Managing leukapheresis in adult and pediatric patients eligible for chimeric antigen receptor T-cell therapy: suggestions from an Italian Expert Panel.

Chimeric antigen receptor (CAR) T-cell therapy relies on T cells engineered to target specific tumor antigens such as CD-19 in B-cell malignancies. In this setting, the commercially available products have offered a potential long-term cure for both pediatric and adult patients. Yet manufacturing CAR T cells is a cumbersome, multistep process, the success of which strictly depends on the characteristics of the starting material, i.e., lymphocyte collection yield and composition. These, in turn, might be affected by patient factors such as age, performance status, comorbidities, and previous therapies. Ideally, CAR T-cell therapies are a one-off treatment; therefore, optimization and the possible standardization of the leukapheresis procedure is critical, also in view of the novel CAR T cells currently under investigation for hematological malignancies and solid tumors. The most recent Best Practice recommendations for the management of children and adults undergoing CAR T-cell therapy provide a comprehensive guide to their use. However, their application in local practice is not straightforward and some grey areas remain. An Italian Expert Panel of apheresis specialists and hematologists from the centers authorized to administer CAR T-cell therapy took part in a detailed discussion on the following: 1) pre-apheresis patient evaluation; 2) management of the leukapheresis procedure, also in special situations represented by low lymphocyte count, peripheral blastosis, pediatric population <25 kg, and the COVID-19 outbreak; and 3) release and cryopreservation of the apheresis unit. This article presents some of the important challenges that must be faced to optimize the leukapheresis procedure and offers suggestions as to how to improve it, some of which are specific to the Italian setting.

Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients.

Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T-cell lymphoma, and it is currently used off-label for graft-versus-host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off-line technique. Since no gold-standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty-one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8-methoxy-psoralen plus ultraviolet A; the variation (ΔAPOPTOSIS ) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, ΔAPOPTOSIS was >15% (the "alerting" threshold for ΔAPOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test-being faster and easier than the proliferation test-could be a reliable way to validate ECP procedures.

Screening of related donors and peripheral blood stem cell collection practices at different Italian apheresis centres.

BACKGROUND: Recommendations on eligibility criteria for donation of haematopoietic stem cells, management of collection of the cells and follow-up mainly concern unrelated donors. The aim of this study was to analyse the screening of related donors and collection practices at different Italian apheresis centres. MATERIALS AND METHODS: A questionnaire regarding eligibility criteria for related haematopoietic stem cell donors, their peripheral blood collections and early follow-up was sent to several apheresis units. Data from the full charts of 500 candidates, screened between May 2005 and December 2009, were retrospectively evaluated. RESULTS: The donors' records, eligibility criteria, collections and follow-up are managed differently in each centre. Of the 500 evaluable candidates (51.2% male, 49.8% female; median age 47 years, range 13-77), 26.4% underwent thorough screening according to Italian Bone Marrow Donor Registry standards, while local protocols were applied to 73.6%; 91 candidates (18.2%) proved ineligible for donation. In the end, 352 donors (53.4% male, 46.6% female; median age 45 years, range 16-76) underwent 508 leukaphereses. Central venous catheters were used in 8.0% of donors, mainly in one centre. Unsuitable pre-apheresis peripheral blood parameters were reported in 38.7% of the aphereses. Leukapheresis-related adverse events were recorded in 23.0% of the procedures, with a drop-out rate of 0.2% for severe events. No donation-related fatalities occurred. The CD34+ cell yield was <2×10(6)/kg of recipient's body weight from 1.1% of donors ≥70 years old. DISCUSSION: More uniformity in donor screening procedures, management of peripheral blood collection and follow-up should be planned at a national level to maximise the safety of related donors.

Peripheral blood stem cell collection in multiple myeloma: a retrospective analysis of 6 years leukapheresis activity in 109 patients treated at the Istituto Nazionale dei Tumori of Milan.

Double autologous stem cell transplantation is the standard treatment in newly diagnosed multiple myeloma (MM) patients younger than 65 years; therefore, optimization of leukapheresis is crucial. We performed a retrospective analysis of 297 leukaphereses comparing semiautomated (V4.7 in 20% of collections) versus automated (V6.0 in 80%) Caridian (COBE) Spectra versions and analyzing the influence of M-protein on the outcome. Both methods gave comparable collection efficiencies (CE%) (53.4% vs. 55.7% in V6.0 and V4.7, respectively) with similar leukapheresis time and processed volume. Harvest volume was higher in V4.7 (P < 0.0001) with similar contamination of red blood cells (RBCs) (P = 0.77) and platelets (P = 0.09) when compared with V6.0. In patients with higher peripheral white blood cells (WBCs), V6.0 with adjusted harvest volume (<700 mL), achieved similar CD34(+) CE% (P = 0.39) and better enrichment of nucleated cells (P < 0.0,002) but higher RBCs (P < 0.0,001) and platelets contamination (P = 0.001), when compared with a larger cycle volume in patients with lower WBCs. In hard to mobilize patients, CD34(+) CE% was significantly more efficient with V4.7 than V6.0 (P < 0.0,001). CD34(+) CE% was unaffected by serologic M-protein, but platelet CE% was higher in the absence of M-protein (P = 0.0,003), without any reduction in peripheral patients platelets. We, therefore, conclude that in the setting of MM patients with a high WBCs count and/or low percentage of peripheral CD34(+) cells, collections with V4.7 or adjusted cycle volume V6.0 gave comparable result in CD34(+) CE%. RBCs and platelets contamination is higher if low cycle volume is chosen. In hard to mobilize patients, V4.7 is advisable.

