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Product catalog No: 2533200
ReaPan 3 8 G
Manufactured by
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ReaPan 3 8 G Reagent
1. INTENDED USE
The ReaPan 3 8 G reagent is a two color immunofluorescence staining reagent for
the enumeration of absolute counts of total Tlymphocytes (CD3+) and Suppressor/
cytotoxic (CD8+) TCells. This reagent is intended for flow cytometry based
analysis in lyzed human whole blood samples, preferably on the Guava PCA
System1.
2. BACKGROUND
The ReaPan 3 8 G reagent contains fluorescently labeled antibodies that bind to
CD3 and CD8 antigens found on the surface of circulating leukocytes in peripheral
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blood samples.
The CD3 antigen is a complex of at least six proteins known collectively as the T
cell receptor (TCR) complex. The antibody used in this reagent binds to the 20kDa
ε chain of this complex.
The CD8 antigen is a complex consisting of two disulfide linked subunits. The
antibody used in this reagent binds to the 32KDa ά subunit of the complex.CD8
interacts with class I major histocompatibity complex molecules.
3. REAGENT
The ReaPan 3 8 G reagent is formulated in buffered saline with sodium azide and
stabilizers. It contains RPhycoerythrin (PE)– labeled antiCD8 monoclonal
antibody, clone LT8; RPhycoerythrin (PE)Dyomics 649 – labeled antiCD3
monoclonal antibody, clone UCHT1; The monoclonal antibodies used in the
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ReaPan 3 8 G were assigned these specificities at the 8th International Workshop
on Human Leukocyte Differentiation Antigens. The ReaPan 3 8 G reagent enables
a singleplatform enumeration of absolute CD8+ (and CD3+) and total CD3+ T
cell counts. The ReaPan 3 8 G reagent is provided in drieddown format and
dispensed in Guava Flow Cytometer compatible sample tubes with each tube
containing one readytouse test.
Precautions
1) Warning: The ReaPan 3 8 G reagent contains Sodium azide. Sodium
azide is harmful if swallowed. Wear suitable protective clothing. If swallowed,
seek medical advice immediately. Contact with acids liberates toxic gas. Azides
should be flushed with large amounts of water during disposal to avoid deposits
in lead or copper plumbing.
2)Warning: All blood specimens are considered biohazards. Handle them as if they
are capable of transmitting infection and dispose off with proper precautions and
accordance with governmental regulations.
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3) The addition of the precise volume of blood is critical to obtain correct
results. Use a calibrated pipette and operate according to the manufacturer’s
instructions.
Storage and Handling
1) Store the reagent at room temperature in a dry place. Do not use the reagent
after the expiry date on the label.
2) Do not freeze the Four Color reagent.
3) The ReaPan 3 8 G reagent is light sensitive. Do not expose to direct light either
during storage or when mixed with blood.
4. INSTRUMENT
The ReaPan 3 8 G reagent has been tested on the Guava PCA™ systems
manufactured by Guava Technologies, Inc.1. ReaMetrix recommends running this
reagent on this instrument. Instruments should be calibrated for setting
photomultiplier tube voltages, fluorescence compensation, and checking instrument
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sensitivity according to the manufacturer’s guidelines.
It is the responsibility of the user to optimize the performance of the reagent for use
in flow cytometers other than that mentioned above. The flow cytometers used along
with this reagent should be equipped with one excitation lasers Green laser 532nm
and two fluorescence detection channels yellow/orange ~580nm to 583nm , red ~675
to 680 nm and Forward scatter1.
5. SPECIMEN COLLECTION
The blood sample should be collected in a sterile blood collection tube containing
K3EDTA. Follow the collection tube manufacturer’s guidelines for the minimum
volume of blood to be collected.
The anticoagulated blood must be stored at room temperature (20°C 25°C) and
should be stained and analyzed ideally within 24 hours of draw.
Refrigerated, hemolyzed, and previously fixed blood specimens can yield erroneous
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results and should be rejected.
6. PROCEDURE
6.1 Reagent Provided
ReaPan 3 8 G in a dried format.
6.2 Reagents and materials required but not provided
1) Blood collection tube containing K3EDTA
2) Calibrated pipettes
3) Vortex mixer
4) ReaLyse Lysing Solution (ReaMetrix Catalog No.2523700) or similar blood
fix/lyse solution
5) Guava® Check Beads KitFor verifying the performance of the Guava systems
6.3 Assay Protocol
1) Mix blood sample (invert blood tube at least 10 times) and pipette 50µL of
blood into the correctly labeled tube that contains the dry down reagent.
