Counting circulating endothelial cells in allo-HSCT: an ad hoc designed polychromatic flowcytometry-based panel versus the CellSearch System

Abstract

Physio-pathologic interrelationships between endothelial layer and graft-versus-host disease (GVHD) have been described leading to assess the entity "endothelial GVHD" as the early step for clinical manifestations of acute GVHD. The availability of the CellSearch system has allowed us to monitor Circulating Endothelial Cells (CEC) changes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as useful tool to help clinicians in GVHD diagnostic definition. We have compared CEC counts generated by an ad hoc designed polychromatic-flowcytometry (PFC) Lyotube with those of the CellSearch system. CEC were counted in parallel at 5 timepoints in 50 patients with malignant hematologic disorders undergoing allo-HSCT (ClinicalTrials.gov, NCT02064972). Spearman rank correlation showed significant association between CEC values at all time points (p = 0.0001). The limits of agreement was demonstrated by Bland Altman plot analysis, showing bias not significant at T1, T3, T4, while at T2 and T5 resulted not estimable. Moreover, Passing Bablok regression analysis showed not significant differences between BD Lyotube and CellSearch system. We show that CEC counts, generated with either the CellSearch system or the PFC-based panel, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as a suitable tool to assist clinicians in GVHD diagnosis.

Conflict of interest statement

K.B. is employee of Janssen Diagnostics. G.L.R. is employee of Becton Dickinson Biosciences Italia.

Figures

Figure 1

Flow cytometry identification and count of CEC in PB samples of a representative patient. CEC were analyzed and counted in an AML patient (57 years), conditioned with Fludarabine/Bu4 regimen and receiving HPC-A from a matched unrelated donor (engraftment at day +28, skin GVHD diagnosed at day +45). Panel A: (a) Events showing lympho-monocyte morphology were gated in a FSC/SSC plot. (b) Dead cells were excluded because 7-AAD positive and afterwards (c) nucleated events were gated. Cells resulting from the logical combination of the three gates intersection were analyzed for CD45 and CD34 expression. Panel B–F: Two subpopulations, showing different levels of CD34 surface expression, were separately gated: CD34 positive cells, being CD45dim, represent the hematopoietic stem cell compartment (green dots), and CD34 bright cells, resulting CD45 negative (blue dots). Both subpopulations were than analyzed for CD146 expression (right column): CEC resulted CD34 bright/CD45 negative/CD146 positive (blue dots). The plot analysis are shown at the different timepoints during allo-HSCT (panel B: T1 pre-conditioning; panel C: T2 pre-Transplant; panel D: T3 engraftment; panel E: T4 GVHD onset; panel F: T5 1 week after steroids). The corresponding results of CEC calculations are shown in Supplemental Table S2.

Figure 2

CEC counts during allogeneic hematopoietic stem cell transplantation. CEC counts performed with CellSearch (V) and with polychromatic flow cytometry (PFC) are shown on right and left column, respectively. CEC values at T1 (pre-conditioning) are shown in row (A) according to diagnosis, in row (B) to disease status, while CEC values at T2 (pre-transplant) are shown in row (C) according to conditioning, in row (D) to HPC donor, and CEC values at T3 (engraftment) are shown in row (E) according to GVHD prophylaxis.

Figure 3

CEC counts in patients with and without GVHD. CEC counts performed with PFC Lyotube (panel A) and with CellSearch System (panel B) at T3 (engraftment) and T4 (GVHD onset or day +28) timepoints.

Figure 4

Linear correlation of CEC counts performed with CellSearch system and BD Lyotube (Custom cat # 623920). Linear correlation (Passing Bablok regression analysis) of CEC counts performed with CellSearch versus PFC (Lyotubes; BD Biosciences) at the different timepoints. Black line shows the regression line, dashed line the 95% C.I. of regression line, while dotted line represents the identity line (x = y).

Figure 5

Linear correlation of CEC counts performed with CellSearch system and BD Lyotube (Custom cat # 623920) at T2 timepoint. Panel A shows the absence of correlation in the first 24 patients, in which the unreliability of lymphocytes absolute numbers obtained by standard cell counter in a very deep leukopenia phase, heavily affected the CEC counts determined by PFC. Intercept A = −905,7(−9816,8 to −112,6); slope B = 20,2 (6,5 to 155,6). Panel B shows the linear correlation from the 25th patient onwards, in which lymphocytes absolute numbers was PFC determined (CD45pos events in lympho-monocyte gate). Intercept A = 5,4 (−34,1 to 71,9); slope B = 1,8(1,2–2,37); not significant deviation from linearity (p > 0,05). Black line shows the regression line, dashed line the 95% C.I. of regression line, while dotted line represents the identity line (x = y).