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1 Test per tube

Product Catalog No: 25197-00

Dry-Tri T-STAT

CD3/CD4/CD45Reagent

Manufactured by

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Dry-Tri T-STAT CD3/CD4/CD45

Reagent1. INTENDED USE

The Dry-Tri T-STAT CD3/CD4/CD45 reagent is a three color

immunofluorescence stain for the labeling, identification, and enumeration of

mature human total T-lymphocytes (CD3+), and helper/inducer T-

lymphocytes (CD3+CD4+) combined with a precise number of fluorescentcounting beads for absolute CD4+ and CD3+ T-Cell counts and

percentages. This reagent is intended to be used in a lyse-no wash

protocol for flow cytometry analysis of erythrocyte-lysed human whole blood.

2. SUMMARY AND EXPLANATION

The Dry-Tri T-STAT CD3/CD4/CD45 reagent contains fluorescently labeled

antibodies that bind to CD45, CD3, and CD4 antigens found on the surface

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of circulating leukocytes. 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

(1).

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) (2, 3).

The CD45 antigen is a complex 180-220kDa transmembrane glycoprotein

which is expressed in high levels on leukocyte cell surfaces and its presencedistinguishes leukocytes from non-haematopoietic cells (1).

Clinical relevance

Identification of abnormal levels of CD4+ T-cells and corresponding % CD4

have been associated with certain types of immunodeficiency disease. For

example infection with human immunodeficiency virus (HIV), results in

profound immunosuppression due predominantly to a selective depletion of

the CD4+ lymphocytes that express the receptor for the virus. Progressive

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clinical and immunologic deterioration generally correlates with a decreasing

CD4+ lymphocyte count. (5-8).

3. PRINCIPLE OF THE PROCEDURE

The Dry Tri T-STAT CD3/CD4/CD45 reagent consists of murine monoclonal

antibodies that specifically recognize the human leukocyte surface antigens,

CD3, CD4, and CD45. Each of the monoclonal antibodies is labeled with a

unique fluorochrome. Specific cell subsets are stained when blood is

combined with the reagent and each monoclonal antibody binds to the binds

to the cell determinant molecules on the cell surface. Specific cell subsetsare identified when passed through a flow cytometer laser beam.

The Dry Tri T-STAT CD3/CD4/CD45 reagent also contains a precise number

of fluorescent beads. When the reagent is combined with a known volume

of blood the reagent provides for the single platform determination of the

absolute cell concentrations of the stained subsets. The volume of sampleanalyzed can be determined by multiplication of the total sample volume by

the fraction of total beads that were detected during the analysis.

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4. REAGENT

The Dry-Tri T-STAT CD3/CD4/CD45 reagent is formulated in buffered saline

with sodium azide and stabilizers. It contains Atto488 labeled CD4

monoclonal antibody, clone RPA-T4; Phycoerythrin (PE) labeled CD45monoclonal antibody, clone HI30; and PEDyomics649 labeled CD3

monoclonal antibody, clone UCHT1. The monoclonal antibodies used in the

Dry-Tri T-STAT CD3/CD4/CD45 were assigned these specificities at the 8th

International Workshop on Human Leukocyte Differentiation Antigens (9). A

precise number of fluorescent counting beads are included in the Dry-Tri T-

STAT CD3/CD4/CD45 reagent to allow single-platform determination ofabsolute CD4+ and CD3+ T-cell counts. The Dry-Tri T-STAT

CD3/CD4/CD45 reagent is provided in dried form and dispensed in flow

cytometer compatible sample tubes with each tube containing one ready-to-

use test. Fifty (50) tests are supplied in each Dry Tri T-STAT

CD3/CD4/CD45 package.

Precautions

1. Warning: The Dry-Tri T-STAT CD3/CD4/CD45 reagent

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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 accordingto the manufacturers 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 or refrigerate Dry-Tri T-STAT CD3/CD4/CD45

reagent.

