Patients

Between June 2014 and October 2015, we prospectively analyzed 50 patients with malignant hematological disorders undergoing allo-HSCT. CEC counts were performed in parallel by means of two different methodologies: (1) polychromatic flow cytometry (PFC) using, all along the study, a single batch of pre-formatted lyophilized-reagent tubes (Becton Dickinson, San Jose, CA, USA; Lyotube, Custom cat # 623920)^18,19; (2) CellSearch system (Janssen Diagnostics LLC, Raritan, NJ, USA). The local research and ethics committee (Comitato Etico della Provincia di Brescia, document NP 1574 of the 14^th January 2014 and Comitato Etico interaziendale AOU Città della Salute e della Scienza di Torino, document 0037975 of the 10^th April 2014) approved the study protocol and all patients and controls provided written informed consent, in accordance with the Declaration of Helsinki. The trial was registered on ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT02064972","term_id":"NCT02064972"}}NCT02064972).

CEC counting was scheduled before and after conditioning regimen, at time of hematopoietic engraftment, at day +28 in the absence of GVHD, at time of GVHD onset and 1 week after steroid treatment. Day +28 was selected as a CEC counting timepoint in patients without GVHD, since the median time of GVHD onset in our previous series was 27 days¹⁴. Therefore, CEC values at day +28 in patients without GVHD were compared with CEC values at GVHD onset. Thus, CEC count during study was performed at the following five time points: T1 (pre-conditioning), T2 (pre-transplant), T3 (engraftment), T4 (GVHD onset or day +28), T5 (1 week after steroids).

During study period, levofloxacin prophylaxis was continued until neutrophil recovery, and fluconazole/itraconazole until immunosuppressive drugs has been suspended, while trimethoprim-sulphamethoxazole was used for Pneumocystis jirovecii prevention. Cytomegalovirus was weekly PCR monitored, and patients testing positive have received ganciclovir/foscarnet treatment. Fungal infections have been diagnosed according to published revised criteria²¹. GVHD diagnosis and grading were defined according to commonly accepted criteria²².