Management of poor peripheral blood stem cell mobilization: incidence, predictive factors, alternative strategies and outcome. A retrospective analysis on 2177 patients from three major Italian institutions.

CD34+ peripheral blood hematopoietic stem cells (HSC) are usually collected following mobilization therapy accomplished by using growth factors (GF) such as rHuG-CSF or rHuGM-CSF with or without chemotherapy. A target dose of yielded CD34+ is usually prescribed by the attending physician depending on different protocols, which may include single or double transplantation. HSC collection usually is performed when at least 20 CD34+ HSC/microL are detected by means of flow cytometry. A cumulative dose of at least 2 x 10(6)/Kg/bw CD34+ HSC has been considered as the threshold to allow a prompt and persistent hematopoietic recovery. Unfortunately, this goal is not achieved by the totality of patients undergoing mobilization regimen. In fact, 5-46% of patients who underwent mobilization therapy fail HSC collection due to very low peripheral blood HSC CD34+ count. Patients' characteristics, including age, sex, stage of the underlying disease (complete or partial remission), diagnosis, previously administered radio/chemotherapy regimens, time-lapse from last chemotherapy before mobilization and mobilization schedule (including dose of GF) were considered as possibly predictive of poor or failed mobilization. We performed a retrospective analysis in 2177 patients from three large Italian academic institutions to assess the incidence of poor mobilizers within our patients' series. Therefore, a patient who fails a first mobilization (and when an HLA-compatible related on unrelated donor is not available) could undergo a second attempt either with different mobilization schedule or by using different GF, such as stem cell factor, growth hormone (GH), or more recently newly introduced drugs such as AMD3100, alone or in combination with rHuG- or -rHuGM-CSF. Thus, we investigated the fate of those who failed a first mobilization and subsequently underwent a second attempt or alternative therapeutic approaches.

Peripheral blood stem cell collection in pediatric patients: feasibility of leukapheresis under anesthesia in uncompliant small children with solid tumors.

Leukapheresis demands patient's compliance and adequate vascular accesses, which can require invasive methods in very small children whose treatment protocol includes hemopoietic stem cell collection for myeloablative chemotherapy and stem cell rescue. Since 1998, at the Istituto Nazionale Tumori of Milan, in selected uncompliant small children, the placement of peripheral vascular accesses and leukapheresis have been performed at the same time under general anesthesia. Peripheral venous cannulas were positioned for blood collection, while blood was returned through either peripheral cannulas or mono-lumen central catheters previously installed for chemotherapy. A continuous-flow cell separator was used for leukapheresis. Between 1998 and 2003, 47 children with solid tumors underwent anesthesia for a total of 54 leukaphereses. The patients' age ranged from 12.7 to 93 months (median 30.3) and their weight ranged from 7 to 20 kg (median 14.1). Neither metabolic nor anesthesiological complications were recorded. In 89% of cases, the CD 34(+) cell target was achieved at a single harvest; the median number of CD 34(+) cells was 10.8 x 10(6)/kg/leukapheresis (range 1-117) and the median collection efficiency was 63.4% (range 25-100.6). Leukapheresis under anesthesia is feasible and safe in very low-weight children whose compliance is lacking due to age and disease.

Integration between point-of-care cardiac markers in an emergency/cardiology department and the central laboratory: methodological and preliminary clinical evaluation.

To achieve rapid assessment of chest pain in emergency/cardiology departments, a short turnaround time for cardiac marker testing is necessary. Nevertheless, Total Quality Management principles must be incorporated into the management of point-of-care testing (POCT); in this setting we implemented the Stratus CS assay as POCT for cardiac markers in our emergency/cardiology department. The analytical performance of the troponin I method was evaluated; information connectivity between the Stratus CS data management system and the laboratory information system was implemented and practical training of testing personnel was carried out at the POCT site. A total of 41 non-ST-segment elevation patients admitted to the hospital were followed to evaluate the appropriateness of hospital admission, formulated on the basis of the cardiac troponin-I level measured at the POCT site by clinical staff. Our preliminary clinical data suggest that the high sensitivity of the Stratus CS troponin method could play an important role in the early identification of patients with acute myocardial infarction in a low to intermediate-risk population for acute coronary syndrome. Our POCT model suggests that the central laboratory could ensure that the POCT program remains in compliance with quality requirements. Nevertheless, our comparison studies suggest that the implementation of POCT requires a high level of integration between cardiologists and pathologists to guarantee appropriate interpretation of the monitoring results for suspected ACS patients.