Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

Abstract

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Sources of Variability in the Response of Labeled Microspheres and B Cells during the Analysis by a Flow Cytometer

“…The values of the absorption cross-section and the quantum yield would have to be changed to values appropriate for the R-PE label. The radius of the cell would have to replace the radius of the bead, and some of the flow cytometer parameters would have to be adjusted to yield the expected number of labeled CD19 antibodies on reference B cells, approximately 8000 antibodies bound per cell (ABCs) [ 15 , 16 ]. The modified model, with inclusion of electronic noise, can be used to estimate the limit of detection of R-PE label on B cells, which is expected to be on the order of 10 R-PE molecules [ 17 ].…”

“…The voltage pulse was digitized using an analog-to-digital converter (ADC) which sampled the voltage pulse in each of the 2 µs time bins and put out a sequence of binary digits, FI, representing the voltage. The relationship between the voltage and the FI was FI = voltage × 2 16 /5, where 2 16 /5 means that the ADC was able to split 5 V into 2 16 distinct digits. This resulted in a dynamic range of about 10 4 .…”

“…Figure 5 shows a histogram for a sample of 1028 beads, with each bead producing a voltage pulse similar to that shown in Figure 2. The blue circles in Figure 5 were generated with model parameters: N lb = 364//19, N bg = 3.6/1.2, QY = 0.95, detector solid angle = 3.53 steradian, QE = 0.8, PMT electron gain G = 1.0 × 10 5 , current-to-voltage converter (CVC) gain = 0.125 × 10 6 , and analog-to-digital converter (ADC) resolution = 5 V into 2 16 distinct binary digits. The orange circles in Figure 5 were generated with a constant value N lb = 364, and all other parameters the same as used to generate the blue circles.…”

Reproducibility in Cytometry: Signals Analysis and its Connection to Uncertainty Quantification

“…Physically, inequality (5) implies an event in which we track a particle starting before it enters the laser and ending after it fully exits; the corresponding function f (t) is padded on the left and right by zeros. 3 Because L 0 is constant, the locations at which we start and stop tracking the particle are fixed. Taking t = 0 as the time when the particle is at the (arbitrary) "center" z = 0 of the laser, f (t) is defined on the interval [−τ, τ ], where τ = L 0 /v.…”

“…Second, the objective has rapid oscillations due to the forms of X(v) and g(k; R). Third, in the limit that R → 0 for R/R 0 constant, the ratio g(k; R 0 )/g(k; R) → (R 0 /R) 3 , so that L Ns develops a connected set of solutions. That is, only the ratio of R 0 to R can be estimated from L Ns .…”