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1 Test per tube

Product catalog No: 25244-00

ReaPan 3 4 G

Manufactured by

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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 T-lymphocytes (CD3+) and

helper/inducer (CD4+) T-Cells. This reagent is intended for flow cytometry basedanalysis 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

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 R-Phycoerythrin (PE) labeled anti-CD4 monoclonal antibody,

clone RPA-T4; R-Phycoerythrin (PE)-Dyomics 649 labeled anti-CD3 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 single-platform

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enumeration of absolute CD4+ (and CD3+) and total CD3+ T-cell counts. The

ReaPan 3 4 G reagent is provided in dried-down format and dispensed in Guava Flow

Cytometer compatible sample tubes with each tube containing one ready-to-use test.

Precautions

1) Warning: The ReaPan 3 4 G reagent contains Sodium azide. Sodium azide isharmful 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 manufacturers instructions.

Storage and Handling

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1) Store the reagent at room temperature in a dry place. Do not use the reagentafter 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 eitherduring 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 settingphotomultiplier tube voltages, fluorescence compensation, and checking instrument

sensitivity according to the manufacturers 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 alongwith 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 manufacturers guidelines for the minimum

volume of blood to be collected.

The anti-coagulated blood must be stored at room temperature (20C - 25C) 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.25237-00) 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 Kit-For verifying the performance of the Guava systems

6.3 Assay Protocol

1) Mix blood sample (invert blood tube at least 10 times) and pipette 50L ofblood into the correctly labeled tube that contains the dry down reagent.

2) Vortex each tube vigorously for 30 seconds. Incubate for 30 minutes atroom temperature. Protect the tube from direct light.

3) Add 450L of 1X ReaLyse Lysing solution (a 10X solution is typicallysupplied 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 themanufacturers 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) ClickGuava EasyCD4 from the main menu. Allow the Guava PCA towarm 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 befound 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.

ClickSettings.

a) To adjust instrument settings, click Adjust Settings.

b) To retrieve instrument settings, click Retrieve Settings. Select asettings file and click Open. The settings are automatically downloaded to the

Guava PCA.

c) A message appears prompting you to load the control specimen.Ensure the tube is loaded and clickOK.

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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 onceevents start to appear on the screen:

f)Set the Refresh Rate to the number of events you want to display. The default is2000 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 L/s). Youmay also select Low (0.2 L/s).

h) Use the FSC Gain settings to reduce or amplify the FSC signal so that thewhite 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 theFSC axis of the FSC vs CD3-PE-Dyomics 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 negativepopulation is positioned below 10e1 on the CD4-PE (PM1) vs. CD3-PE

Dyomics649 (PM2) plot.

k) Adjust the PM1 voltage so that the negative population is positioned below10e1 on the CD4-PE (PM1) vs. CD3-PE-Dyomics 649 (PM2) plot. Drag to

adjust FSC threshold.

l) Adjust the CD3 gate on the FSC vs. CD3- PE-Dyomics 649 (PM2) plot toinclude 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 CD3-PE-Dyomics 649 (PM2) plot.

7) When you are finished adjusting settings, click Next Stepto advance to the dataacquisition 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.

9) If you want to identify individual specimens or sets of specimens, enter anoptional ID in the Specimen ID field.

10) The specimen ID may be any text up to 40 characters long, such as thename of the specimen you are testing.

11) Mix the first specimen and load it on the Guava PCA. ClickAcquireNext 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 loadeach tube. You do not need to clickAcquire Next Specimen. Autostart Acquisition is

automatically disabled if you click any of the following: Settings(followed byAdjust

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orRetrieve), Quick Clean, orBackflush. You must recheck the box to continue using

the feature.

If the acquisition rate appears to slow dramatically, the fluid pathway may be blocked.

ClickAbort, load a tube of 20% bleach, then click Backflush. When the backflush iscomplete, 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.

ClickSave 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 clickQuick 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 datafor 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.

