Leukapheresis Safely and Effectively Yields Lymphocyte Populations Sufficient for Chronic Gvhd Research

The pathophysiology of chronic Graft-versus-Host Disease (cGVHD) is complex and poorly understood. Storage of peripheral blood mononucleated cells (PBMC) for research in patient cohorts is vital. However, conventional peripheral blood draws are often insufficient for larger scale immunology studies; in particular, cGVHD patients are frequently lymphopenic, which further limits an ability to study the role of relatively rare populations such as Treg cells. To address this limitation, we evaluated the safety of collecting large quantities of lymphocytes in cGVHD patients by steady-state peripheral blood leukapheresis. In total, 132 patients with cGVHD underwent PBMC collection for research purposes via leukapheresis performed from 2004 to 2014 on NCI cGVHD Natural History Protocol, NCT00092235. Various apheresis machines were used, namely: Baxter CS3000 (n=63, 47%), Haemonetics MCS-P (n=18, 14%), and Terumo SPECTRA (n=52, 39%). Median whole blood/anticoagulant (ACDA) ratio was 12:1; median whole blood flow rate (WBFR) was 60 ml/min (35-85); and median product volume was 147 ml (59-450). Median age was 50 years (18-68). NIH cGVHD Global scores were moderate (n=46, 35%) or severe (n=82, 62%). Mean absolute circulating PBMC level pre-leukapheresis was 2.4 K/µL (0.3-11.6). 22 patients (17%) had an absolute lymphocyte count <1.0 K/µl. A subset of patients (17/132) had sufficiently low pre-leukapheresis peripheral counts that required special approval for the procedure (Hb <9.0 g/dL, n=3; WBC < 3.0 K/µL, n=5; platelets < 120 K/µL, n=9). A two-arm continuous flow apheresis procedure was used in 108 patients (81%) whereas a one-arm procedure was used in 25 patients (8%). Only nine patients underwent leukapheresis via central venous catheter (the remainder were pheresed using peripheral access). 94 (71%) achieved the goal collection of 2 x 109 PBMCs, with a mean volume processed of 4.6 L.  Median total run time was 88 minutes. Mean number of cells collected and efficiencies were: (1) lymphocytes, 3.7 x 109, 66.5%; (2) monocytes, 1.1 x 109, 51.1%; and (3) PBMCs: 4.8 x 109, 59.7%. Mild decreases in absolute peripheral counts were observed, including (median change 1 hour post-leukapheresis; each value significant, p<0.0001):  Hgb: -8%; Plt: -22%; lymphocytes: -26%; and monocytes: -17%. Grade 1 AEs (CTCAE v4.03) were experienced by 21 patients (16%): hypotension (n=1, 1%), oral dysesthesia (n=10, 8%), paresthesias (n=6, 5%), anxiety (n=1, 1%), localized bleeding (n=2, 2%), and nausea (n=1, 1%). In conclusion, we found that steady-state PBMC leukapheresis represents a safe and effective method for collecting high numbers of immune cells in patients with moderate-to-severe cGVHD. Wider utilization of leukapheresis in the cGVHD setting should accelerate immunology research into the pathogenesis of cGVHD, particularly relating to the role of relatively rare cell populations.