R7-31001 6-Color TBNK-SL Reagent
Product Information
The Cytek 6-Color TBNK-SL Reagent is a product of Cytek
Biosciences, Inc. It is a fluorescently labeled monoclonal antibody
reagent intended for use in flow cytometry analysis of human
lymphocytes. The reagent contains antibodies specific to CD3, CD4,
CD8, CD19, CD16, and CD56 antigens which allow for the
identification and quantification of T, B, and NK lymphocyte
subsets. The product is stable until the expiration date indicated
on the label when stored at 2-8°C and protected from light.
Product Usage
- Intended Use: The Cytek 6-Color TBNK-SL
Reagent is intended for use in flow cytometry analysis of human
lymphocytes to identify and quantify T, B, and NK lymphocyte
subsets. - Components: The reagent contains fluorescently
labeled monoclonal antibodies specific to CD3, CD4, CD8, CD19,
CD16, and CD56 antigens. - Storage and Handling: The product should be
stored at 2-8°C and protected from light until the expiration date
indicated on the label. Do not freeze. - Specimen Requirements: After collection,
samples should be stored at room temperature away from light for no
more than 36 hours. After staining with the reagent, samples should
be stored at 2-8°C away from light and analyzed by flow cytometry
within 24 hours. Samples with microbial contamination or
coagulation should be avoided. - Sample Staining: Samples should be stained
with the reagent for 15 minutes at room temperature in the dark.
After staining, the samples should be stored at 2-8°C away from
light and analyzed by flow cytometry within 24 hours. - Flow Cytometry: The Cytek 6-Color TBNK-SL
Reagent can be used with the Cytek NL-CLC flow cytometer with
various laser and software configurations. Refer to the instrument
and software User’s Guide for details. - Quality Control: Quality control measures
should be implemented according to laboratory standards. - Warnings: Refer to the product label and
User’s Guide for warnings. - Representative Data Analysis: Refer to the
instrument and software User’s Guide for representative data
analysis procedures. - Results: The results are reported as the
percentage of positive cells in total lymphocytes and the number of
positive cells per microliter of blood (absolute count) for each
cell population, which can be exported in the Statistics table from
the software.
cFluor® 6-Color TBNK-SL
Instructions For Use
Catalog No. R7-31001
Test/Vial 50
Product Name cFluor® 6-Color TBNK-SL
Copyright and Trademarks © 2022 Cytek Biosciences, Inc. All rights reserved. Cytek, the Cytek logo, cFluor and Northern Lights are trademarks or registered trademarks of Cytek Biosciences, Inc. All other service marks, trademarks and tradenames are the property of their respective owners.
Cytek Biosciences, Inc 47215 Lakeview Blvd. Fremont, CA 94538 USA 1.877.92.CYTEK (1.877.922.9835) [email protected] cytekbio.com
Emergo Europe Prinsessegracht 20 2514 AP The Hague Netherlands
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1. Intended Use
cFluor® 6-Color TBNK-SL reagent is intended for in vitro diagnostic use with a suitable Cytek flow cytometer, to identify and enumerate the percentages and absolute counts of human peripheral blood lymphocyte subsets, including total T cells, CD4+ helper/inducer T cells, CD8+ suppressor/ cytotoxic T cells, B cells, and natural killer (NK) cells. cFluor® 6-Color TBNK-SL reagent is intended for use in countries where the regulatory approval has been obtained from the local regulatory authorities.
2. Application
Human lymphocytes are categorized into three major subsets based on their immunologic function and cellular antigen expression: T lymphocytes (CD3+), B lymphocytes (CD19+), and NK lymphocytes (CD3-CD16+ and/or CD56+). T lymphocytes are further classified into helper/inducer T lymphocytes (CD3+CD4+) and suppressor/cytotoxic T lymphocytes (CD3+CD8+).
