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Product catalog No: 2524400
ReaPan 3 4 G
Manufactured by
ReaPan 3 4 G Reagent
1. INTENDED USE
The ReaPan 3 4 G reagent is a two color immunofluorescence staining reagent for
the enumeration of absolute counts of total Tlymphocytes (CD3+) and
helper/inducer (CD4+) TCells. This reagent is intended for flow cytometry based
analysis in lyzed human whole blood samples, preferably on the Guava PCA
System1. Please get in touch with ReaMetrix before running the reagents on other
Flow Cytometer systems.
2. BACKGROUND
The ReaPan 3 4 G reagent contains fluorescently labeled antibodies that bind to CD3
and CD4 antigens found on the surface of circulating leukocytes in peripheral blood
samples.
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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 CD4 antigen is a 59kDa protein. It interacts with class II molecules of the major
histocompatibility complex and is the primary receptor for the Human
Immunodeficiency Virus (HIV).
Cells that are CD3+ & CD4+ are identified as helper/inducer lymphocytes.
Decreased CD4+CD3+ cell counts have been associated with some forms of
immunodeficiency.
3. REAGENT
The ReaPan 3 4 G reagent is formulated in buffered saline with sodium azide and
stabilizers. It contains RPhycoerythrin (PE)– labeled antiCD4 monoclonal antibody,
clone RPAT4; RPhycoerythrin (PE)Dyomics 649 – labeled antiCD3 monoclonal
antibody, clone UCHT1; The monoclonal antibodies used in the ReaPan 3 4 G were
assigned these specificities at the 8th International Workshop on Human Leukocyte
Differentiation Antigens. The ReaPan 3 4 G reagent enables a singleplatform
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enumeration of absolute CD4+ (and CD3+) and total CD3+ Tcell counts. The
ReaPan 3 4 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 4 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.
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
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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 4 G reagent is light sensitive. Do not expose to direct light either
during storage or when mixed with blood.
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4. INSTRUMENT
The ReaPan 3 4 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
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.
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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
results and should be rejected.
6. PROCEDURE
6.1 Reagent Provided
1. ReaPan 3 4 G in a dried format.
2. ReaLyse Lysing Solution (ReaMetrix Catalog No.2523700) or similar blood
fix/lyse solution
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6.2 Reagents and materials required but not provided
1) Blood collection tube containing K3EDTA
2) Calibrated pipettes
3) Vortex mixer
4) 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.
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.
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4)Vortex sample tube thoroughly (at low speed) and load onto cytometer for
analysis.
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
CD4 Cytosoft 2.2 software Guava check™ software). Open the Easy CD4
Cytosoft 2.2 software and connect to the cytometer.
1)DETERMINING THE ABSOLUTE CD4 CONCENTRATION:
2) Click Guava EasyCD4 from the main menu. Allow the Guava PCA to
warm up for 15 minutes before acquiring specimens.
3)Select EasyCD4 from the Reagent Type menu.
4) Enter the reagent kit lot # and expiration date. This information can be
found on the ReaPan 3 4 G Reagent pouch.
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5) 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.
6)Select the reagent type.
7)Mix the normal control specimen and load it on the Guava PCA.
Click Settings.
a) To adjust instrument settings, click Adjust Settings.
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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
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amount of debris is eliminated below the threshold. Set the threshold 2 to 3 mm
to the left of the CD3+ cells.
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 CD4PE (PM1) vs. CD3PE
Dyomics649 (PM2) plot.
k) Adjust the PM1 voltage so that the negative population is positioned below
10e1 on the CD4PE (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 CD4 gate on the CD4
PE (PM1) vs CD3PEDyomics 649 (PM2) plot.
7) When you are finished adjusting settings, click Next Step to advance to the data
acquisition screen.
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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.
10) The specimen ID may be any text up to 40 characters long, such as the
name of the specimen you are testing.
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
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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.
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.
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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.
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 EasyCD4TM icon from the main menu.
Click Go to Analysis from the Acquisition screen.
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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 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
CD4 GATE:
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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/CD4 Plot. When All Events is selected, all of the events to the right of the
FSC threshold are displayed in the CD3/CD4 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 CD4 gate.
RECTANGULAR MARKER:
To set a gate on the CD4 Tcell population, click and drag the handles so that all of
the CD3+/CD4+ 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
handle’s current x and y coordinates. Enter coordinates to set gate. Rectangular
markers selecting CD4+ 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:
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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.
Click Next under Specimen List Navigation in the Specimen Information control
panel or Unit Control panel. You can also click on the next specimen in the list, or use
the keyboard arrow keys to select specimens.
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.
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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 changes you made by exiting Analysis or clicking Open Data Set. A dialog
box appears prompting you to save the changes. Click Yes and either 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:
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You can view all the events or CD3+ gated events only, by selecting either All Events
or CD3+ Gated Events under CD3/CD4 Plot (or CD3/CD8 Plot) to the left of the dot
plot. Guava recommends selecting All Events when viewing the results for CD4 (or
CD8) 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
The CD4 gate is used to determine the concentration of CD4 T cells.
CD3+CD4+ cells x dilution factor = CD4 T cell concentration
Volume of specimen acquired
The summary of each quadrant is outlined in the table below:
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CD4
Figure 1. Gating CD3 and CD4 cell population with Cytosoft (version 2.2)
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FIGURE 1A FIGURE 1B
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11.PERFORMANCE CHARACTERISTICS
Accuracy
The absolute counts for CD4+ and CD3+ TCells determined using ReaPan 3 4 G
reagent were compared with Guava Easy CD4 Reagent using Guava PCA
Instrument. The absolute counts for CD3+ determined by the ReaPan 3 4 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 > 0.98) as well as for CD4 counts
(R2 > 0.97, R > 0.97). This is comparable and is some cases better than correlations
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determined across reagents in literature2,4.
CD3 Counts:
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Guava Reagents on PCA - 6000
y =1.027x +15.844
5000 2
R =0.9718
CD3 Counts
4000
3000
2000
1000
0
0 1000 2000 3000 4000 5000 6000
RMX Reagents on PCA - CD3 Count
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CD4 Counts:
1600
Guava Reagents on PCA -
1000
800
600
400
200
0
0 200 400 600 800 1000 1200 1400 1600
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Precision
The precision on a set of dispensed reagents was tested using Immunotrol Cells3
(Beckman Coulter, US) stained with three tubes of dried reagents. The precision on CD4
counts was seen to be within 2.5% and that on CD3 counts was seen to be within 1.4%.
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 CD4, Cytosoft 2.2 are all registered trade
names of Guava Technologies.
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14. REFERENCES
4.Kovit Pattanapanyasat, Yuwadee PhuangNgern, Surada Lerdwana, Punneeporn
Wasinrapee, Natthaga Sakulploy, Egarit Noulsri, Charin Thepthai, Janet M.
McNicholl, Evaluation of a singleplatform microcapillary flow cytometer for
enumeration of absolute CD4+ Tlymphocyte counts in HIV1 infected Thai
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patients, Volume 72B, Issue 5, Pages 38739.
Manufactured by ReaMetrix India Pvt. Ltd.
Manufacturing License Number: KTK25/519/2006
50B, II Phase, Peenya Industrial Area
Peenya, Bangalore 560058, India
Ph: +918028378693/5, Fax: +918041172451
Email: info@reametrix.com,
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www.reametrix.com
Rev No. 2.0, 28Apr09
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