3. The Dry-Tri T-STAT CD3/CD4/CD45 reagent is light sensitive.

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Do not expose to direct light either during storage or when

mixed with blood.

4. Use the ziplock to reseal the reagent pouch after you have

removed the desired number of tests.

Indication of Instability

The Dry Tri T-STAT CD3/CD4/CD45 reagent is a dry glassy coating at the

bottom of the reaction tube. Do not use if the reagents appears to be moist

or it has become dislodged from the bottom of the reaction tube.

5. INSTRUMENT

The Dry-Tri T-STAT CD3/CD4/CD45 reagent is designed to be used on flow

cytometers equipped with a 488nm laser, that are capable of detecting

forward and side scatter, and detecting three color fluorescence in threeranges: 515-545nm, 562-607nm, and >650nm. Examples of flow cytometers

with the above features are the BD FACScan, BD FACSCalibur, Coulter

EPICS-XL and Accuri flow cytometer systems. Calibrate the instrument

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for setting photomultiplier tube voltages, fluorescence compensation, and

checking instrument sensitivity according to the manufacturers guidelines.

Refer to the manufacturers instructions for setting up three color

immunophenotyping, principles of operation, operating instructions, andoperating precautions, limitations, and hazards.

6. SPECIMEN COLLECTION

The blood sample should be collected in a sterile blood collection tube

containing K3EDTA. Follow the collection tube manufacturers guidelines forthe 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.

Interfering conditionsRefrigerated, hemolyzed, and previously fixed blood specimens can yield

erroneous results and should be rejected. Aged or partially clotted blood

specimens can affect flow cytometer performance.

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7. PROCEDURE

Reagent Provided: Dry-Tri T-STAT CD3/CD4/CD45 reagent in a dried

format.

Reagents and Materials required but not provided

1. Blood collection tube containing K3EDTA

2. Calibrated pipettes

3. Vortex mixer

4. Lysing SolutionA or similar blood fix/lyse solution

5. Sheath fluidB

6. 0.2m filtered, deionized distilled water or Milli-Q waterC

7. Flow cytometer Calibration BeadsD

A. ReaLyse Lysing solution: ReaMetrix Catalog No: 25237-00

B. Sheath fluid: BD Catalog No.342003/ BC Catalog No. 8546859 or equivalent

C. Accuri Flow cytometer uses Milli-Q water/Distilled deionized water instead of

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Sheath fluid

D. Calibration Beads: BD Catalog No. 340486/ BC Catalog No. 6605359 or

equivalent

Assay Protocol

1. Start flow cytometer according to manufacturers instructions.(In the case of FACScan or FACSCalibur, the instrument should

be setup 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 by

Beckman Coulter. For Accuri flow cytometer instruments, theperformance of the instrument should be checked by Validation

beads provided by Accuri.)

3. Mix blood sample (invert blood tube at least 10 times) and

reverse pipette 50L of blood into the correctly labeled tube

that contains the dry down reagent.

3. Gently vortex each tube for 30 seconds. Incubate for 30minutes at room temperature. Protect the tube from direct

light.

4. Add 450L of 1X ReaLyse lysing solution to each tube and

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View 3: CD4 Atto488 (FL1) versus CD45 PE fluorescence

(FL2) of all events from first view. View-3 will have a gate (R2)

to capture the fluorescent reference beads. The beads have a

high FL1 and FL2 Mean Fluorescence Intensity.

2. Set the instrument Threshold on FL2. Before acquisition adjust theFL-2 threshold to minimize debris.

3. Set the number of events to capture at 3000 for R-2 gate bead

events (Figure 1; View 3) and start data acquisition. When 3000

beads are acquired in the gate, acquisition stops.

4. Gate the brightest CD45+ cell cluster in View-1(Figure 1) and label

the population as R-1. This CD45+ cell cluster is that population

which has the lower SSC intensity and higher FL-2 mean

fluorescence intensity. It represents the lymphocyte population. The

Gate R-1 is applied on View-2.

