Mobilization, Collection, Infusion and Granulocyte Recovery of PBSC in Type 1 Diabets Mellitus Pacients after High Dose Immunosupression.
Authors: Prata Karen L. , De Santis Gil C. , Oliveira Maria Carolina B. , Moraes Daniela A. , Júnior Benedito P.A. Prado , Orellana Maristela D. , Palma Patricia V.B. , Stracieri Ana Beatriz P.L. , Pieroni Fabiano , Coutinho Marina A. , Simões Belinda P. , Covas Dimas T. , Voltarelli Júlio C. 
Published: October 2019

Abstract
Background. Type 1 diabetes mellitus (T1DM) is an autoimmune disease. A safe induction of an autoimmunity-free status may become a promising therapy. We hypothesize that intense immuno- and myelosuppression followed by autologous hematopoietic stem cell (HSC) rescue is an option to establish a lasting immunetolerance by eliminating auto-reactive lymphocytes and thus facilitate a possible recovery of autologous insulin production. Aims. Evaluate the HSC mobilization, infusion and engraftment in T1DM patients.
Patients and Methods. Between January of 2004 and July of 2006 15 T1DM patients (12 male/3 female), with no more than 6 weeks after the first episode of hyperglycemia, were selected and enrolled. Their median age was 17 years old (range 14–31). HSC were mobilized into the peripheral blood (PB) by cyclophosphamide (Cy) 2g/m2 and filgrastim (G-CSF) 10 μg/kg/day. Peripheral blood stem cells (PBSC) were collected by leukapheresis using COBE Spectra (Gambro BCT, Lakewood, CO) blood cell separator. Collected PBSC were mixed with a cryoprotectant solution (autologous plasma and 10% dimethyl-sulfoxide (DMSO)), then frozen and stored in a mechanical freezer (−80°C). The CD34+ cell were counted using the ISHAGE protocol by a FACSort flow cytometer and CellQuest software packages (Becton Dickinson, San Jose, CA, USA). The conditioning regimen was Cy 200mg/kg and anti-lymphocyte globulin (4.5mg/kg). The protocol and the consent form were approved by the institution and the national ethics committees.
Results. All results are expressed as median (range), except when specified. The collection was done on the 7th day (7–9) after the chemotherapy. The pre-apheresis values were: WBC (×103/μL) 7.9 (2.6–56.0); Hct (%) 37.4 (30.9–44.7); platelets (×103/μL) 177 (87–295); CD34+ (μL) 80.9 (36.5–167.6). The blood volemia processed and apheresis duration (min) were 2.8 (2.0–3.1) and 235 (177–280), respectively. Five patients (33.3%) experienced mild adverse reactions related to G-CSF administration (osteo-muscular pain and headache) and six (40%) related to citrate infusion (paresthesia and tremors), although thirteen (86%) had received prophylactic intravenous calcium infusion. The final yield values were: volume (mL) 210 (170–273); WBC (×108/Kg) 6.0 (3.0–14.9); Hct (%) 5.0 (3.3–9.0); CD34+ (×106/kg) 9.6 (5.8–22.5). Fourteen patients have already been transplanted. The time between cryopreservation and infusion was 21 days (13–35). The dose of DMSO infused was 0.4 mL/kg (0.0–0.5) (one patient received washed PBSC). All patients presented mild complications related to infusion: 13 (93%) nausea or vomiting, 8 (57%) flushing, 5 (36%) abdominal pain, 4 (29%) headache and 3 (21%) dyspnea, but only one needed oxygen supplementation. None of the patients presented complications like renal failure, hepatic insufficiency or cardiac arrhythmias. The time to reach granulocytes recovery (500/μL) was 9 days (7–10).
Conclusions. The present data suggest that HSC mobilization in T1DM patients are very effetive, precocious and provide a good collection of CD34+ cells. The infusion of PBSC is a safe and reliable procedure with a low incidence of severe side effects and early engraftment. These results could be explained by a good bone marrow function as these patients had never been submitted to any kind of myelo or immunosuppressive therapy.