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Dry Tri T-STAT (CD3/CD4/CD8) Reagent

INTENDED USE

The Dry Tri T-STAT (CD3/CD4/CD8) reagent is a three color immuno-fluorescencestain for the labeling, identification, and enumeration of helper/inducer (CD3+CD4+)and cytotoxic/suppressor (CD3+CD8+) T lymphocytes combined with a precisenumber of fluorescent counting beads for absolute CD4+ and CD8+ T-Cell counts.This reagent is intended to be used in a no wash protocol for flow cytometricanalysis of erythrocyte-lysed human whole blood .

SUMMARY AND EXPLANATION

The Dry-Tri T-STAT reagent contains fluorescently labeled antibodies that bind toCD3, CD4, and CD8 antigens found on the surface of circulating leukocytes. TheCD3 antigen is a complex of at least six proteins known collectively as the T-cellreceptor (TCR) complex. The antibody used in this reagent binds to the 20kDachain of this complex (1). The CD4 antigen is a 59kDa protein. It interacts with class

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fluorochrome. Specific cell subsets are stained when blood is combined with thereagent and each monoclonal antibody binds to the binds to the cell determinantmolecules on the cell surface. Specific cell subsets are identified when passedthrough a flow cytometer laser beam.

The Dry Tri T-STAT (CD3/CD4/CD8) reagent also contains a precise number offluorescent beads. When the reagent is combined with a known volume of bloodthe reagent provides for the single platform determination of the absolute cellconcentrations of the stained subsets. The volume of sample analyzed can bedetermined by multiplication of the total sample volume by the fraction of total beads

that were detected during the analysis.

REAGENT

The Dry-Tri T-STAT reagent is formulated in buffered saline with sodium azide andstabilizers. It contains ATTO 488 labeled CD4 monoclonal antibody, clone RPA-

T4; phycoerythrin (PE) labeled CD8 monoclonal antibody, clone LT8; and PE-DY-649 labeled CD3 monoclonal antibody, clone UCHT1. The specificities of themonoclonal antibodies used in the Dry Tri T-STAT (CD3/CD4/CD8) were confirmedby the Human Leukocyte Differentiation Antigens (HLDA) Workshops (9). A precise

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number of fluorescent counting beads are included in the Dry Tri T-STAT reagent toallow single-platform determination of absolute CD4+ and CD8+ T-cell counts. TheDry-Tri T-STAT reagent is provided in dried form and dispensed in flow cytometercompatible sample tubes with each tube containing one ready-to-use test. Fifty (50)

tests are supplied in each Dry Tri T-STAT (CD3/CD4/CD8) package.

The absolute number of counting beads in each test was set by determining thenumber of beads in a representative number of tubes after the reagent wasdispensed. The reagent was solubilized with a precise volume of fluid andtransferred to a chamber where the number of beads per unit volume dould be

counted.ATTO 488 (Atto-Tec GmbH, Germany) is a water soluble fluorescent dye withexcitation/emission maxima of 501nm/523nm and spectral qualities similar to FITC.DY-649 (Dyomics GmbH, Germany) is a water soluble fluorescent dye withexcitation/emission maxima of 655nm/676nm and spectral qualities similar to AlexaFlour 647.

Precautions1. Warning: The Dry-Tri T-STAT reagent contains sodium azide. Sodium

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azide is harmful if swallowed. Wear suitable protective clothing. Ifswallowed, seek medical advice immediately. Contact with acidsliberates toxic gas. Azides should be flushed with large amounts ofwater during disposal to avoid deposits in lead or copper plumbing.

2. Warning: All blood specimens are considered biohazards. Handlethem as if they are capable of transmitting infection and dispose offwith proper precautions and accordance with governmental

regulations.

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

Storage and Handling1. 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 or refrigerate Dry-Tri T-STAT (CD3/CD4/CD8)

reagent.3. The Dry-Tri T-STAT (CD3/CD4/CD8) reagent is light sensitive. Do not

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expose to direct light either during storage or when mixed withblood .

4. Use the ziplock to reseal the reagent pouch after you have removedthe desired number of tests. When stored below 40C sealed in the

ziplock pouch and protected from moisture and light the reagent isstable for 12 months.

Indication of Instability

The Dry Tri T-STAT (CD3/CD4/CD8) reagent is a dry glassy coating at the bottom ofthe reaction tube. Do not use if the reagents appears to be moist or it has becomedislodged from the bottom of the reaction tube.