CD3+CD4+ percentages or counts and total T and B lymphocytes are used to characterize and monitor human immunodeficiency and autoimmune diseases. A steady decrease of CD3+CD4+ lymphocyte counts has been observed in individuals infected with HIV. Decreased CD3+CD4+ and/ or CD3+CD8+ percentages and counts in COVID-19 patients are associated with severe diseases and unfavorable outcomes. NK lymphocytes (CD3-CD16+ and/or CD56+) have been shown to mediate cytotoxicity against certain tumors and virus infection.
3. Components
cFluor® 6-Color TBNK-SL reagent is a cocktail supplied in phosphate-buffered saline, pH 7.2, containing 0.09% sodium azide and 0.2% BSA (BSA Country of Origin USA). cFluor® 6-Color TBNK-SL
reagent contains the following fluorescent labeled monoclonal antibodies.
Antibody specificity Clone Immunoglobulin subtype Species and genus Fluorescent dye
Excitation wavelength Emission peak
CD45 2D1 IgG1, kappa Mouse cFluor® B690 488 nm
690 nm
CD3 SK7 IgG1, kappa Mouse cFluor® B520 488 nm
520 nm
CD4 SK3 IgG1, kappa Mouse cFluor® BYG781 488 nm
781 nm
CD8 SK1 IgG1, kappa Mouse cFluor® BYG61012 488 nm
CD19 SJ25C1 IgG1, kappa Mouse cFluor® BYG6672 488 nm
CD16
3G8
IgG1, kappa
Mouse cFluor® BYG575
488 nm
610 nm
667 nm 575 nm
CD56 5.1H11 IgG1, kappa Mouse cFluor® BYG575 488 nm
575 nm
4. Storage and Handling
This product is stable until the expiration date shown on the label when stored away from light at 2 ~ 8 . Do not freeze.
5. Other Materials required but not supplied
· RBC lysing solution (compatible with lyse no wash method) · Pipettes and pipette tips of 20 µL, 100 µL and 1000 µL · 12 x 75 mm tube
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· Vortex mixer · Flow cytometer (Cytek® Northern LightsTM-CLC) · SpectroFlo® QC Beads
6. Specimen Requirements
1 Require peripheral blood of no less than 500µL collected by venipuncture in EDTA anticoagulant tube. Refer to manufacturer’s instructions for blood collection.
2 After collection, the samples should be stored at room temperature (18 ~ 25 ) away from light for no more than 36 hours.
3 After staining, store the samples at 2 ~ 8 away from light and analyze by flow cytometry within 24 hours.
4 Avoid samples with microbial contamination or coagulation.
7. Interfering Conditions
A study was conducted to evaluate the specimen interfering conditions with 6-Color TBNK-SL reagent. No interference was detected under the following conditions: · Bilirubin concentration is < 275 µmol/L or 16.1 mg/dL · Hemolysis · Lipidemia · Jaundice
8. Sample Staining
1 By reverse pipetting, add 50 µL well-mixed EDTA anticoagulated whole blood to the bottom of the tube. Avoid blood touching the side of the tube.
2 Add 20 µL of cFluor® 6-Color TBNK-SL reagent to the bottom of a 12 x 75 mm tube. 3 Mix well by vortex and incubate for 15 minutes at room temperature away from light. 4 Add 450 µL of 1 X lysis buffer into the tube, mix briefly by vortex, and incubate for at least 15
minutes at room temperature in the dark. 5 Store the samples at 2 ~ 8 away from light and analyze by flow cytometry within 24 hours.
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9. Flow Cytometry
cFluor® 6-Color TBNK-SL reagent is designed for use on Cytek® Northern LightsTM-CLC (NL-CLC) flow cytometers. The recommended instrument models and software versions are listed below. Cytek® Automated Sample Loader (ASL) can be used with this product.
Flow Cytometer Cytek NL-CLC
Configurations
3 Laser: V16, B14, R8 2 Laser: V16, B14 2 Laser: B14, R8 1 Laser: B14
Setup Beads SpectroFlo® QC Beads
Software
SpectroFlo® CLC v1.0.1 or higher
· Refer to the manufacturer’s User’s Guide for instrument operation and software application.