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2) Vortex each tube vigorously for 30 seconds. Incubate for 30 minutes at
room temperature. Protect the tube from direct light.
3) Add 450µL of 1X ReaLyse Lysing solution (a 10X solution is typically
supplied with the kit) to each tube and vortex for 20 seconds. Return tubes to
the dark for at least 15 minutes.
6.4 Flow Cytometer Acquisition and Analysis
This protocol assumes that the flow cytometer has been setup according to the
manufacturer’s instructions (For example, In the case of Guava PCA™, the
instrument should be setup and calibrated using Guava® Check Kit and Easy CD8
Cytosoft 2.2 Software Guava check™ software). Open the Easy CD8 Cytosoft
2.2 software and connect to the cytometer.
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g. DETERMINING THE ABSOLUTE CD8 CONCENTRATION:
1) Click Guava EasyCD8 from the main menu. Allow the Guava PCA to warm
up for 15 minutes before acquiring specimens.
2) Select EasyCD8 from the Reagent Type menu.
3) Enter the reagent kit lot # and expiration date. This information can be found on
the ReaPan 3 8 G Reagent pouch.
4) Click New Data Set. Select the folder where you want to save the file, and enter a
file name for this session. Click Save. The same file name you enter for the FCS
file will also be used for the spreadsheet (.csv) file. If you wish, you may select
an existing data file and either overwrite it or append it with the data from this
session.
5) Select the reagent type.
6) Mix the normal control specimen and load it on the Guava PCA.
Click Settings.
a) To adjust instrument settings, click Adjust Settings.
b) To retrieve instrument settings, click Retrieve Settings. Select a
settings file and click Open. The settings are automatically downloaded to
the Guava PCA.
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c) A message appears prompting you to load the control specimen. Ensure
the tube is loaded and click OK.
d)Enter a file name for this data set and click Save. The Adjust Settings screen
appears. The system automatically sets the threshold to exclude background
fluorescence.
e) To fine tune the settings, you can make the following adjustments
once events start to appear on the screen.
f) Set the Refresh Rate to the number of events you want to display. The
default is 2000 events appearing on the display at any time. To view fewer
events, select 100.To view all events, select Cumulative.
g) Set the Flow Rate. The recommended flow rate is Medium (0.5
µL/s). You may also select Low (0.2 µL/s).
h) Use the FSC Gain settings to reduce or amplify the FSC signal so
that the white blood cell (WBC) population is positioned between 10e2 and
10e3.
i) To adjust the FSC threshold, click and drag the threshold marker up or down
the FSC axis of the FSC vs CD3PEDyomics 649 (PM2) dot plot until the
desired amount of debris is eliminated below the threshold. Set the threshold
2 to 3 mm to the left of the CD3+ cells.
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j) Adjust the PM2 voltage (using the slider or the arrow keys on the keyboard)
to optimize the separation between CD3– and CD3+ cells. Make sure the
negative population is positioned below 10e1 on the CD8PE (PM1) vs.
CD3PE Dyomics649 (PM2) plot.
k) Adjust the PM1 voltage so that the negative population is positioned
below 10e1 on the CD8PE (PM1) vs. CD3PEDyomics 649 (PM2) plot.
Drag to adjust FSC threshold.
l) Adjust the CD3 gate on the FSC vs. CD3 PEDyomics 649 (PM2) plot to
include all CD3+ cells. The CD3 gate is used as a counting gate. Events that
are included in the gate are counted toward the number of Events to Acquire.
For example, if the Events to Acquire is set to 2000, acquisition is complete
when 2000 events have passed through the CD3 gate. Set the CD8 gate on
the CD8PE (PM1) vs CD3PEDyomics 649 (PM2) plot.
7) When you are finished adjusting settings, click Next Step to advance to the data
acquisition screen.
8) Open the Specimen Information control panel and enter the number of events to
acquire and dilution factor. The default number of events to acquire is 2000.
The default dilution factor is 20. If you set a CD3 gate to include CD3+ cells
only, as many events will be acquired as is necessary to satisfy 2000 CD3+
events.
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9)If you want to identify individual specimens or sets of specimens, enter an
optional ID in the Specimen ID field.
10) The specimen ID may be any text up to 40 characters long, such as the name
of the specimen you are testing.