INSTRUMENT

The Dry-Tri T-STAT (CD3/CD4/CD8) reagent is designed to be used on flow

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cytometers equipped with a 488nm laser, that are capable of detecting forward andside scatter, and detecting 3 color fluorescence: 515-545nm, 562-607nm, and>650nm. Examples of flow cytometers with the above features are the FACScan ,FACSCalibur and EPICS-XL systems. Calibrate the instrument for setting

photomultiplier tube voltages, fluorescence compensation, and checking instrumentsensitivity according to the manufacturers guidelines.

Refer to the manufacturers instructions for setting up three colorimmunophenotyping, principles of operation, operating instructions, and operatingprecautions, limitations, and hazards.

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 - 25 C) andshould be stained and analyzed within 24 hours of draw.

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Interfering conditionsRefrigerated or previously fixed blood specimens can yield erroneous results andshould be rejected. Hemolyzed, aged, or partially clotted blood specimens can affectflow cytometer performance.

PROCEDURE

Reagent ProvidedDry Tri T-STAT (CD3/CD4/CD8) reagent in a dried format

Reagents and materials required but not provided1. Blood collection tube containing K 3EDTA2. Calibrated pipettes3. Vortex mixer4. Lysing Solution A or similar blood fix/lyse solution

5. Sheath fluid B6. Flow cytometer Calibration Beads C

A. Lysing solution:

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(BD Catalog No. 349202 / BC Catalog No. A11895) or equivalentB. Sheath Fluid:

(BD Catalog No.342003/ BC Catalog No. 8546859) or equivalentC.Calibration Beads:

(BD Catalog No. 340486/ BC Catalog No. 6605359) or equivalent

Assay Protocol1. Start flow cytometer according to manufacturers instructions.2. In the case of FACScan or FACSCalibur , the instrument should

be set up and calibrated using CaliBrite beads and FACSComp software. For Beckman Coulter EPICS instruments, the instrument

should be set up using the Flow-Check fluorospheres provided byBeckman Coulter.3. Mix blood sample (invert blood tube at least 10 times) and pipette

50 L of blood into the correctly labeled tube that contains the reagent.4. Gently vortex each tube for 30 seconds. Incubate for 30 minutes

at room temperature . Protect the tube from direct light.

5. Add 450 L of lysing solution to each tube and vortex for 30 seconds.Return tubes to the dark for at least 15 minutes.

6. Vortex sample tube thoroughly (at low speed) and load ontocytometer for analysis.

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Note Once the sample has been stained and processed it can be held of 3 daysbefore analysis on the flow cytometer

Quality ControlCommercially available quality control cells can be run each day. Establishedvalues can be used to assess reagent and system performance.

RESULTS

For BD instruments

1. Draw three dot-plot views2. View 1 :

Display FL3 [Anti-CD3-PE-Dy-649 fluorescence] vs SSC [sidescatter]. Refer Figure 1.View 2:Display FL1 vs FL2. This view will have events gated as CD3+ fromView 1. Refer Figure 2.

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View 3:Display FL1 [Anti-CD4-Atto 488 fluorescence] vs FL2 [Anti-CD8-PEfluorescence]. This is not gated. Refer Figure 3.

3. Set the instrument Threshold on FL3. Before acquisition, adjust the

FL3 threshold to minimize debris. Ideally, the CD3+ cells in the firstview should account for one third of total events .4. Set the number of events to capture 10,000 events and start data

acquisition. In the case of low CD4 counts, it is recommended that atleast 3000 bead events are captured.

5. Gate the CD3+ cells in View 1 (Refer Figure 1) as R1. The CD3+ cells

will have low SSC intensity and high mean fluorescence intensity onFL3.6. Apply the gate R1 in View 2 (Refer Figure 2) to identify the

CD4+CD3+ and CD8+CD3+ cell populations. The CD4+CD3+ T-cellscan be identified as the population with the highest meanfluorescence intensity on FL1. The CD8+CD3+ T-cells can be

identified as the population with the highest mean fluorescenceintensity on FL2.7. Gate the absolute counting beads in View 3 (Refer Figure 3) as R2.

The beads will show high mean fluorescence intensities on FL2 and

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FL1.8. The absolute CD4+CD3+ and CD8+CD3+ T-cells counts can be

calculated using the following equation.

Absolute Cell Count per L = Gated Cell Count x Bead Count per testGated Bead Count x Test volume (in L)

where the Gated Cell count and Bead count are obtained from the flowcytometer analysis, the bead count per test from the test kit provided

Example:If the bead count per Dry Tri T-STAT CD3/CD4/CD8 test is 50,000, thevolume of blood tested is 50 l, the number of gated beads is 3,000 (Figure3 gated events), and the number of gated CD4+ T-cells is 1,500 (Figure 2LR quadrant) then the absolute CD4+ T-cell count is 500 cell/ l.