· Make sure the instrument is properly set up and daily QC has passed before analyzing the samples.
10. Quality Control
· Instrument QC: Use the manufacturer recommended controls according to the model of the flow cytometer.
· Refer to the instrument User’s Guide for instrument maintenance.
· Process control: Recommend using Streck CD-Chex Plus and CD-Chex Plus CD4 Low.
11. Warnings
· This reagent contains traces of sodium azide. Do not pipette by mouth.
· Use appropriate personal protective equipment per the safety data sheet when using this product.
· Follow biosafety practice in compliance with federal, state, and local regulations to handle all biological samples and materials in contact with them.
· Contact Cytek Support or refer to cytekbio.com for details on troubleshooting.
12. Representative Data Analysis
The following is an example of the analysis for a peripheral blood sample from a normal adult. Refer to the instrument and software User’s Guide for details.
1 Display all events on FSC-A vs. SSC-A plot, place a gate on events with very low FSC and SSC to remove debris.
2 Show events that are not debris on CD45 vs. SSC-A plot and gate the low SSC and high CD45 expression lymphocytes.
3 Display CD45+ lymphocytes in CD3/SSC scatter plot, gate the CD3+ cell population.
4 Display CD3+ population in CD4/CD8 scatter plot and create the quadrant gates.
5 Display CD3- population in CD19/CD16+56 scatter plot and create the quadrant gates.
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The following table shows the cFluor® 6-Color TBNK-SL gating schema and populations of interest.
Plot Number 1 2
Plot X vs. Y FSC-A vs. SSC-A CD45 vs. SSC-A
Population of interest White blood cells Lymphocytes
3
CD3 vs. SSC-A
4
CD8 vs. CD4
5
CD(16+56) vs. CD19
CD3- and CD3+ cells
CD3+ subset CD4+CD8CD4-CD8+ CD4+CD8+ CD4-CD8-
CD3- subset CD19+ CD(16+56)+
The following figure shows a visual presentation of the gating schema and example data.
13. Results
The results are reported as the percentage of positive cells in total lymphocytes and the number of positive cells per microliter of blood (absolute count) for each cell population, which can be exported in the Statistics table from the software. The absolute count is calculated by multiplying
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cell population counts per microliter in the final test sample to the sample dilution factor that is derived from the following equation:
Final sample volume (20µL + 50µL + 450µL) Sample dilution factor =
blood volume (50µL)
14. Reference Intervals
Reference intervals for this product were determined in a study including 209 hematological normal adults between the ages of 18-65 from Wuxi, China, as listed in the table below.
Cell Population Lymphocytes CD3+
Sample Size 209 209
CD3+CD4+
209
CD3+CD8+
209
CD3-CD19+
209
CD3-CD(16+56)+ 209
Unit cells/µL % cells /µL % cells /µL % cells /µL % cells /µL % cells /µL
Mean 2528 66.35 1677 35.32 891 26.07 662 11.33 292 19.17 482
95% Range 1453-4089 50.53-82.18 856-2929 24.26-50.93 518-1600 12.14- 40.01 171-1152 5.04-21.10 113-687 6.23-35.76 146-1003
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15. Performance Characteristics
15.1. Accuracy Three replicates of the control blood specimen (Lyophilized Human T/B/NK Lymphocytes) were stained with 6-Color TBNK-SL reagent and analyzed on an NL-CLC flow cytometer. The lymphocyte subset results were within the target range provided by the manufacturer.
