ClearLLab Control Cells – a Process Control for ClearLLab 10C Application
Li Yang, Lin Jiang, Brittany Kuhl, Xiangyang Dong, Jin Zhang, Lidice Lopez Research and Development, Beckman Coulter Life Sciences, Miami, FL 33196
Introduction
This white paper is to introduce ClearLLab Control Cells. Flow cytometry immunophenotyping is a commonly used technology as an aid in diagnosis of hematolymphoid malignancies. ClearLLab 10C Panels are composed of 4 independent immunophenotyping reagents including 27 unique makers which are consistent with WHO and Bethesda guidelines. To be compliant, the performance of reagents and staining procedures needs to be verified using positive control materials. ClearLLab Control Cells were developed to support ClearLLab 10C Panels and include Normal Control and Abnormal Control Cells. ClearLLab Control Cells are whole blood based products that can consistently provide process/positive controls for all the markers/antigens in ClearLLab 10C Panels.
Methods
Multiple lots of both fresh (*T0M) and aged (*T3M+Op-1M) Control cells were stained with each of the four ClearLLab 10C Panels to assess the staining signal, resolution of each antigen as well as specimen stability. In addition, unique gating strategies have been developed for Control Cell data acquisition and analysis to provide an easy, simple and consistent assessment for each relevant cell population.
Figure 1. Control Cells follow the same sample prep and cell acquisition procedure as for the whole blood specimens defined in ClearLLab 10C application. Data is analyzed using Kaluza C Software.
Normal or Abnormal Control Cells
- Pre-wash control cells three times with PBS+2% FBS (0.45mL:12mL)
- Stain washed cells (100μL) with ClearLLab 10C T, B, M1 or M2 Cell Tubes
- Lyse cells with IOTest3 Lysing plus Fixative Solution (2mL per sample)
- Spin and aspirate, wash again with 1x PBS (3mL per sample)
- Spin and aspirate, suspend cell pellet with 1x PBS (0.5mL per sample)
- Acquire sample with Navios or Navios EX Cytometer Instrument
- Analyze Data with Kaluza C Software
Results
ClearLLab Control Cells are liquid preparations of stabilized human erythrocytes and leukocytes (lymphocytes, monocytes, and granulocytes) that have lysing, light scatter, antigen expression, and antibody staining properties representative of those found in human whole blood specimens: The erythrocytes function as the lysable component of ClearLLab Control Cells. The leukocytes function as the positive cell component having surface antigens present on the targeted cells that bind to the antibodies included in ClearLLab 10C Panels, including CD34, CD117 and CD123 (Figure 2).
Figure 2. ClearLLab 10C Panels. The four ClearLLab 10C Panels (B, T, M1 or M2 Cell Tubes) contain 27 different markers/antigens.
1Pacific Blue. 2APC-Alexa Fluor 700. 3APC-Alexa Fluor 750.
Alexa Fluor and Pacific Blue are registered trademarks of Molecular Probes, Inc.
Figure 3. ClearLLab Control Cells Gating Strategy for B, T , M1 or M2 Cell Tubes: The gating strategies are developed specifically for control cells to provide easy, simple and consistent assessment of every relevant cell population. The same population/marker is gated in a similar way. ClearLLab Control Cells include Normal and Abnormal controls - the only difference is that the Abnormal Control has a blast-like population that is CD45dim, and expresses CD34, CD123 and CD117. The same gating strategy is applied to both Normal and Abnormal, B Cell Tube (3A); T Cell Tube (3B), M1 Cell Tube (3C) and M2 Cell Tube (3D). Gating strategy using Kaluza C Analysis Software.
3A: B Cell Tube
3B: T Cell Tube
3C: M1 Cell Tube
3D: M2 Cell Tube
Figure 4. ClearLLab Control Cells Staining Profile: Representative dot plots of ClearLLab Control Cells Normal and Abnormal: No significant difference was observed between fresh (T0M) and aged (T3M+Op-1M) ClearLLab Control Cells specimen except that aged cells have dropped side scatters and decreasing separation of blast and mono population. Neither impacts ClearLLab Control Cells application. The results had confirmed the consistent staining performance of ClearLLab Control Cells.
Conclusions
- The ClearLLab Control Cells Normal and Abnormal showed prominent expression of the 27 antibodies contained in the ClearLLab 10C Panels, with acceptable cell population resolution.
- The Control Cells have demonstrated similar property as whole blood specimens in terms of antibody staining performance, erythrocyte lysing and flow cytometric analysis as process control for ClearLLab 10C application.
- The gating strategies specific for ClearLLab Control Cells provide easy, efficient and consistent assessment of relevant cell populations/markers of ClearLLab 10C Panels.
Acknowledgments
We are grateful for the following support: Robert Magari, Karen Lo & all members of the ClearLLab 10C project team.
*Fresh (T0M): Control cell specimen was tested within 2 weeks of post production date; Aged (T3M+ Op-1M): Control Cell was tested 3-month close-vial plus a 1-Month open-vial post production date.
Navios EX is CE-IVD marked under Regulation (EU) 2017/746 for 10-color in-vitro diagnostic use. In U.S., Navios EX is intended for use as an in vitro diagnostic device for immunophenotyping with Navios tetra software and CYTO-STAT tetraCHROME reagents and ClearLLab 10C system. All other uses are for research use only.
ClearLLab 10C Panels and ClearLLab Control Cells are CE-IVD marked under Regulation (EU) 2017/746. For In Vitro Diagnostic Use. For Non-Hodgkin’s lymphoma only.
IOTest 3 Lysing Solution, IOTest3 Fixative Solution, and Kaluza C are CE-IVD marked under Regulation (EU) 2017/746. For In Vitro Diagnostic Use.
