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1 Test per tube

Product catalog No: 25332-00ReaPan 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 forthe enumeration of absolute counts of total T-lymphocytes (CD3+) and Suppressor/ cytotoxic (CD8+) T-Cells. This reagent is intended for flow cytometry basedanalysis in lyzed human whole blood samples, preferably on the Guava PCASystem 1.

2. BACKGROUND

The ReaPan 3 8 G reagent contains fluorescently labeled antibodies that bind toCD3 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

chain of this complex.

The CD8 antigen is a complex consisting of two disulfide linked subunits. Theantibody used in this reagent binds to the 32KDa subunit of the complex.CD8 interacts with class I major histocompatibity complex molecules.

Cells that are CD3+ and CD8+ are identified as suppressor/cytotoxic Tlymphocytes. Decreased CD8+CD3+ cell counts have been associated with someforms of immunodeficiency.

3. REAGENT

The ReaPan 3 8 G reagent is formulated in buffered saline with sodium azide andstabilizers. It contains R-Phycoerythrin (PE) labeled anti-CD8 monoclonalantibody, clone LT8; R-Phycoerythrin (PE)-Dyomics 649 labeled anti-CD3monoclonal 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 Workshopon Human Leukocyte Differentiation Antigens. The ReaPan 3 8 G reagent enablesa single-platform enumeration of absolute CD8+ (and CD3+) and total CD3+ T-cell counts. The ReaPan 3 8 G reagent is provided in dried-down format and

dispensed in Guava Flow Cytometer compatible sample tubes with each tubecontaining one ready-to-use test.

Precautions

1) Warning: The ReaPan 3 8 G reagent contains Sodium azide. Sodiumazide is harmful if swallowed. Wear suitable protective clothing. If swallowed,seek medical advice immediately. Contact with acids liberates toxic gas. Azidesshould be flushed with large amounts of water during disposal to avoid depositsin lead or copper plumbing.

2)Warning: All blood specimens are considered biohazards. Handle them as if theyare capable of transmitting infection and dispose off with proper precautions andaccordance with governmental regulations.

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3) The addition of the precise volume of blood is critical to obtain correctresults. Use a calibrated pipette and operate according to the manufacturersinstructions.

Storage and Handling

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 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 systemsmanufactured by Guava Technologies, Inc. 1. ReaMetrix recommends running thisreagent on this instrument. Instruments should be calibrated for settingphotomultiplier tube voltages, fluorescence compensation, and checking instrument

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sensitivity according to the manufacturers guidelines.

It is the responsibility of the user to optimize the performance of the reagent for usein flow cytometers other than that mentioned above. The flow cytometers used alongwith this reagent should be equipped with one excitation lasers Green laser 532nmand two fluorescence detection channels yellow/orange ~580nm to 583nm , red ~675

to 680 nm and Forward scatter 1.

5. SPECIMEN COLLECTION

The blood sample should be collected in a sterile blood collection tube containingK3EDTA. 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 (20 C - 25C) andshould 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 K 3EDTA2) Calibrated pipettes

3) Vortex mixer4) ReaLyse Lysing Solution (ReaMetrix Catalog No.25237-00) or similar blood

fix/lyse solution5) 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 50 L 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 atroom temperature. Protect the tube from direct light.

3) Add 450 L of 1X ReaLyse Lysing solution (a 10X solution is typicallysupplied with the kit) to each tube and vortex for 20 seconds. Return tubes tothe dark for at least 15 minutes.

4)Vortex sample tube thoroughly (at low speed) and load onto cytometer foranalysis.

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 CD8Cytosoft 2.2 Software Guava check software). Open the Easy CD8 Cytosoft2.2 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 warmup 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 selectan existing data file and either overwrite it or append it with the data from thissession.

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 asettings file and click Open. The settings are automatically downloaded tothe Guava PCA.

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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 thenegative population is positioned below 10e1 on the CD8-PE (PM1) vs.CD3-PE Dyomics649 (PM2) plot.

k) Adjust the PM1 voltage so that the negative population is positionedbelow 10e1 on the CD8-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 thatare 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 completewhen 2000 events have passed through the CD3 gate. Set the CD8 gate onthe CD8-PE (PM1) vs CD3-PE-Dyomics 649 (PM2) plot.

7) When you are finished adjusting settings, click Next Step to advance to the dataacquisition 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+ cellsonly, 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 anoptional ID in the Specimen ID field.

10) The specimen ID may be any text up to 40 characters long, such as the nameof 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 theresults. 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 click Acquire Next Specimen . Autostart Acquisition isautomatically disabled if you click any of the following: Settings (followed by

Adjust or Retrieve ), Quick Clean , or Backflush . You must recheck the box tocontinue using the feature.

If the acquisition rate appears to slow dramatically, the fluid pathway may beblocked. Click Abort , load a tube of 20% bleach, then click Backflush. When thebackflush 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 acquisitionperiod, 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 beforeclicking Save and Close Current Specimen .