5. View-2 (Figure 1) cell clusters are separated using quadrant gating.

The CD4+CD3+ cell populations can be identified in View-2 (Figure

1) in the upper right quadrant as double positives. Total CD3+

events can be identified by adding up the cell events from both the

upper quadrants (Upper Left and Upper Right quadrants).

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6. The absolute count for each cell type can be calculated using the

following equation, where the Gated Cell count and Bead count are

obtained from the Flow Cytometer analysis and the Bead count per

test from the test kit provided.

inVolume(TestxCount)Bead(Gated

tperCount(BeadxCount)Cell(GatedL/CountCellAbsolute =

Figure 1. Dot plot views: View 1: CD45-PE fluorescence (FL2 log scale) versus

side scatter (SSC Linear scale) View 2: CD4-Atto488 (FL1 log scale) versus

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CD3-PE-Dyomics649 fluorescence (FL3 log scale) of events gated as CD45+

R1 from the first view. View 3: CD4-Atto488 (FL1) versus CD45-PE

fluorescence (FL2) of all events from first view. View-3 will have a gate R2 to

capture the fluorescent reference beads.

For Beckman-Coulter Flow Cytometer instruments (EPICS series)

1. Draw five dot-plot views Refer Figure 2.

View 1: Display CD45 PE fluorescence (FL2 - log scale)

versus side scatter (SSC Linear scale)

View 2: side scatter (SSC Linear scale) versus CD3 PE-

Dyomics649 (FL4 - log scale)

View 3: CD4 Atto488 (FL1 log scale) versus CD3 PE-

Dyomics649 fluorescence (FL4 log scale) of events gated as

A (CD45+) from the first view.


(FL2) of all events from first view. View-4 will have a gate ( D) tocapture the fluorescent reference beads. The beads have a


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D (Beads) from the fourth view. View-5 will have a gate (E) to

capture the fluorescent reference beads

2. Set the instrument Discriminator on FL2. Before acquisition adjustthe FL-2 Discriminator to minimize debris.

3. Set the number of events to capture at 3000 for E gate bead events

(Figure 2; View 5) and start data acquisition. When 3000 beads are

acquired in the gate E, acquisition stops.


the population as A. This CD45+ cell cluster is that population

which has the lower SSC intensity and higher FL-2 mean

fluorescence intensity. It represents the lymphocyte population. The

Gate A is applied on View-2 and 3.

5. View-2 (Figure 2); CD3+ cell clusters are separated using gate B.

The CD3+ cell population has the lower SSC intensity and higher

FL-4 mean fluorescence intensity.

6. View-3 (Figure 2) cell clusters are separated using quadrant gating

C. The CD4+CD3+ cell populations can be identified in View-3

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(Figure 2) in the upper right quadrant (C2) as double positives.

7. View-4 (Figure 2); fluorescent reference beads are separated using

gate D. The beads have a high FL1 and FL2 Mean Fluorescence

Intensity. The gate D is applied on View-5.

8. View-5 (Figure 2); fluorescent reference beads are separated usinggate E. Set the number of events to capture at 3000 for E gate

bead events.

9. Enter the Calibrator (CAL) value in the Region E on the template to

generate absolute cell counts. The Calibrator value is calculated

using bead count per test from the test kit provided.

