CYTEK R7-31001 6-Color TBNK-SL Reagent Instruction Manual

CYTEK 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

1. 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.
2. Components: The reagent contains fluorescently labeled monoclonal antibodies specific to CD3, CD4, CD8, CD19, CD16, and CD56 antigens.
3. 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.
4. 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.
5. 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.
6. 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.
7. Quality Control: Quality control measures should be implemented according to laboratory standards.
8. Warnings: Refer to the product label and User’s Guide for warnings.
9. Representative Data Analysis: Refer to the instrument and software User’s Guide for representative data analysis procedures.
10. 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.

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)
products@cytekbio.com
cytekbio.com
Emergo Europe
Prinsessegracht 20
2514 AP The Hague
Netherlands

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.

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.

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.

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.

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
• Vortex mixer
• Flow cytometer (Cytek® Northern Lights™-CLC)
•  Spectro Flo® QC Beads

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.

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

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.

 Flow Cytometry

cFluor® 6-Color TBNK-SL reagent is designed for use on Cytek® Northern Lights™-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.

2 Laser: V16, B14
2 Laser: B14, R8
1 Laser: B14| SpectroFlo® QC Beads| 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.

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.

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.

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.

The following table shows the cFluor® 6-Color TBNK-SL gating schema and populations of interest.

The following figure shows a visual presentation of the gating schema and example data. 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 cell population counts per microliter in the final test sample to the sample dilution factor that is derived from the following equation:

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.

 Performance Characteristics

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.

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 (%)| CDN Mean (%)| CDN SD (%)| CDL Mean (%)| CDL SD (%)
---|---|---|---|---
CD3+| 76.57| 1.24| 61.35| 1.35
CD3+CD4+| 50.93| 1.38| 11.38| 0.77
CD3+CD8+| 23.16| 0.98| 43.89| 1.29
CD3-CD19+| 10.59| 0.67| 19.43| 0.95
CD3-CD (16+56)+| 11.27| 0.90| 17.55| 1.10
Subset Absolute Count| CDN Mean (cells/μL)| CDN CV| CDL Mean (cells/μL)| CDL CV
---|---|---|---|---
CD3+| 1788| 3.69%| 887| 3.39%
CD3+CD4+| 1189| 4.15%| 165| 5.87%
CD3+CD8+| 541| 4.81%| 635| 3.80%
CD3-CD19+| 247| 6.63%| 281| 4.67%
CD3-CD (16+56)+| 263| 6.92%| 254| 5.98%

Sample and Staining Stability
Sample and staining stability was evaluated with EDTA anticoagulated whole blood stained with 6-Color 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.

Linearity
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.

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 TBNK-SL reagent and analyzed on NL-CLC flow cytometer. For CD4+ percentage and absolute counts, the results were shown to be linear.

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.

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.
• The percentage and absolute counts obtained are not comparable between laboratories using different reagents or instruments.

 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;297:2349–50.
•  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;36:1028–33.
• 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 594™.
• 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.

References

• Full Spectrum Flow Cytometry Systems | Discover the Advantages | Cytek Biosciences

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