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Product Catalog No: 2523900
ReaPan 34845
Reagent
Manufactured by
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ReaPan 34845 Reagent
INTENDED USE
The ReaPan 34845 reagent is a four color immunofluorescence stain for
the labeling and identification of helper/inducer (CD4+)
cytotoxic/suppressor (CD8+) cells and total Tlymphocytes (CD3+)
combined with a precise number of fluorescent counting beads for absolute
and percentages of CD4+, CD8+ and CD3+ TCells and absolute
lymphocyte counts. This reagent is intended to be used in a “lyseno wash”
protocol for flow cytometry analysis of erythrocytelysed human whole
blood.
SUMMARY AND EXPLANATION
The ReaPan 34845 reagent contains fluorescently labeled antibodies that
bind to CD45, 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
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the Tcell receptor (TCR) complex. The antibody used in this reagent binds
to the 20kDa ε chain of this complex (1).
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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 CD8 antigen is a complex consisting of two disulfide linked subunits.
The antibody used in this reagent binds to the 32kDa α subunit of the
complex. CD8 interacts with class I major histocompatibility complex
molecules (2,3).
The CD45 antigen is a complex 180220kDa transmembrane glycoprotein
which is expressed in high levels on leukocyte cell surfaces and its
presence distinguishes leukocytes from nonhaematopoietic cells (1).
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
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cases of immunodeficiency (8)
PRINCIPLE OF THE PROCEDURE
The ReaPan 34845 reagent consists of murine monoclonal antibodies that
specifically recognize the human leukocyte surface antigens CD3, CD4,
CD8 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 subsets are
identified when passed through a flow cytometer laser beam.
The ReaPan 34845 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 sample analyzed can
be determined by multiplication of the total sample volume by the fraction
of total beads that were detected during the analysis.
REAGENT
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The ReaPan 34845 reagent is formulated in buffered saline with sodium
azide and stabilizer. It contains Allophycocyanin (APC)– labeled antiCD4
monoclonal antibody, clone RPAT4; RPhycoerythrin(PE)– labeled anti
CD8 monoclonal antibody, clone LT8; Atto™488– labeled antiCD3
monoclonal antibody, clone UCHT1; and Peridininchlorophyllprotein
Complex (PerCP)– labeled antiCD45 monoclonal antibody, clone
HI30.The monoclonal antibodies used in the ReaPan 34845 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 ReaPan 34845 reagent to allow singleplatform
determination of absolute CD4+, CD8+, CD3+ Tcell and absolute
lymphocyte counts. The ReaPan 34845 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
ReaPan 34845 reagent package.
Precautions
1. Warning: The ReaPan 34845 reagent contains Sodium azide.
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Sodium azide is harmful if swallowed (R22). Wear suitable
protective clothing (S36). If swallowed, seek medical advice
immediately (S46). Contact with acids liberates toxic gas (R32).
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 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 mentioned on the label.
2. Do not freeze or refrigerate the ReaPan 34845 reagent.
3. The ReaPan 34845 reagent is light sensitive. Do not expose to
direct light either during storage or when mixed with blood.
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4. Use the zip lock to reseal the reagent pouch after you have
removed the desired number of tests. Do not use the tubes
beyond the expiration date indicated on the packaging.
Indication of Instability
The ReaPan 34845 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
The user is responsible to optimize the performance of the reagent for use
in flow cytometers other than that mentioned above. For other flow
cytometers, set up the cytometer for fourcolor acquisition following the
manufacturer’s recommendations. Setup should include setting or checking
PMT voltages, fluorescence compensation, and instrument sensitivity.
SPECIMEN COLLECTION
Interfering conditions
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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
ReaPan 34845 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. 0.2µm filtered, deionized distilled water or MilliQ waterC
7. Flow cytometer Calibration BeadsD
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A. ReaLyse Lysing solution: ReaMetrix Catalog No: 2523700
B. Sheath fluid: BD Catalog No.342003
C. Accuri Flow cytometer uses MilliQ water/Distilled deionized water instead of
Sheath fluid
D. Calibration Beads: BD Catalog No. 340486
Assay Protocol
1. Mix blood sample (invert blood tube at least 10 times) and
reverse pipette 50µL of blood into the correctly labeled tube
that contains the dry down reagent.
2. Gently vortex each tube for 30 seconds. Incubate for 30
minutes at room temperature. Protect the tube from direct
light.
3. Add 450µL of 1X ReaLyse lysing solution to each tube and
vortex for 10 seconds. Return tubes to the dark for at least 15
minutes.
4. Vortex sample tube thoroughly (at low speed) and load onto
cytometer for analysis.
