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Product catalog #: 2511800
Dry Tri TSTAT
(CD3/CD4/CD8)
Reagent
For In Vitro Diagnostic Use
Manufactured by
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Dry Tri TSTAT (CD3/CD4/CD8) Reagent
INTENDED USE
The Dry Tri TSTAT (CD3/CD4/CD8) reagent is a three color immunofluorescence
stain for the labeling, identification, and enumeration of helper/inducer (CD3+CD4+)
and cytotoxic/suppressor (CD3+CD8+) T lymphocytes combined with a precise
number of fluorescent counting beads for absolute CD4+ and CD8+ TCell counts.
This reagent is intended to be used in a “no wash” protocol for flow cytometric
analysis of erythrocytelysed human whole blood.
SUMMARY AND EXPLANATION
The DryTri TSTAT reagent contains fluorescently labeled antibodies that bind to
CD3, CD4, and CD8 antigens found on the surface of circulating leukocytes. The
CD3 antigen is a complex of at least six proteins known collectively as the Tcell
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
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II molecules of the major histocompatibility complex and is the primary receptor for
the Human Immunodeficiency Virus (HIV) (2, 3).
The CD8 antigen is a complex consisting of two disulfide linked subunits. The
antibody used in this reagent binds to the 32kDa subunit of the complex. CD8
interacts with class I major histocompatibility complex molecules (4).
Clinical relevance
Cells that are both CD3+ and CD4+ are identified as helper/inducer lymphocytes.
Decreased CD4+CD3+ Tcell counts have been associated with some forms of
immunodeficiency (such as HIV). Suppressor/cytotoxic lymphocytes are the subset
of cells that have both CD3 and CD8 receptors (57). Increased CD8+CD3+ Tcell
counts have been observed in some cases of immunodeficiency (8).
PRINCIPLES OF THE PROCEDURE
REAGENT
The DryTri TSTAT reagent is formulated in buffered saline with sodium azide and
stabilizers. It contains ATTO 488 – labeled CD4 monoclonal antibody, clone RPA
T4; phycoerythrin (PE) – labeled CD8 monoclonal antibody, clone LT8; and PEDY
649 – labeled CD3 monoclonal antibody, clone UCHT1. The specificities of the
monoclonal antibodies used in the Dry Tri TSTAT (CD3/CD4/CD8) were confirmed
by 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 TSTAT reagent to
allow singleplatform determination of absolute CD4+ and CD8+ Tcell counts. The
DryTri TSTAT reagent is provided in dried form and dispensed in flow cytometer
compatible sample tubes with each tube containing one readytouse test. Fifty (50)
tests are supplied in each Dry Tri TSTAT (CD3/CD4/CD8) package.
The absolute number of counting beads in each test was set by determining the
number of beads in a representative number of tubes after the reagent was
dispensed. The reagent was solubilized with a precise volume of fluid and
transferred to a chamber where the number of beads per unit volume dould be
counted.
ATTO 488 (AttoTec GmbH, Germany) is a water soluble fluorescent dye with
excitation/emission maxima of 501nm/523nm and spectral qualities similar to FITC.
DY649 (Dyomics GmbH, Germany) is a water soluble fluorescent dye with
excitation/emission maxima of 655nm/676nm and spectral qualities similar to Alexa
Flour® 647.
Precautions
1. Warning: The DryTri TSTAT reagent contains sodium azide. Sodium
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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 precise volume of blood is critical to obtain correct
results. Use a calibrated pipette and operate according to the
manufacturer’s 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 DryTri TSTAT (CD3/CD4/CD8)
reagent.
3. The DryTri TSTAT (CD3/CD4/CD8) reagent is light sensitive. Do not
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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. When stored below 40ºC sealed in the
ziplock pouch and protected from moisture and light the reagent is
stable for 12 months.
Indication of Instability
The Dry Tri TSTAT (CD3/CD4/CD8) 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.
INSTRUMENT
SPECIMEN COLLECTION
The blood sample should be collected in a sterile blood collection tube containing
K3EDTA. Follow the collection tube manufacturer’s guidelines for the minimum
volume of blood to be collected.
The anticoagulated blood must be stored at room temperature (20°C 25°C) and
should be stained and analyzed within 24 hours of draw.
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Interfering conditions
Refrigerated or previously fixed blood specimens can yield erroneous results and
should be rejected. Hemolyzed, aged, or partially clotted blood specimens can affect
flow cytometer performance.
PROCEDURE
Reagent Provided
Dry Tri TSTAT (CD3/CD4/CD8) 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. Flow cytometer Calibration BeadsC
A. Lysing solution:
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(BD Catalog No. 349202 / BC Catalog No. A11895) or equivalent
B. Sheath Fluid:
(BD Catalog No.342003/ BC Catalog No. 8546859) or equivalent
C. Calibration Beads:
(BD Catalog No. 340486/ BC Catalog No. 6605359) or equivalent
Assay Protocol
1. Start flow cytometer according to manufacturer’s 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 FlowCheck™ fluorospheres provided by
Beckman Coulter.