Absolute CD4+ T-cell count = 1500 x 50,000 = 500 cells/ l3,000 x 50

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Figure 1. Scatter plot of FL3 vs. side scatter. This view allows the gating of the CD3+ population

Figure 2. Scatter plot of FL1 versus FL2(gated). This view is used to determine theabsolute count of CD4+CD3+ and CD8+CD3+T-cells.

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Figure 3 . Scatter plot of FL1 versus FL2 (ungated). This view can be used to determine the absolute number of beads

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For Beckman-Coulter instruments (Epics series)

1. Draw three dot-plot views2. View 1:

Display FL4 [Anti-CD3-PE-Dy-649 fluorescence] vs SSC [sidescatter]. Refer Figure 4.View 2:Display FL1 vs FL2. This view will have events gated as CD3+ fromView 1. Refer Figure 5.View 3:Display FL1 [Anti-CD4-Atto 488 fluorescence] vs FL2 [Anti-CD8-PEfluorescence]. This is not gated. Refer Figure 6.

3. Before acquisition, adjust the FL4 threshold to minimize debris.Ideally, the CD3+ cells in the first view should account for one third oftotal events .

4. Set the number of events to 10,000 and start data acquisition. In thecase of low CD4 counts, it is recommended that at least 3000 beadevents are captured.

5. Gate the CD3+ cells in View 1 (Refer Figure 4) as R1. The CD3+ cells

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will have low SSC intensity and high mean fluorescence intensity onFL4.

6. Apply the gate R1 in View 2 (Refer Figure 5) to identify theCD4+CD3+ and CD8+CD3+ cell populations. The CD4+CD3+ T-cells

can be identified as the population with the highest meanfluorescence intensity on FL1. The CD8+CD3+ T-cells can beidentified as the population with the highest mean fluorescenceintensity on FL2.

7. Gate the absolute counting beads in View 3 (Refer Figure 6) as R2.The beads will show high mean fluorescence intensities on FL2 and

FL1.8. The absolute CD4+CD3+ and CD8+CD3+ T-cells counts can becalculated using the following equation.

Absolute Cell Count per L = Gated Cell Count x Bead Count per testGated Bead Count x Test volume (in L)

where the Gated Cell count and Bead count are obtained from the flowcytometer analysis and the bead count per test is obtained from the test kitprovided

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Figure 6 . Scatter plot of FL1 versus FL2 (ungated). This view can be used to determine the absolute number of beads

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( F L 2 ) C D 8

- P E

(FL1) CD4-Atto488

( F L 2 ) C D 8

- P E

(FL1) CD4-Atto488

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LIMITATIONS1. The Dry Tri T-STAT CD3/CD4/CD8 reagent has only been validated

with K3EDTA treated whole blood.2. Laboratories should establish their own reference ranges for the

absolute counts obtained using the Dry Tri T-STAT CD3/CD4/CD8reagent.

EXPECTED RESULTS

The absolute CD4+ and CD8+ T lymphocyte concentrations were measured for anormal population of adults in southern India, unselected gender and ages rangingfrom 21 to 80 years old, using the Dry Tri T-STAT (CD3/CD4/CD8) reagent. Valuesobtained from this diverse population are displayed below and consistent with thereported normal range (10).

We recommend that each laboratory establish its own expected range of valuesfrom the local population of normal donors.

nMinimu

m Maximum Mean SD

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CD3+CD4+ T-cells 114 457 2888 1015 348CD3+CD8+ T-cells 114 195 1402 645 256

PERFORMANCE CHARACTERISTICS

Correlation

The absolute CD3+CD4+ and CD3+CD8+ T-cells concentrations for 96 bloodsamples determined using the Dry Tri T-STAT (CD3/CD4/CD8) reagent werecompared to those values determined using the Beckman Coulter Cyto-Stat tetraCHROME and Becton Dickinson TriTest reagents. The Deming linearregression analysis reported in the table below indicates substantial equivalence.

