Automating flow cytometry sample preparation without centrifugation

While flow cytometry has become a prominent technology in both research and clinical laboratories, the sample preparation workflow is often still highly manual.  Variabiability in this workflow, as well as in instrument setup and data analysis, can create reproducibility issues that impair the utility of this otherwise powerful platform.

In this presentation, I will review major sources of variability in flow cytometry and some known solutions for reducing variability within specific steps.  These include optimizing and standardizing pre-analytical variables, antibody panels, and staining protocols; and minimizing batch effects through use of stabilized reagents, standarized instrument setup, and uniform/automated gating.  Less attention has historically been placed on the automation of sample preparation, in part because this generally involves large and complex robotics.  Here, I present a small-footprint liquid handling solution that simplifies sample preparation automation by eliminating the use of a centrifuge, in favor of gravity-based settling and precise pipetting to create laminar flow washing of settled cells.  Our laboratory has tested this system in the context of (1) a protocol requiring both cell-surface and intracellular stainging steps, and (2) a protocol performing high-parameter (28-color) cell-surface staining.  For the latter, automated antibody cocktailing was also employed.  I report on viable cell recovery, staining patterns and stain index, and reproducibility for automated vs. manual preparation in the context of these two protocols.

Going forward, we hope to automate protocols for all of our lab’s typical flow cytometry assays.  This should both reduce technician time and increase reproducibility of results for better reliability of our flow and mass cytometry data.

Learning Objectives:

1. List the major sources of variability in flow cytometry, and known strategies to mitigate them.

2. Describe how flow cytometry sample preparation can be automated without centrifugation.

3. Define the major criteria by which to evaluate an automated sample preparation protocol.