Specimen: Lyophilized Human T/B/NK Lymphocytes
Subset Percentage (%) CD3+ CD3+CD4+ CD3+CD8+ CD3-CD19+ CD3-CD (16+56)+ Subset Absolute Count CD45+ CD3+ CD3+CD4+ CD3+CD8+ CD3-CD19+ CD3-CD (16+56)+
Target range (%) 73.1-82.6 36.5-45.4 26.6-32.6 6.7-10.3 7.4-16.0 Target Range (cells/L) 2202-3662 1720-2831 894-1501 667-1062 180-313 214-463
Replicate R1 77.15 40.68 27.75 9.28 11.45 R1 2804 2163 1141 778 260 321
R2 76.74 40.37 28.24 8.68 12.3 R2 2862 2196 1155 808 249 352
R3 76.76 40.19 28.35 9.06 12.03 R3 2836 2177 1140 804 257 341
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15.2. Precision The precision was evaluated using CD-Chex Plus Normal (CDN) and CD-Chex Plus CD4 Low (CDL) control blood specimen by two operators on 4 NL-CLC cytometers with different configurations for 21 days with 2 runs per day. Samples were stained using three batches of 6-Color TBNK-SL reagent. The overall mean of percentage and absolute count, standard deviation (SD) of percentage, and coefficient of variation (CV) of absolute count for each cell population are listed below.
Subset Percentage (%) CD3+ CD3+CD4+ CD3+CD8+ CD3-CD19+ CD3-CD (16+56)+
CDN Mean (%) 76.57 50.93 23.16 10.59 11.27
CDN SD (%) 1.24 1.38 0.98 0.67 0.90
CDL Mean (%) 61.35 11.38 43.89 19.43 17.55
CDL SD (%) 1.35 0.77 1.29 0.95 1.10
Subset Absolute Count
CD3+ CD3+CD4+ CD3+CD8+ CD3-CD19+ CD3-CD (16+56)+
CDN Mean (cells/L)
1788 1189 541 247 263
CDN CV
3.69% 4.15% 4.81% 6.63% 6.92%
CDL Mean (cells/L)
887 165 635 281 254
CDL CV
3.39% 5.87% 3.80% 4.67% 5.98%
15.3. Sample and Staining Stability
Sample and staining stability was evaluated with EDTA anticoagulated whole blood stained with 6Color TBNK-SL reagent. Whole blood samples were tested up to 72 hours after draw and stored at room temperature (20~25°C). Stained samples were tested up to 72 hours after staining and stored at 2~8°C in the dark away from light. Based on the results from this study, we recommend staining samples within 36 hours of draw and analyzing samples within 24 hours of staining.
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15.4. Linearity
15.4.1. Sample Dilution Linearity The control blood specimen was serial diluted into five levels (undiluted, 2X, 4X, 8X, 16X). Four replicate tubes at each dilution level were stained with the same batch of 6-Color TBNK-SL reagent and analyzed on NL-CLC flow cytometer. For each subset percentage, the median of each dilution level was compared to the overall median, the relative difference calculated is shown below.
Dilution Factor
1X 2X 4X 8X 16X
Relative Difference of Subset Percentage (%)
CD3+ -0.43% -0.47% 0.52% -0.19% 0.56%
CD3+CD4+ -0.53% 0.11% -0.10% 0.54% -0.43%
CD3+CD8+ 2.26% -0.57% -0.48% -2.88% 0.22%
CD3-CD19+ 1.18% 6.06% -0.15% -1.87% -3.89%
CD3-CD (16+56)+ 3.47% -0.96% -1.14% 0.37% -1.19%
15.4.2. Linear Range
The linear range was evaluated at the range of 160-1135 CD4+ cells/µL. CD-Chex Plus Normal and CD-Chex Plus CD4 Low control blood were mixed in different proportions to prepare 11 concentration levels, then the lowest and third lowest levels were mixed into another 7 levels. Three replicates from each concentration level were stained with the same batch of 6-Color TBNKSL reagent and analyzed on NL-CLC flow cytometer. For CD4+ percentage and absolute counts, the results were shown to be linear.