3. Mix blood sample (invert blood tube at least 10 times) and pipette
50µL 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 450µL 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 onto
cytometer for analysis.
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Note – Once the sample has been stained and processed it can be held of 3 days
before analysis on the flow cytometer
Quality Control
Commercially available quality control cells can be run each day. Established
values can be used to assess reagent and system performance.
RESULTS
For BD instruments
1. Draw three dotplot views
2. View 1 :
Display FL3 [AntiCD3PEDy649 fluorescence] vs SSC [side
scatter]. Refer Figure 1.
View 2:
Display FL1 vs FL2. This view will have events gated as CD3+ from
View 1. Refer Figure 2.
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View 3:
Display FL1 [AntiCD4Atto 488 fluorescence] vs FL2 [AntiCD8PE
fluorescence]. 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 first
view 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 at
least 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 on
FL3.
6. Apply the gate R1 in View 2 (Refer Figure 2) to identify the
CD4+CD3+ and CD8+CD3+ cell populations. The CD4+CD3+ Tcells
can be identified as the population with the highest mean
fluorescence intensity on FL1. The CD8+CD3+ Tcells can be
identified as the population with the highest mean fluorescence
intensity 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+ Tcells counts can be
calculated using the following equation.
Absolute Cell Count per µ L = Gated Cell Count x Bead Count per test
Gated Bead Count x Test volume (in µ L)
where the Gated Cell count and Bead count are obtained from the flow
cytometer analysis, the bead count per test from the test kit provided
Example:
If the bead count per Dry Tri TSTAT CD3/CD4/CD8 test is 50,000, the
volume of blood tested is 50µl, the number of gated beads is 3,000 (Figure
3 gated events), and the number of gated CD4+ Tcells is 1,500 (Figure 2
LR quadrant) then the absolute CD4+ Tcell count is 500 cell/µl.
Absolute CD4+ Tcell count = 1500 x 50,000 = 500 cells/µl
3,000 x 50
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Figure 1. Scatter plot of FL3 vs. side Figure 2. Scatter plot of FL1 versus FL2
scatter. This view allows the gating of the (gated). This view is used to determine the
CD3+ population absolute count of CD4+CD3+ and CD8+CD3+
Tcells.
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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 BeckmanCoulter instruments (Epics™ series)
1. Draw three dotplot views
2. View 1:
Display FL4 [AntiCD3PEDy649 fluorescence] vs SSC [side
scatter]. Refer Figure 4.
View 2:
Display FL1 vs FL2. This view will have events gated as CD3+ from
View 1. Refer Figure 5.
View 3:
Display FL1 [AntiCD4Atto 488 fluorescence] vs FL2 [AntiCD8PE
fluorescence]. 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 of
total events.
4. Set the number of events to 10,000 and start data acquisition. In the
case of low CD4 counts, it is recommended that at least 3000 bead
events 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 on
FL4.
6. Apply the gate R1 in View 2 (Refer Figure 5) to identify the
CD4+CD3+ and CD8+CD3+ cell populations. The CD4+CD3+ Tcells
can be identified as the population with the highest mean
fluorescence intensity on FL1. The CD8+CD3+ Tcells can be
identified as the population with the highest mean fluorescence
intensity 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+ Tcells counts can be
calculated using the following equation.
Absolute Cell Count per µ L = Gated Cell Count x Bead Count per test
Gated Bead Count x Test volume (in µ L)
where the Gated Cell count and Bead count are obtained from the flow
cytometer analysis and the bead count per test is obtained from the test kit
provided
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Example:
If the bead count per Dry Tri TSTAT CD3/CD4/CD8 test is 50,000, the
volume of blood tested is 50µl, the number of gated beads is 3,000 (Figure
6 gated events), and the number of gated CD4+ Tcells is 1,500 (Figure 5
LR quadrant) then the absolute CD4+ Tcell count is 500 cell/µl.
Absolute CD4+ Tcell count = 1500 x 50,000 = 500 cells/µl
3,000 x 50
(FL2) CD8PE
Side Scatter
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(FL4) CD3PEDy649 (FL1) CD4Atto488
Figure 6. Scatter plot of FL1 versus FL2 (ungated). This view can be used to determine the
absolute number of beads
(FL2) CD8PE
(FL1) CD4Atto488
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LIMITATIONS
1. The Dry Tri TSTAT 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 TSTAT CD3/CD4/CD8
reagent.
