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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.
View 4: CD4 Atto488 (FL1) versus CD45 PE fluorescence
(FL2) of all events from first view. View-4 will have a gate ( D) tocapture the fluorescent reference beads. The beads have a
high FL1 and FL2 Mean Fluorescence Intensity.
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View 5: CD4 Atto488 (FL1 log scale) versus CD3 PE-
Dyomics649 fluorescence (FL4 log scale) of events gated as
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.
4. Gate the brightest CD45+ cell cluster in View-1(Figure 2) and label
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.
View 3: CD4 Atto488 (FL1 log scale) versus CD3 PE-
Dyomics649 fluorescence (FL3 log scale) of events gated as
P1 (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 P2 to
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capture the fluorescent reference beads. The beads have a
high FL1 and FL2 Mean Fluorescence Intensity.
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.
6. Gate the brightest CD45+ cell cluster in View-1(Figure 3) and label
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 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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