High-Content Flow Cytometry and Temporal Data Analysis for Defining a Cellular Signature of Graft-Versus-Host Disease

Abstract

Acute graft-versus-host disease (GVHD) is diagnosed by clinical and histologic criteria that are often nonspecific and typically apparent only after the disease is well established. Because GvHD is mediated by donor T cells and other immune effector cells, we sought to determine whether changes within a wide array of peripheral blood lymphocyte populations could predict the development of GvHD. Peripheral blood samples from 31 patients undergoing allogeneic blood and marrow transplant were analyzed for the proportion of 121 different subpopulations defined by 4-color combinations of lymphocyte phenotypic and activation markers at progressive time points posttransplant. Samples were processed using a newly developed high content flow cytometry technique and subjected to a spline- and functional linear discriminant analysis (FLDA)-based temporal analysis technique. This strategy identified a consistent posttransplant increase in the proportion and extent of fluctuation of CD3+CD4+CD8beta+ cells in patients who developed GVHD compared to those that did not. Although larger prospective clinical studies will be necessary to validate these results, this study demonstrates that high-content flow cytometry coupled with temporal analysis is a powerful approach for developing new diagnostic tools, and may be useful for developing a sensitive and specific predictive test for GVHD.

Automation in high‐content flow cytometry screening

“…Full details regarding patient demographics and the methods used to obtain the data are given in (6). In this section, we describe the most essential aspects of the data.…”

“…Table 1 lists the 9 antibody combinations that we analyze. Additional details on these antibodies are given in (6). Figure 1 illustrates the nature of the data by plotting a subset.…”

“…Recently, (6) conducted a sophisticated analysis of FC-HCS data in an effort to identify cellular signatures for acute graft-versus-host-disease (GvHD). The data were obtained from blood samples of 31 patients undergoing blood and marrow transplant.…”

“…Working with the same data, we develop a statistically based processing algorithm that aims to mimic the manual analyses of (6). Except for the choice of a small number of tuning parameters, the algorithm takes the raw FC-HCS data and automatically produces cellular signatures similar to those given in their Figure 3.…”

“…A recent contribution of which we are aware is (7). These authors develop a mixture model approach to automatic gating and also apply it to the data of (6).…”