

air filter —This filter cleans the air that cools the laser.

Specifications

EXCITATION

laser:                                        15-mW, 488-nm, air-cooled, argon-ion laser.

                                                Life expectancy >5,000 hours.

beam geometry:                       Prismatic expander and spherical lens provide

                                                20 x 64-µm elliptical beam.

OPTICS

alignment :                                Fixed—no user adjustments necessary or available.

dichroics:                                 560/22.5° (blue/orange-red)

                                                640 LP (orange/red

filters:                                       FL1: 530/30

                                                FL2: 585/42

                                                FL3: 650LP

FLUIDICS

flow rates:                                LO (12 µL ±3 µL/min)

                                                HI (60 µL ±7 µL/min)

quartz cuvette:                         Internal cross-section is a 430 x 180-µm rect-

                                                angle. External surfaces are antireflection­coated.

air pressure:                             Internal air pump provides sheath pressure of

                                                4.5 psig and sample pressures of 4.6 and 5.0 psig.

acquisition speed:                    Acquires up to 10,000 cells/s.

SIGNAL PROCESSING

measurement resolution:         256 or 1024 channels an all five parameters

                                                (seven when acquiring with DDM).

signal modes:                          Any combination of linear or logarithmic

                                                selections for each detector.

dynamic range:                        Four decades are provided by logarithmic

                                                amplifiers for each of the five parameters.

fiuorescence sensitivity:          1,000 molecules of equivalent soluble fluorescein.

fluorescence resolution:          Coefficient of variation (CV) is <3% fall peak for propidium iodide-stained chicken erythro­cyte nuclei for FL2-A.

INSTALLATION REQUIREMENTS

power :                                      120 VAG, 50/60 Hz ±10%, minimum of 20 A.

For installations outside the US , a power transformer/conditioner is provided to accommodate 110 V ±10%, 220 V ±10%, 240 V ±10%, 50-60 Hz ±2 Hz.

size:                                         width — 76 cm (30 in)

                                                depth — 60 cm (23.5 in)

                                                height — 51 cm (20 in)

weight:                                      61 kg (135 Ib)

temperature:                            16-29°C (60-85°F)