ClickGuava EasyCD4

TM

icon from the main menu.

ClickGo to Analysis from the Acquisition screen.

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ClickOpen Data Set. Select an FCS file for analysis and clickOpen.

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 upper-left 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 lower-right handle. Position the bottom of the gate between the

CD3and 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 recommendsselecting All Events when setting the CD4 gate.

RECTANGULAR MARKER:

To set a gate on the CD4 T-cell 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 thecoordinates in the Marker Position fields and clicking Set. Gate set on CD3+ T

lymphocytes.Click and drag handles to manually set gate. A pop-up label displays

handles 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

pop-up label displays handles 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.

ClickNext 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 pop-up label displays handles current x and y coordinates.Select type of

markers.

Select data to view. Enter coordinates and angle to set markers. Click and dragintersection 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. Holddown 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, clickSave 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

Quadrant Staining Population Color

lower left CD4, CD3 PMN cells, B cells, NK black

lower right CD4+, CD3 monocytes black

upper right CD4+, CD3+ helper/inducer T pink

upper left CD4, CD3+ suppressor/cytotoxic T black

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

The performance data for ReaPan 3 4 G Reagent were generated by performing

clinical validation against the predicate Guava Reagents on the same Guava PCA

platform

Accuracy

The absolute counts for CD4+ and CD3+ T-Cells 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 werecompared 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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y =1.027x +15.844

R2= 0.9718

0

1000

2000

3000

4000

5000

6000

0 1000 2000 3000 4000 5000 6000

RMX Reagents on PCA - CD3 Count

Gua

vaReagentso

nPC

CD3Counts

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CD4 Counts:

y =1.0364x +8.1047

R2=0.9745

0

200

400

600

800

1000

1200

1400

1600

0 200 400 600 800 1000 1200 1400 1600

RMX Reagents on PCA - CD4 Count

GuavaReagen

tsonPC

CD4Co

unt

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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

1. The ReaPan 3 4 G reagent has only been validated withK3EDTA treated whole blood.

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2. Laboratories should establish their own reference ranges for theabsolute counts obtained using the ReaPan 3 4 G reagent.

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. ReaMetrixs

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

1.Guava Technologies Website, The Guava Personal Cell Analysis (PCA)Platform, http://guavatechnologies.com/

2.A. J. Kandathil, R. Kannangai, S. David, G. Nithyanandam, S. Solomon, P.Balakrishnan, O. C. Abraham, S. Subramanian, P. Rupali, V. P. Verghese, S.

Pulimood, and G. Sridharan, Comparison of Microcapillary Cytometry

Technology and Flow Cytometry for CD4+ and CD8+ T-Cell Estimation,

Clin Diagn Lab Immunol. 2005 August; 12(8): 10061009.

3.Immunotrol Cell Controls from Beckman Coulter,http://www.beckmancoulter.com. Part Number 6607077.

4.Kovit Pattanapanyasat, Yuwadee Phuang-Ngern, Surada Lerdwana, Punneeporn

Wasinrapee, Natthaga Sakulploy, Egarit Noulsri, Charin Thepthai, Janet M.McNicholl, Evaluation of a single-platform microcapillary flow cytometer for

enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai

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http://guavatechnologies.com/http://www.beckmancoulter.com/http://www.beckmancoulter.com/http://guavatechnologies.com/http://www.beckmancoulter.com/

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patients, Volume 72B, Issue 5, Pages 387-39.

Manufactured by ReaMetrix India Pvt. Ltd.

Manufacturing License Number: KTK25/519/200650-B, II Phase, Peenya Industrial Area

Peenya, Bangalore 560058, India

Ph: +91-80-28378693/5, Fax: +91-80-41172451

E-mail: info@reametrix.com,

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www.reametrix.com

Rev No. 2.0, 28-Apr-09

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http://www.reametrix.com/http://www.reametrix.com/