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Deming Linear Regression 95% CIParameter Slope Y-intercept X-intercept Slope Y-intercept

Site 1CD3+CD4+ T-cell

Regression 0.868 -3.28 3.78 0.841 - 0.896 -16.5 10.0

CD3+CD8+ T-cellRegression 0.899 35.0 -38.9 0.845 0.954 -30.7 100


Regression0.970 -8.47 8.73 0.917 1.02 -34.7 17.7

CD3+CD8+ T-cellRegression 1.044 -58.3 55.8 0.978 1.11 -136 19.3


Regression 0.982 5.85 -5.95 0.922 1.04 -20.0 31.7


Regression 0.901 12.99 -14.42 0.871 0.931 -2.67 28.7

CD3+CD8+ T-cellRegression 0.884 2.49 -2.82 0.831 - 0.936 -64.3 69.3

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Linearity

Assay linearity was determined by testing six point dilution series, 64-1680CD3+CD4+ T-cells/ l and 32-1640 CD3+CD8+ T-cells/ l. The measurement ateach level was performed in triplicate. The assay was determined to be linear forboth CD3+CD4+ and CD3+CD8+ T-cells with correlation coefficients (R 2) of 0.995and 0.999, respectively.

The two dilution series spanned the specified reportable ranges of 65-1500CD3+CD4+ T-cells/ l and 40-1500 CD3+CD8+ T-cells/ l.

Precision

The repeatability of measurement was determined for three levels of CD3+CD4+and CD3+CD8+ T-cells counts analyzed in replicates of ten. The results aretabulated below.

Level Mean SD %CVCD3+CD4+ T- Low 29 1.7 6

Medium 607 23.6 3.9

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cellsHigh 1173 66.3 5.7

CD3+CD8+ T-cells

Low 655 45.1 6.9Medium 770 37.1 4.8

High 1557 30 1.9

SpecificityThe antigen specificities for the CD3, CD4, and CD8 monoclonal antibodies used inthe Dry Tri T-STAT (CD3/CD4/CD8) reagent were confirmed by the HumanLeukocyte Differentiation Antigens (HLDA) Workshops.After conjugation of the antibodies no additional cross-reactivity was observed.

BIBLIOGRAPHY

1. Knapp W, Dorken B, Gilks WR, Rieber EP, Schmidt RE, Stein H, & von demBorneAEGKr, eds. Leukocyte typing IV. White cell differentiation antigens.Oxford university press 1989

2. Reinherz EL, Meuer SC, and Schlossman SF. The delineation of antigen

receptors on human T lymphocytes. Immunology Today, 1983, 4:5-8.3. Fauci AS: The human deficiency virus: Infectivity and mechanism of

pathogenesis. Science, 1988, 239:617-622.4. Reinherz EL and Schlossman SF. The differentiation and function of human T

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lymphocytes. Cell, 1980, 19:821-827.5. Phillips A, Lee C, Elford J, et al. Serial CD4 lymphocyte counts and the

development of AIDS. Lancet 1991, 337:389-392.6. Nicholson J. Use of flow cytometry in the evaluation and diagnosis of primary

and secondary immunodeficiency diseases. Arch. Pathol. Lab. Med. 1989,113:598-605.

7. Yarchoan R, Venzon D, Pluda J, et al. CD4 count and the risk of death inpatients infected with HIV receiving antiretroviral therapy. 1991, 115:184-189.

8. Giorgi J & Detels R. T-cell subset alterations in HIV-infected homosexual men:NIAID multicenter AIDS cohort study. 1989, 52:10-18.

9. Heddy Z, Swart B, Nicholson I, & Voss E, eds. Leukocyte and stromal cellmolecules; the CD markers. John Wiley & Sons 2007

10. Uppal SS, Verma S, Dhot PS. Normal values of CD4 and CD8 lymphocytesubsets in healthy Indian adults and the effects of sex, age, ethnicity, andsmoking. Cytometry Part B 2003, 52B:32-36

WARRANTY

This product is warranted only to conform to the quantity and contents stated onthe label at the time of delivery to the customer. There are no warranties,

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expressed or implied, that extend beyond the description on the label of theproduct. ReaMetrixs sole liability is limited to replacement of the product.Reametrix is not liable for property damage, personal injury, or economic losscaused by the product.

Note: FACScan, CaliBRITE, FACSLyse, FACSComp, FACSCalibur are all registered trade names of Becton-Dickinson . Alexa Fluor 647 is a registered trade nameof Invitrogen.

ReaMetrix Inc.1585 Industrial Rd.San Carlos, CA 94070

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(650)620-9253

Manufactured by ReaMetrix India Pvt. Ltd.Manufacturing License Number: KTK/25/519/2006

50-B, II Phase, Peenya Industrial AreaPeenya, Bangalore 560058, IndiaPh: +91-80-28378693/5, Fax: +91-80-41172451E-mail: [email protected],www.reametrix.com

Rev No. 4.0, 08-May-09

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PI-25118-00-DTTS (CD3CD4CD8)