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Quality Control
Commercially available quality control whole blood control cells can be run
each day. Established values can be used to assess reagent and system
performance.
FLOW CYTOMETER ACQUISITION AND ANALYSIS
Start flow cytometer according to manufacturer’s instructions (In the case
of FACSCalibur™, the instrument should be setup and calibrated using
CaliBRITE™ beads and FACSComp™ software; For Accuri™ flow
cytometer instruments, the performance of the instrument should be
checked by Validation beads provided by Accuri.)
For BD FACSCalibur™ Flow Cytometer Instruments
1. Draw five dotplot views – Refer Figure 1
• View 1: AntiCD45 PerCP (FL3) versus side scatter (SSC–
Linear scale)
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• View 2: AntiCD3 Atto488 fluorescence (FL1) versus side
scatter (SSC– Linear scale) of events gated as CD45+ (R1)
from the View 1.
• View 3: CD4 APC (FL4) versus CD3 Atto 488 (FL1) of
events gated as CD45+ (R1) from the View 1.
• View 4: CD8 PE (FL2) versus CD3 Atto 488 (FL1) of events
gated as CD45+ (R1) from the View 1.
• View 5: AntiCD8 PE (FL2) versus AntiCD3 Atto488 (FL1).
View5 will have a gate (R3) to capture the fluorescent
reference beads. The beads have a high FL1 and FL2 Mean
Fluorescence Intensity
2. Before acquisition adjust the FL3 threshold to minimize debris.
3. Set the number of events to capture at 3000 for R3 gate (View
5) and start data acquisition. When 3000 beads are acquired in
the gate R3, acquisition stops.
4. Gate the brightest CD45+ cell cluster in View1 and gate the
population as R1. This CD45+ cell cluster is that population
which has the lower SSC intensity and higher FL3 mean
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fluorescence intensity. It represents the lymphocyte population.
The Gate R1 is applied on View 2, 3 & 4.
5. Total CD3+ events can be identified in View 2 in R2.
6. The CD4+ and CD8+ cell populations can be identified in View 3
and View 4 in the upper right quadrants.
7. 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.
Example:
If the bead count per ReaPan 34845 test is 50,000, the volume of
blood tested is 50µl, the number of gated beads is 3,000 (Figure 1,
View 5, R3 gated events), and the number of gated CD4+ Tcells is
1,500 (Figure 1, View 3, UR quadrant) then the absolute CD4+ Tcell
count is 500 cell/µl.
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1500 x 50000
Absolute CD4 CellCount= = 500cells / µL
3000 x 50
View 1 View 2
View 5
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View 3 View 4
For Accuri™ Flow Cytometer Instruments
1. Draw five dotplot views – Refer Figure 2.
• View 1: CD45 PerCP (FL3Log scale) versus side scatter
(SSC – Linear scale)
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• View 2: CD3 Atto488 fluorescence (FL1Log scale) versus
side scatter (SSC – Linear scale) of events gated as CD45+
(P1) from the View 1.
• View 3: CD4 APC (FL4 Log scale) versus CD3 Atto 488
(FL1 Log scale) of events gated as CD45+ (P1) from the View
1.
• View 4: CD8 PE (FL2 Log scale) versus CD3 Atto 488
(FL1 Log scale) of events gated as CD45+ (P1) from the View
1.
• View 5: CD8 PE (FL2 Log scale) versus CD3 Atto488
(FL1 Log scale) of all events from View 1. View5 will have a
gate (P2) to capture the fluorescent reference ReaCount beads.
Following table (Table1) represents the Plot Specification Value to set
the scales:
Table1: Dot Plot View X & YAxis Scale Specifications
Dot Plot Views Axis Scale Min Value Max value
View 1 XAxis FL3Log scale 500 200,000
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YAxis SSCLinear scale 0 175,000
XAxis FL1Log scale 500 500,000
View 2
YAxis SSCLinear scale 0 120,000
XAxis FL4Log scale 50 1,000,000
View 3
YAxis FL1Log scale 200 1,000,000
XAxis FL1Log scale 50 500,000
View 4
YAxis FL2Log scale 500 500,000
XAxis FL1Log scale 50 500,000
View 5
YAxis FL2Log scale 500 500,000
2. In the Run Limit section of the Instrument template panel, set the
instrument Threshold on FL3 and set the value to 1000.
Before/After acquisition adjust the FL3 Threshold value to minimize
debris.
3. Click on the Set Colour Compensation to open the compensation
matrix and set the compensation values as per Figure 3.
4. 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 2; View 5)
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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 View1 and adjust the P1
gate around the lymphocyte population. This CD45+ cell cluster is
that population which has the lower SSC intensity and higher FL3
mean fluorescence intensity. It represents the lymphocyte
population. The Gate P1 is applied on View 2, 3 and 4.