(L)VolumeTest

TestperCountBead(CAL)Calibrator =

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Figure 2. Dot plot views: View 1: CD45-PE fluorescence (FL2 log scale) versus sidescatter (SSC Linear scale) View 2: CD3-PE-Dyomics649 fluorescence (FL4 log

scale) versus side scatter (SSC Linear scale) View 3: CD4-Atto488 (FL1 log scale)

versus CD3-PE-Dyomics649 fluorescence (FL4 log scale) of events gated as A

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(CD45+) from the first view. View 4: CD4-Atto488 (FL1) versus CD45-PE

fluorescence (FL2) of all events from first view. View-4 will have a gate D to capture

the fluorescent beads View 5: CD4-Atto488 (FL1 log scale) versus CD45-PE

fluorescence (FL2) of events gated as D (Beads) from the fourth view. View-5 will

have a gate E to capture the fluorescent reference beads

For Accuri Flow Cytometer Instruments

1. Draw four dot-plot views Refer Figure 3.

View 1: Display CD45 PE fluorescence (FL2 - log scale)

versus side scatter (SSC Linear scale) View 2: Side scatter (SSC Linear scale) versus CD3 PE-

Dyomics649 (FL3 - log scale) of events gated as P1 (CD45+)

from the first view.



P1 (CD45+) from the first view.


(FL2) of all events from first view. View-4 will have a gate P2 to

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capture the fluorescent reference beads. The beads have a


Following table (Table-1) represents the Plot Specification Value to

set the scales:

Table-1: Dot Plot View X & Y-Axis Scale Specifications

Dot Plot

ViewsAxis Scale Min Value Max value

View 1X-Axis FL2-Log scale 800 1,000,000

Y-Axis SSC-Linear scale 0 180,000

View 2

X-Axis FL3-Log scale 300 600,000

Y-Axis SSC-Linear scale 0 150,000

View 3X-Axis FL1-Log scale 1000 200,000

Y-Axis FL3-Log scale 100 5,000,000

View 4X-Axis FL1-Log scale 1000 10,000,000

Y-Axis FL2-Log scale 500 2,000,000

2. In the Run Limit section of the Instrument template panel, set theinstrument Threshold on FL2 and set the value to 2000.

Before/After acquisition adjust the FL-2 Threshold value to

minimize debris.

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3. In the Run Limit section of the Instrument template panel, set the

fluidics flow rate on MEDIUM. Set the number of events to capture

at 3000 for P2 gate bead events (Figure 3; View 4).

4. Click on the Set Colour Compensation to open the compensation

matrix and set the compensation as per Figure 4.5. Vortex sample tube thoroughly at low speed and load onto flow

cytometer for acquisition. When 3000 bead events are acquired in

the gate P2, acquisition stops.


the population as P1. This CD45+ cell cluster is that population

which has the lower SSC intensity and higher FL2 meanfluorescence intensity. It represents the lymphocyte population. The

Gate P1 is applied on View-2 and 3.

7. CD3+ cell clusters are separated using gate R1 (View-2, Figure 3).

The CD3+ cell population has the lower SSC intensity and higher

FL3 mean fluorescence intensity.

8. In View-3 (Figure 3) cell clusters are separated using quadrant gate

Q1. The CD4+CD3+ cell populations can be identified in View-3

(Figure 3) in the upper right quadrant Q1-UR as double positives.

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9. View-4 (Figure 3); fluorescent reference beads are separated using

gate P2. The beads have a high FL1 and FL2 Mean Fluorescence

Intensity.

10. The absolute count for each cell type can be calculated using the

following equation, where the Gated Cell count and Bead count areobtained from the Flow Cytometer analysis and the Bead count per

test from the test kit provided.

)LinVolume(TestxCount)Bead(Gated

test)perCount(BeadxCount)Cell(GatedL/CountCellAbsolute =

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View 1 View 2

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View 3 View 4

Figure 3. Dot plot views: View 1: CD45-PE fluorescence (FL2 log scale) versus side

scatter (SSC Linear scale) View 2: CD3-PE-Dyomics649 fluorescence (FL3 log

scale) versus side scatter (SSC Linear scale) View 3: CD4-Atto488 (FL1 log scale)

versus CD3-PE-Dyomics649 fluorescence (FL3 log scale) View 4: CD4-Atto488 (FL1

log scale) versus CD45-PE fluorescence (FL2 log scale)

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Figure 4: Accuri Flow cytometer Compensation settings for Dry-Tri T-STAT