When you are finished, load a tube of Guava ICF and click Quick Clean . Followwith a second Quick Clean running deionized water to rinse.

9. ANALYSISUse the Analysis screen to analyze specimens, print results, log comments, or viewthe event log from a data set that was saved previously. You can also export data toFCS 2.0 format or a spreadsheet file. You can save changes made to the gate ormarkers within Analysis by overwriting the existing file or saving a new file. Guavadoes not recommend overwriting files.

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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 eventlog. However, you cannot change analysis settings (gates and markers) from the

analysis screen during acquisition. Any analysis settings you wish to change duringacquisition should be done from the Acquisition screen.

Click Guava EasyCD8 TM 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 settingsappear as they were when the specimen was acquired. To see a list of all specimensin the data set, click the title bar of the Analysis Specimen List control panel.

CD3 GATEThe CD3 gate is used as a counting gate. Events that are included in the gate arecounted toward the number of Events to Acquire. To set a gate on the CD3population, position the cursor over the upper-left 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 anyCD3+ cells. Repeat with the lower-right handle. Position the bottom of the gatebetween the CD3and CD3+ cells. Be sure to include all CD3+ cells. ExcludingCD3+ 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 byentering 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 theevents 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+ GatedEvents is selected, only those events within the CD3+ gate are displayed. Guavarecommends selecting All Events when setting the CD8 gate.

RECTANGULAR MARKER:To set a gate on the CD8 T-cell 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 thecoordinates in the Marker Position fields and clicking Set . Gate set on CD3+ Tlymphocytes.Click and drag handles to manually set gate. A pop-up label displays

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handles current x and y coordinates. Enter coordinates to set gate. Rectangularmarkers 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. Apop-up label displays handles 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, thenclick and drag to the desired location. You may also set the markers by entering thecoordinates in the Marker Position fields and clicking Set . If necessary, you canadjust 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 themarker to the desired angle. You may also angle the markers by entering the degreesin the Marker Position Angle fields and clicking Set .

Click Next under Specimen List Navigation in the Specimen Information controlpanel or Unit Control panel. You can also click on the next specimen in the list, oruse the keyboard arrow keys to select specimens.

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Select data to view. Enter coordinates to set gate. Click and drag handles to manuallyset 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 ycoordinates and angles. Select type of markers.

You can apply gate and/or marker settings from one specimen to anotherspecimen(s), whether you have made changes or the specimens were acquired withdifferent settings. Select the specimens to which you want to apply the settings fromthe Analysis Specimen List. Be sure the original specimen, whose setting you wishto 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 saveany analysis change you made by exiting Analysis or clicking Open Data Set . Adialog 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 automaticallyexported 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 LogComment 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 leftof the dot plot. Guava recommends selecting All Events when viewing the resultsfor 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 leftCD8, CD3+

helper/inducer T lymphocyteslymphocytes

black

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 performingclinical validation against the predicate Guava Reagents on the same Guava PCAplatformAccuracy

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The absolute counts for CD8+ and CD3+ T-Cells determined using ReaPan 3 8 Greagent were compared with Guava Easy CD8 Reagent using Guava PCAInstrument. 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 100sample comparison for CD3 counts (R 2 > 0.97, R > 0.99) as well as for CD8 counts(R 2 > 0.96, R > 0.98). This is comparable and is some cases better than correlationsdetermined across reagents in literature 2,4.

CD8:

R 2 = 0.9634

0

500

1000

1500

2000

2500

3000

3500

0 500 1000 1500 2000 2500 3000 3500

RMX ReaPan 3 8 G Reagent- CD8 Count

G u a v a E a s y C D 8 R e

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CD3:

R 2 = 0.9713

0

500

1000

1500

2000

2500

3000

3500

4000

0 500 1000 1500 2000 2500 3000 3500 4000

RMX ReaPan 3 8 G Reahent- CD3 Count

G u a v a E a s y C D 8 R e a

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R = 0.98

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12. LIMITATIONS

1. The ReaPan 3 8 G reagent has only been validated with K 3EDTAtreated whole blood.

2. Laboratories should establish their own reference ranges for theabsolute counts obtained using the ReaPan 3 8 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 thelabel 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. ReaMetrixssole liability is limited to replacement of the product. ReaMetrix is not liable forproperty 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

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 MicrocapillaryCytometry Technology and Flow Cytometry for CD4+ and CD8+ T-CellEstimation, 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 flowcytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patients, Volume 72B, Issue 5, Pages 387-39.

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

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Manufactured byReaMetrix India Pvt. Ltd. Manufacturing License Number: KTK25/519/2006 50-B, II Phase, Peenya Industrial AreaPeenya, Bangalore 560058, India

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Ph: +91-80-28378693/5, Fax: +91-80-41172451E-mail: info@reametrix.com,www.reametrix.com

Rev No. 1.0, 28-Apr-09

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