11) Mix the first specimen and load it on the Guava PCA. Click Acquire Next
Specimen. The system acquires the specimen and automatically displays the
results. Click to automatically start acquisition when a tube is loaded.
8. ACQUISITION NOTES
If you select Autostart Acquisition, acquisition automatically starts when you load
each tube. You do not need to click Acquire Next Specimen. Autostart Acquisition is
automatically disabled if you click any of the following: Settings (followed by
Adjust or Retrieve), Quick Clean, or Backflush. You must recheck the box to
continue using the feature.
If the acquisition rate appears to slow dramatically, the fluid pathway may be
blocked. Click Abort, load a tube of 20% bleach, then click Backflush. When the
backflush is complete, load a tube of DI water and click Quick Clean.
Reload the specimen and click Acquire Next Specimen.
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The progress bar provides an estimate of the target event count during the acquisition
period, which times out after 4 minutes.
You may set or fine tune the gates immediately after acquisition from the
Acquisition screen.
Click Save and Close Current Specimen.
You may still enter or change the Specimen ID for the current specimen before
clicking Save and Close Current Specimen.
When you are finished, load a tube of Guava ICF and click Quick Clean. Follow
with a second Quick Clean running deionized water to rinse.
9. ANALYSIS
Use the Analysis screen to analyze specimens, print results, log comments, or view
the event log from a data set that was saved previously. You can also export data to
FCS 2.0 format or a spreadsheet file. You can save changes made to the gate or
markers within Analysis by overwriting the existing file or saving a new file. Guava
does not recommend overwriting files.
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If you access the Analysis screen during data acquisition you can view or print data
for any specimens already acquired. You may also log comments or view the event
log. However, you cannot change analysis settings (gates and markers) from the
analysis screen during acquisition. Any analysis settings you wish to change during
acquisition should be done from the Acquisition screen.
Click Guava EasyCD8TM icon from the main menu.
Click Go to Analysis from the Acquisition screen.
Click Open Data Set. Select an FCS file for analysis and click Open.
The data and results for the first specimen in the data set appear. The marker settings
appear as they were when the specimen was acquired. To see a list of all specimens
in the data set, click the title bar of the Analysis Specimen List control panel.
CD3 GATE
The CD3 gate is used as a counting gate. Events that are included in the gate are
counted toward the number of Events to Acquire. To set a gate on the CD3
population, position the cursor over the upperleft handle. Click and drag the handle
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to a new location. Make sure the left side of the rectangle does not eliminate any
CD3+ cells. Repeat with the lowerright handle. Position the bottom of the gate
between the CD3–and CD3+ cells. Be sure to include all CD3+ cells. Excluding
CD3+ cells from the CD3 gate will affect results. Events that fall within the center
rectangle and appear in red are included in the gate. You may also set the gate by
entering the coordinates in the Marker Position fields and clicking Set
CD8 GATE:
You can set rectangular markers or quadrant markers. You can choose to view all the
events acquired or only the CD3+ gated events by clicking the appropriate option
under CD3/CD8 Plot. When All Events is selected, all of the events to the right of
the FSC threshold are displayed in the CD3/CD8 Dot Plot. When CD3+ Gated
Events is selected, only those events within the CD3+ gate are displayed. Guava
recommends selecting All Events when setting the CD8 gate.
RECTANGULAR MARKER:
To set a gate on the CD8 Tcell population, click and drag the handles so that all of
the CD3+/CD8+ cells are within the gate. You may also set the gate by entering the
coordinates in the Marker Position fields and clicking Set. Gate set on CD3+ T
lymphocytes.Click and drag handles to manually set gate. A popup label displays
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handle’s current x and y coordinates. Enter coordinates to set gate. Rectangular
markers selecting CD8+ T cells (pink) and excluding all other cell populations
(black).Enter coordinates to set gate. Click and drag handles to manually set gate. A
popup label displays handle’s current x and y coordinates. Select data to view.
Select type of markers.
QUADRANT MARKERS:
To set quadrant markers, position the cursor over the handle at the intersection, then
click and drag to the desired location. You may also set the markers by entering the
coordinates in the Marker Position fields and clicking Set. If necessary, you can
adjust the angle of the markers ±44° from their original location.
Click and drag the handle (Solid Square) towards the end of the marker and tilt the
marker to the desired angle. You may also angle the markers by entering the degrees
in the Marker Position Angle fields and clicking Set.
