Immuno-hematological monitoring after allogeneic stem cell transplantation: a single-center, prospective study of 104 patients.

BACKGROUND: The impact of ABO incompatibility on the outcome of hematopoietic stem cell transplantation (HSCT) is still debated. We report the results of a prospective, single-center study evaluating the impact of ABO mismatch on the development of immediate and late immuno-hematological complications, and the efficacy of the protocol used at the "Sapienza" University (Rome, Italy) to manage ABO incompatibility in patients undergoing HSCT. MATERIALS AND METHODS: From January 2013 to December 2016, we prospectively analyzed all patients undergoing HSCT. Graft manipulation or desensitization strategies were used according to ABO incompatibility, donor sex and donor transfusion history. Red blood cell and platelet transfusions were given based on immunohematological features. RESULTS: From January 2013 to December 2016, 104 consecutive patients underwent HSCT from a matched related donor (29.81%), matched unrelated donor (53.58%), cord blood (1.9%) or haploidentical donor (14.42%). Forty-nine patients (47%) were ABO-identical and 55 (53%) ABO-incompatible (23 major, 25 minor, 7 bidirectional). Donor engraftment, graft failure or other complications did not differ between ABO compatible or incompatible patients. ABO incompatibility did not show a significant impact on graft-versus-host disease, overall survival or disease-free survival. Factors associated with the need for prolonged red blood cell support were ABO incompatibility (p=0.0395), HLA disparity between donor and recipient (p=0.004) and the onset of hemorrhagic cystitis (p=0.015). In multivariate analysis HLA disparity was the only statistically significant condition (p=0.004). DISCUSSION: ABO incompatibility does not represent a barrier to allogeneic HSCT. It is, however, associated with prolonged transfusion requirements. Close immunohematological monitoring, as a shared standard procedure, allows appropriate transfusion support to be provided and limits post-HSCT immuno-hematological complications.

A good manufacturing practice method to ex vivo expand natural killer cells for clinical use.

BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

HLA typing strategies in a cord blood bank.

BACKGROUND AND OBJECTIVES: Although widely used, serological typing of HLA loci does not always produce uequivocal results. This may be particularly the case for cord blood since samples may be of small volume and poor quality, and contaminated. DESIGN AND METHODS: We typed 220 cord blood units (CBU) for HLA class I antigens using the serological technique. For those samples giving doubtful results we repeated the HLA typing by polymerase chain reaction with sequence specific primers (PCR-SSP). RESULTS: Results were satisfactory for 181 samples (82.3%). For the remaining 39 (17.7%) we had a doubtful antigen assignment for A locus in 9/39 cases (23.1%) and for B locus in 22/39 cases (56.4%). Eight of the 39 samples (20.5%) could not be analyzed by serology due to the high mortality of the cell suspension. Using PCR-SSP we obtained clear definition of class I antigens in all cases. All CBU were typed for HLA class II alleles by PCR-SSP with clear results in 100% of cases. INTERPRETATION AND CONCLUSIONS: In our experience, PCR-SSP can resolve the limitations of serology but, at the moment, it cannot substitute the latter in routine practice. The best strategy, in cord blood typing, is to perform both serological and molecular typing in order to obtain an accurate and clear result.

Enhanced macrophagic attack on beta-thalassemia major erythroid precursors.

BACKGROUND AND OBJECTIVES: In beta-thalassemia major (Cooley's anemia), ferrokinetic studies show that 60-80% of erythroid precursors die in the marrow or extramedullary sites. However, study of marrow aspirates does not reveal huge numbers of dead and dying erythroid precursors. We explored this apparent discrepancy with the hypothesis that enhanced phagocytosis of thalassemic erythroid precursors was a likely explanation. Prior studies had reported on an increase in thalassemic marrow macrophages and their enhanced state of activation. Therefore this study explored the characteristics of thalassemic erythroid precursors which might lead to enhanced susceptibility to phagocytosis. We have shown that enhanced erythroid apoptosis parallels the extent of ineffective erythropoeisis in thalassemic patients, and apoptotic cells are rapidly phagocytosed. Thus, increased apoptosis and perhaps other features of thalassemic erythroid precursors might be the cause of their enhanced phagocytic removal. DESIGN AND METHODS: Erythroid precursors were isolated from normal and beta-thalassemia major marrow, and incubated with uniform cultures of murine macrophages. The extent of phagocytosis was measured and then specific inhibitors were added to identify some of the messages effete erythroid precursors use to signal their condition to macrophages. RESULTS: Beta-thalassemia major erythroid precursors are phagocytosed twice as effectively as normal erythroid precursors. INTERPRETATION AND CONCLUSIONS: Experiments using inhibitors of phagocytosis showed that enhanced apoptosis is certainly responsible for part of the increased phagocytosis of thalassemic erythroid precursors. Interestingly, normal erythroid precursors are also subject to phagocytosis by qualitatively similar mechanisms.