Concentration Levels 11 levels
8 levels
CD4+ Subset Absolute Counts (cells/L) Percentage (%) Absolute Counts (cells/L) Percentage (%)
Concentration Range 160-1135 10.7-48.6 160-355 10.7-21.3
R2 0.9936 0.9988 0.9781 0.9942
16. Analytical Specificity
A study was conducted to evaluate the analytical specificity. It was demonstrated that CD45, CD3, CD4, CD8, CD19, and CD16+CD56 monoclonal antibodies in 6-Color TBNK-SL reagent can specifically bind the target antigens.
17. Limitations
· The test results of this reagent are for clinical reference only. Patient history, other laboratory tests, and treatment response should also be considered for diagnosis.
· Incorrect results can occur if the flow cytometer is set up or used incorrectly. · This product is not intended for screening of the lymphocyte subsets in leukemia patients.
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· The percentage and absolute counts obtained are not comparable between laboratories using different reagents or instruments.
18. References
· Mellors JW, Margolick JB, Phair JP, Rinaldo CR, Detels R, Jacobson LP, Munoz A. Prognostic value of HIV -1 RNA, CD4 cell count,And CD4 Cell count slope for progression to AIDS and death in untreated HIV -1 infection. JAMA.2007;297:234950.
· Gebo KA, Gallant JE, Keruly JC, Moore RD. Absolute CD4 vs. CD4 percentage for predicting the risk of opportunistic illness in HIV infection. J Acquir Immune Defic Syndr.2004;36:102833.
· Chen G, Wu D, Guo W, et al. Clinical and immunological features of severe and moderate coronavirus disease 2019. J Clin Invest.2020;130(5):2620-2629.
· Allegra A, Di Gioacchino M, Tonacci A, Musolino C, Gangemi S. Immunopathology of SARS – CoV 2 Infection: Immune Cells and Mediators, Prognostic Factors, And Immune – Therapeutic Implications. Int J Mol Sci.2020;21(13):4782.
· Zhao Y, Nie HX, Hu K, et al. Abnormal immunity of non – survivors with COVID -19: predictors for mortality. Infect Dis Poverty.2020;9(1):108.
· Gaglia J, Kissler S. Anti – CD3 Antibody for the Prevention of Type 1 Diabetes: A Story of Perseverance. Biochemistry.2019;58(40):4107-4111.
· Hao W, Bahnson HT, Speake C, Cerosaletti K, Greenbaum CJ. In – vivo assessment of T cell kinetics in individuals at risk for type 1 diabetes. Clin Exp Immunol.2020;199(1):50-55.
· Hofmann K, Clauder AK, Manz RA. Targeting B Cells and Plasma Cells in Autoimmune Diseases. Front Immunol. 2018;9:835.
· Poli A, Michel T, Theresine M, Andres E, Hentges F, Zimmer J. CD56bright natural killer (NK) cells: an important NK cell subset. Immunology.2009;126(4):458-465.
· Altin JG, Sloan EK. The role of CD45 and CD45- associated molecules in T cell activation. Immunol Cell Biol.1997;75(5):430-445.
· Birnbaum ME, Berry R, Hsiao YS, et al. Molecular architecture of the aß T cell receptor – CD3 complex. Proc Natl Acad Sci U S A.2014;111(49):17576-17581.
· McBride JA, Striker R. Imbalance in the game of T cells: What can the CD4/ CD8 T- cell ratio tell us about HIV and health. PLoS Pathog.2017;13(11):e1006624.
· Wang K, Wei G, Liu D. CD19: a biomarker for B cell development, lymphoma diagnosis and therapy. Exp Hematol Oncol.2012;1(1):36.
1cFluor® BYG610 Anti-Human CD8 (SK1) is manufactured and supplied by BioLegend, Inc. and is commercialized under the trademark PE/Dazzle 594TM. 2cFluor® BYG610 and BYG667 are tandem dyes made with R-PE. Caution: Tandem dyes may show changes in their emission spectra with prolonged exposure to light or fixatives.
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