Sample stability and variability of B‐cell subsets in blood from healthy subjects and patients with systemic lupus erythematosus

Abstract

Objective: Characterization of peripheral leukocytes is an important aspect of monitoring the effect of immunotherapeutic interventions in systemic lupus erythematosus (SLE). We analyzed cell surface markers commonly used to assess patients with SLE, focusing on the effect of holding blood prior to processing/analysis and the relative reliability of the measurements that were conducted.Methods: Healthy volunteers (HV; n 5 20) and patients with SLE (n 5 42) were studied. Whole blood was collected for flow cytometric analysis on days 1, 8, 15, 105, 195, 285, and 375 and held overnight for analysis. A subset of samples was additionally analyzed on the day of collection.Results: Variability arising from overnight storage of whole blood was found to be within 20% for most lymphocyte subsets. There was greater between rather than within subject variability over a 1-year period. As anticipated, the data showed higher CD38 and lower CD19 densities on B cells from patients with SLE compared to HV. Although a higher percentage of cells with markers of plasmablasts/cells were observed in the blood of patients with SLE relative to HV, these measurements were found to be among the least reliable (i.e., most variable).Conclusions: This study provides technical perspectives for those conducting immunophenotypic analyses of B-cells in patients with SLE. We envision that our data, which addresses sample stability issues and presents a method to describe the relative reliability of one measure over another, holds value for clinical assessments of B-cells in SLE and the evaluation of investigational agents designed to modify the B-cell compartment. Numerous B-cell directed therapies are now either marketed or are under active clinical investigation for autoimmune diseases including systemic lupus erythematosus (SLE) (1). These include rituximab, which leads to rapid and profound decrease in circulating B-cells; as well as epratuzumab, an anti-CD22 antibody that also specifically targets B cells but leads to a partial depletion in peripheral B cells. Inhibition of the TNF family cytokine BAFF, either alone or with APRIL, leads to functional inhibition of B-cells and a partial decrease in the number of circulating B-cells over several months (2-4).Changes in numbers and the relative distributions of circulating B-cells and B-cell subsets as a consequence of

Circulating lymphocyte subsets in normal adults are variable and can be clustered into subgroups

“…In the case of small populations, which are given similar weight in standardized lymphocyte fractions, small variations in staining intensity can lead to large VARIABILITY AND CLUSTERING OF LYMPHOCYTE SUBSETS relative changes. Others have also found that variability was proportionally greater for measurements of numerically smaller subsets of lymphocytes (5,6,10,12). In the case of absolute cell counts, additional sources of variation including analytical (e.g., the complete blood count (13), and the use of a dual platform method, as discussed above) and biological [e.g., diurnal shifts (14)], contribute to increased inter-and intraindividual variability.…”

“…These reasons include preanalytical variables such as the source, condition, and manner of preservation of the sample, the time of day that the blood was drawn, subject age, disease state, and medications (5)(6)(7)(8). Analytical variables include instrumentation, instrument settings, the types and quantities of antibody-fluorophore conjugates, and the manner in which lymphocyte subsets are defined and electronically gated (5,9,10).…”

“…When the change in lymphocyte subset percentages was analyzed as a function of the length of time between time points, there was no positive or negative correlation (data not shown). Lymphocyte subsets and their level of variability also differ in subjects with diseases such as SLE (6,(15)(16)(17). Therapy with corticosteroids and other immunomodulatory agents can have profound effects on lymphocyte subset distributions as well (2,(18)(19)(20)(21), further complicating longitudinal data interpretation in the setting of disease.…”

B Cells in Health and Disease – Leveraging Flow Cytometry to Evaluate Disease Phenotype and the Impact of Treatment with Immunomodulatory Therapeutics

“…Therefore, DN memory B cells are also included in Figure 3A. This system is still routinely used to monitor changes in peripheral B cell composition in patients with SLE treated with investigational agents (Belouski et al, 2010 , and CD77 expression in addition to IgD. This resulted in a classification scheme whereby "mature B cells" were binned into categories Bm1 through Bm5 ( Figure 3B) (Pascual et al, 1994 (Bohnhorst et al, 2001).…”

“…However, a cell stabilization formula that truly extends the stability of blood without impacting resolution of dim markers has yet to be brought forward. Peripheral (whole) blood is the specimen of choice in our laboratory because the composition of cells in the unseparated and unfrozen state is most likely to resemble the in vivo state of the blood donor (Belouski et al, 2010). Peripheral blood analysis also has the advantage that the cells are exposed to the biologic matrix throughout the assay.…”