EXPECTED RESULTS
The absolute CD4+ and CD8+ T lymphocyte concentrations were measured for a
normal population of adults in southern India, unselected gender and ages ranging
from 21 to 80 years old, using the Dry Tri TSTAT (CD3/CD4/CD8) reagent. Values
obtained from this diverse population are displayed below and consistent with the
reported normal range (10).
We recommend that each laboratory establish its own expected range of values
from the local population of normal donors.
Minimu
n m Maximum Mean±SD
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CD3+CD4+ Tcells 114 457 2888 1015±348
CD3+CD8+ Tcells 114 195 1402 645±256
PERFORMANCE CHARACTERISTICS
Correlation
The absolute CD3+CD4+ and CD3+CD8+ Tcells concentrations for 96 blood
samples determined using the Dry Tri TSTAT (CD3/CD4/CD8) reagent were
compared to those values determined using the Beckman Coulter CytoStat®
tetraCHROME™ and Becton Dickinson TriTest™ reagents. The Deming linear
regression analysis reported in the table below indicates substantial equivalence.
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Deming Linear Regression 95% CI
Parameter Slope Yintercept Xintercept Slope Yintercept
Site 1
CD3+CD4+ Tcell
0.868 3.28 3.78 0.841 0.896 16.5 – 10.0
Regression
CD3+CD8+ Tcell
0.899 35.0 38.9 0.845 – 0.954 30.7 – 100
Regression
Site 2
CD3+CD4+ Tcell
0.970 8.47 8.73 0.917 – 1.02 34.7 – 17.7
Regression
CD3+CD8+ Tcell
1.044 58.3 55.8 0.978 – 1.11 136 – 19.3
Regression
Site 3
CD3+CD4+ Tcell
0.982 5.85 5.95 0.922 – 1.04 20.0 – 31.7
Regression
Site 4
CD3+CD4+ Tcell
0.901 12.99 14.42 0.871 – 0.931 2.67 – 28.7
Regression
CD3+CD8+ Tcell
0.884 2.49 2.82 0.831 0.936 64.3 – 69.3
Regression
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Linearity
Assay linearity was determined by testing six point dilution series, 641680
CD3+CD4+ Tcells/µl and 321640 CD3+CD8+ Tcells/µl. The measurement at
each level was performed in triplicate. The assay was determined to be linear for
both CD3+CD4+ and CD3+CD8+ Tcells with correlation coefficients (R2) of 0.995
and 0.999, respectively.
The two dilution series spanned the specified reportable ranges of 651500
CD3+CD4+ Tcells/µl and 401500 CD3+CD8+ Tcells/µl.
Precision
The repeatability of measurement was determined for three levels of CD3+CD4+
and CD3+CD8+ Tcells counts analyzed in replicates of ten. The results are
tabulated below.
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 of antigen
receptors on human T lymphocytes. Immunology Today, 1983, 4:58.
3. Fauci AS: The human deficiency virus: Infectivity and mechanism of
pathogenesis. Science, 1988, 239:617622.
4. Reinherz EL and Schlossman SF. The differentiation and function of human T
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lymphocytes. Cell, 1980, 19:821827.
5. Phillips A, Lee C, Elford J, et al. Serial CD4 lymphocyte counts and the
development of AIDS. Lancet 1991, 337:389392.
6. Nicholson J. Use of flow cytometry in the evaluation and diagnosis of primary
and secondary immunodeficiency diseases. Arch. Pathol. Lab. Med. 1989,
113:598605.
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, 115:184189.
8. Giorgi J & Detels R. Tcell subset alterations in HIVinfected homosexual men:
NIAID multicenter AIDS cohort study. 1989, 52:1018.
9. Heddy Z, Swart B, Nicholson I, & Voss E, eds. Leukocyte and stromal cell
molecules; the CD markers. John Wiley & Sons 2007
10. Uppal SS, Verma S, Dhot PS. Normal values of CD4 and CD8 lymphocyte
subsets in healthy Indian adults and the effects of sex, age, ethnicity, and
smoking. Cytometry Part B 2003, 52B:3236
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,
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expressed or implied, that extend beyond the description on the label of the
product. ReaMetrix’s 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: FACScan, CaliBRITE, FACSLyse, FACSComp, FACSCalibur are all registered
trade names of BectonDickinson. Alexa Fluor® 647 is a registered trade nameof Invitrogen.
ReaMetrix Inc.
1585 Industrial Rd.
San Carlos, CA 94070
27
(650)6209253
Manufactured by ReaMetrix India Pvt. Ltd.
Manufacturing License Number: KTK/25/519/2006
50B, II Phase, Peenya Industrial Area
Peenya, Bangalore 560058, India
Ph: +918028378693/5, Fax: +918041172451
Email: info@reametrix.com,
www.reametrix.com
Rev No. 4.0, 08May09
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