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Select data to view. Enter coordinates to set gate. Click and drag handles to manually
set gate. A popup label displays handle’s current x and y coordinates. Select type of
markers.
Select data to view. Enter coordinates and angle to set markers. Click and drag
intersection to set markers. A label displays markers’ x and y coordinates and angles.
Click and drag handle to set marker angle. A label displays markers’ x and y
coordinates and angles. Select type of markers.
You can apply gate and/or marker settings from one specimen to another
specimen(s), whether you have made changes or the specimens were acquired with
different settings. Select the specimens to which you want to apply the settings from
the Analysis Specimen List. Be sure the original specimen, whose setting you wish
to use, is also selected. Then, click Apply Current Settings to Selected Specimens.
Hold down the Shift key while clicking and dragging to select groups of specimens.
Or, hold down the Ctrl key while clicking to select multiple specimens.
When you have finished analyzing the specimens in the current file, you can save
any analysis change you made by exiting Analysis or clicking Open Data Set. A
dialog box appears prompting you to save the changes. Click Yes and either
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overwrite the existing file or save the file with a new name. Results are automatically
exported to a CSV file that is given the same name as the FCS files.
If you wish to view the event log, click View Event Log. You can also enter
comments related to the assay and save these comments to the event log. Click Log
Comment and type in the information. Then, click Save Comments to Log.
10. RESULTS:
You can view all the events or CD3+ gated events only, by selecting either All
Events or CD3+ Gated Events under CD3/CD8 Plot (or CD3/CD4 Plot) to the left
of the dot plot. Guava recommends selecting All Events when viewing the results
for CD8 (or CD4) T cells.
The CD3 gate is used to determine the concentration of CD3 T cells.
CD3+ cells x dilution factor = CD3 T cell concentration
Volume of specimen acquired
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The CD8 gate is used to determine the concentration of CD8 T cells.
CD3+CD8+ cells x dilution factor = CD8 T cell concentration
Volume of specimen acquired
The summary of each quadrant is outlined in the table below:
CD8
Quadrant Staining Population Color
lower left CD8–, CD3– PMN cells, B cells, monocytes black
lower right CD8+, CD3– Natural Killer Cells (NK cells) black
upper right CD8+, CD3+ suppressor/cytotoxic T pink
upper left helper/inducer T lymphocytes
CD8–, CD3+ black
lymphocytes
Figure 1. Gating CD3 and CD8 cell population with Cytosoft (version 2.2)
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FIGURE 1A FIGURE 1B
11. PERFORMANCE CHARACTERISTICS
The performance data for ReaPan 3 8 G Reagent were generated by performing
clinical validation against the predicate Guava Reagents on the same Guava PCA
platform
Accuracy
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The absolute counts for CD8+ and CD3+ TCells determined using ReaPan 3 8 G
reagent were compared with Guava Easy CD8 Reagent using Guava PCA
Instrument. The absolute counts for CD3+ determined by the ReaPan 3 8 G were
compared with a Guava Easy CD3 Reagent. Excellent correlation is seen for a 100
sample comparison for CD3 counts (R2 > 0.97, R > 0.99) as well as for CD8 counts
Guava Easy CD8 Reagent- CD8 Count
(R2 > 0.96, R > 0.98). This is comparable and is some cases better than correlations
determined across reagents in literature2,4.
CD8:
3500
R2 = 0.9634
3000
R = 0.98
2500
2000
1500
1000
500
0
0 500 1000 1500 2000 2500 3000 3500
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R = 0.98
4000
R2 = 0.9713
3500
R = 0.99
3000
2500
2000
1500
1000
500
0
0 500 1000 1500 2000 2500 3000 3500 4000
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12. LIMITATIONS
3. The reagents must only be used with the ReaLyse Lysis Solution
13.WARRANTY
This product is warranted only to conform to the quantity and contents stated on the
label at the time of delivery to the customer. There are no warranties, expressed or
implied, that extend beyond the description on the label of the product. ReaMetrix’s
sole liability is limited to replacement of the product. ReaMetrix is not liable for
property damage, personal injury, or economic loss caused by the product.
Note: Guava Check kit, Guava PCA, Easy CD8, Cytosoft 2.2 are all registered
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trade names of Guava Technologies.
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14. REFERENCES
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Ph: +918028378693/5, Fax: +918041172451
Email: info@reametrix.com,
www.reametrix.com
Rev No. 1.0, 28Apr09
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