7. CD3+ cell clusters are separated using gate R1 (View2, Figure 2).
The CD3+ cell population has the lower SSC intensity and higher
FL1 mean fluorescence intensity.
8. In View3 (Figure 2) cell clusters are separated using quadrant gate
Q1. The CD4+CD3+ cell populations can be identified in View3
(Figure 2) in the upper right quadrant Q1UR as double positives.
9. In View4 (Figure 2) cell clusters are separated using quadrant gate
Q1. The CD8+CD3+ cell populations can be identified in View4
(Figure 2) in the upper right quadrant Q1UR as double positives
10. View5 (Figure 2); fluorescent reference beads are separated using
gate P2. The beads have a high FL1 and FL2 Mean Fluorescence
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Intensity.
11. The absolute count for each cell type can be calculated using the
following equation.
(Gated Cell Count) x(Bead Count per test)
Absolute CellCount / µL =
(Gated Bead Count) x(Test Volumein µL)
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.
Example:
If the bead count per ReaPan 34845 test is 50,000, the volume of blood
tested is 50µl, the number of gated beads is 3,000 (Figure 2, View 5, P2
gated events), and the number of gated CD4+ Tcells is 1,500 (Figure 2,
View 3, Q1UR quadrant) then the absolute CD4+ Tcell count is 500
cell/µl.
1500 x 50000
Absolute CD4 CellCount= = 500cells / µL
3000 x 50
View 1 View 2
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View 5
View 3 View 4
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Figure 2. Dot plot views: View 1: CD45 PerCP fluorescence (FL3 log scale)
versus side scatter (SSC – Linear scale); View 2: CD3 Atto488 fluorescence (FL1
log scale) versus side scatter (SSC– Linear scale) of events gated as CD45+ (P1)
from the View 1; View 3: CD4 APC fluorescence (FL4– log scale) versus CD3 Atto
488 (FL1– log scale) of events gated as CD45+ (P1) from the View 1; View 4: CD8
PE fluorescence (FL2 log scale) versus CD3 Atto488 (FL1 log scale) of events
gated as CD45+ (P1) from the View 1; View 5: CD8 PE fluorescence (FL2 log
scale) versus CD3 Atto488 (FL1 log scale). R3 captures the fluorescent reference
beads
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Figure 3: Accuri Flow cytometer Compensation settings for ReaPan 34845 Reagent
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PERFORMANCE CHARACTERISTICS
The performance data for ReaPan 34845 reagent was generated by
undertaking clinical validation.
Accuracy
The absolute counts for CD4+, CD8+ and CD3+ TCells determined
using ReaPan 34845 reagent were compared with DryTri TSTAT
CD3/CD4/CD8 reagents (Figure 4, Figure 5 and Figure 6 ) using a BD
FACSCalibur™ flow cytometer.
The absolute counts for CD45+ (Absolute Lymphocyte Count – ALC)
determined by the ReaPan 34845 were compared with a hematology
counter (Figure 7).
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Figure4: Correlation of absolute CD4+ TCell counts determined using ReaPan
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34845 compared to a commercially available reagent Dry Tri TSTAT
CD3/CD4/CD8
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Figure 5: Correlation of absolute CD8+ TCell counts determined using the
ReaPan 34845 compared to a commercially available reagent Dry Tri TSTAT CD3/
CD4/CD8
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Figure 6: Correlation of absolute CD3+ TCell counts (Total TCells) determined
using the ReaPan 34845 compared to a commercially available reagent Dry Tri T
STAT CD3/CD4/CD8
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Figure 7: Correlation of CD45+ cell counts (ALC) determined using the ReaPan
34845 compared to the lymphocyte count obtained from a Hematology analyzer
Specificity
The antigen specificities for the CD3, CD4, CD8 and CD45 monoclonal
antibodies used in the ReaPan 34845 reagent were confirmed by the
Human Leukocyte Differentiation Antigens (HLDA) Workshops. After
conjugation of the antibodies no additional crossreactivity was observed.
LIMITATIONS
1. The ReaPan 34845 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 ReaPan 34845 reagent.
BIBLIOGRAPHY
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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 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.
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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.
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. 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: FACSCalibur, CaliBRITE, FACSComp are all registered trade names of
BectonDickinson; 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
50B, II Phase, Peenya Industrial Area
Peenya, Bangalore 560058, India
Ph: +918028378693/5,
Fax: +918041172451
Email: info@reametrix.com,
www.reametrix.com
Rev No. 3.0, 22Sep09
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