Biotechnology company, SOTIO is developing a unique Active Cellular Immunotherapy (ACI) to treat autoimmune diseases and cancer. The company is utilizing an immunotherapy platform to develop novel medical therapies and subsequently to improve the treatment of these diseases.
This innovative platform is based on activated dendritic cells (DC). At present, SOTIO is developing a breakthrough medicinal product called DCVAC/PCa to treat prostate cancer. To this end, clinical studies performed on this product have shown promising results.
In this article, the Vi-CELL™ XR, an automated cell counting and viability analyzer, was assessed as a suitable method for quality control (QC) of DC count and viability for the end product, DCVAC/PCa.
Assessment of Vi-CELL™ Measurement Accuracy
Vi-CELL™ is an automated counting method whose accuracy was ascertained by measuring intra and inter operator CV% (Vi-CELL™ XR versus Burker chamber). The accuracy of Vi-CELL™ was determined through beads of known concentration (1x106/ml). Two operators determined the concentration of beads thrice under the same operating conditions. In order to assess the counting precision, the results acquired with the Vi-CELL™ method were compared to those acquired with the Burker chamber. Coefficient of variation between operators and between each counting did not go beyond 5% for both counting techniques. The end concentration of control beads was similar and found to be in the right range of 0.9x106count/ml - 1.1x106count/ml as specified in the concentration control manual (Figure 1). This end concentration of control beads was determined by automated and manual counting technique.
Figure 1. Accuracy test.
Optimal Setting of Vi-CELL™ XR for Automated Cell Counting
DCVAC/PCa includes various cell populations, but predominantly contains lymphocytes and DC. Vi-CELL™ imaging system is capable of studying different types of cells based on their circularity and size. As a result, circularity and diameter of lymphocyte and DC were established for proper recognition of DC to keep out lymphocytes during Vi-CELL™ counting. NIS Elements BR imaging software and NIKON™ Eclipse microscope at 20x magnification were employed to resolve both size parameters.
Cell circularity was examined on 5 DCVAC/PCa lots measuring 10 lymphocytes and 20 DC for each lot; the average circularity was determined for each lot, as shown in Figure 2A. When the circularity for each type of cell was evaluated, no major difference was found between lymphocytes and DC (P=0.6560; paired t-test) and thus made it difficult to characterize those types of cells based on their circularity.
Figure 2. Cell circularity and diameter determination in DCVAC/PCa.
Next, the diameter of the cell was examined on 19 DCVAC/PCa lots measuring 10 lymphocytes and 20 DC for each lot, as shown in Figure 2B. Then, the diameters of both cell populations were evaluated to ascertain the size range that would help in differentiating DC from lymphocytes, so during Vi-CELL™ counting lymphocytes were not included.
Validation of Cell Counting Using Vi-CELL™ Automated Counter
Given that the size separation between two cell populations was not fully established, two size parameters, 11.5-30µm and 12-30µm, were tested as potential Vi-CELL™ setting parameters for dendritic cell counting. DC viability, number of viable DC, and number of total DC were determined on 20 DCVAC/PCa lots for both size parameters in Vi-CELL™ XR analyzer along with Burker chamber counting. The values obtained by the Vi-CELL™ were then evaluated against Burker counting values by means of paired t-test. This would help determine which size parameter is more appropriate for Vi-CELL™ set up for DC recognition and not considerably different from Burker counting. Conversely, no major difference was found between DC counts in Burker chamber and Vi-CELL™ XR analyzer (Figure 3).
Figure 3. DC count and viability.
Both recommended size parameters were appropriate for DC recognition by the Vi-CELL™ method. However, when compared to Burker chamber counting, the range 11.5-30 µm was less different than the range 12-30 µm (Figures 3A and 3B). In addition, when the variation between a pair of measurements was plotted against their mean, the variations were smaller and found to be more centered around zero in the case of 11.5-30 µm range than that of the 12-30 µm range. This shows that the 11.5-30 µm range is more accurate as a Vi-CELL™ size parameter for DC counting (Figure 4).
Figure 4. XY plot.
The study shows that the Vi-CELL™ XR automated cell count and viability analyzer is suitable for DC counting and similar to the existing QC method, Bürker chamber. No major difference was found between DC counts values acquired by Bürker chamber and Vi-CELL™ XR analyzer.
This information has been sourced, reviewed and adapted from materials provided by Beckman Coulter, Inc. - Particle Characterization.
For more information on this source, please visit Beckman Coulter, Inc. - Particle Size Characterization.