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CD3CD4CD45 Reagent

9. LIMITATIONS1. The Dry-Tri T-STAT CD3/CD4/CD45 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/CD45 reagent.

10. PERFORMANCE CHARACTERISTICS

Accuracy

The absolute counts for CD4+CD3+ and CD3+ T-Cells determined usingDry-Tri T-STAT CD3/CD4/CD45 reagent were compared with a

commercially available ReaPan 34845 reagent. Aliquots of the same blood

samples were analyzed with the two reagents at the same time. Regression

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analysis reported in the Table-2 below and the plots in Figures 5 - 8 indicate

that the results are substantially equivalent.

Table-2. Regression analysis

T-Cell Subset n Slope Intercept R Range

CD3+CD4+ T-Cells (cells/L) 53 1.09 -19 0.99 9 - 1786

Total T lymphocytes - CD3+ (cells/L) 53 1.00 71 0.96 243 - 3577

CD3+CD4+ T-Cells (%) 53 1.00 -1.1 0.99 3.3 55.6

Absolute lymphocyte count (cells/L) 53 1.03 65 0.96 280 - 4069

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Figure 5. Correlation of absolute CD4+CD3+

T-Cell counts determined using the Dry-Tri T-

STAT CD3/CD4/CD45 reagent and comparedto ReaPan 34845 reagent

Figure 6. Correlation of % CD4 determined

using the Dry-Tri T-STAT CD3/CD4/CD45

reagent and compared to ReaPan 34845reagent

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Figure 7. Correlation of absolute CD3+ T-Cell

counts determined using the Dry-Tri T-STAT

CD3/CD4/CD45 reagent and compared toReaPan 34845 reagent

Figure 8. Correlation of Absolute

Lymphocyte counts (ALC) determined using

the Dry-Tri T-STAT CD3/CD4/CD45 reagentand compared to ReaPan 34845 reagent

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Precision

The repeatability of measurement was determined for three samples

representing high, medium and low CD3+CD4+ and CD3+ T-cells counts

were analyzed by performing triplicates per level. These results are

summarized in Table-3.

Table-3. Repeatability of CD4+CD3+ and CD3+ T-Cell enumeration

Cell Type Level Mean SD CV (%)

CD4+CD3+ T-Cells

High 917 67.5 7.3

Medium 378 9.5 2.5

Low 147 4.7 3.1

CD3+ T-Cells

High 1812 104.0 5.7

Medium 891 29.9 3.2

Low 361 31.7 3.1

Specificity

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The antigen specificities for the CD3, CD4, and CD45 monoclonal antibodies

used in the Dry Tri T-STAT CD3/CD4/CD45 reagent were confirmed by the

Human Leukocyte Differentiation Antigens (HLDA) Workshops. After

conjugation of the antibodies no additional cross-reactivity was observed.

11. BIBLIOGRAPHY

1. Knapp W, Dorken B, Gilks WR, Rieber EP, Schmidt RE, Stein H, &

von dem BorneAEGKr, eds. Leukocyte typing IV. White cell

differentiation antigens. Oxford university press 1989

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

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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 in patients 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 cell molecules; the CD markers. John Wiley & Sons 2007

12. 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.

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Note: FACScan, FACSCalibur, CaliBRITE, FACSComp are all registered trade

names of Becton-Dickinson; EPICS, Flow-Checkare all registered trade names of

Beckman Coulter, Accuri C6 flow cytometer is the trade name of Accuri.

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Manufactured by ReaMetrix India Pvt. Ltd.

Manufacturing License Number: KTK/25/519/2006

50-B, II Phase, Peenya Industrial Area

Peenya, Bangalore 560058, IndiaPh: +91-80-28378693/5,

Fax: +91-80-41172451

E-mail: [email protected],

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www.reametrix.com

Rev No. 4.0, 22-Sep-09

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PI-25197-00-DTTS (CD3CD4CD45)