Prognostic factors and outcomes for pediatric patients receiving an haploidentical relative allogeneic transplant using CD3/CD19-depleted grafts.

Haploidentical hematopoietic stem cell transplantation using T-cell-depleted grafts is a valid option for pediatric patients with hematological malignancies in need of an allogeneic transplantation and lacking an HLA-identical donor. Seventy-five transplantations were performed in 70 patients. Thirty-eight patients had ALL, 32 had AML, 3 had advanced myelodysplastic syndromes and 2 juvenile myelomonocytic leukemia; 19 were in first CR, 30 in second CR, 12 in greater than second CR and 14 were considered to be in refractory disease at time of transplantation. Four patients developed graft failure. Among engrafted patients, the median time to neutrophil and platelet recovery was 13 (range 8-20) and 10 days (range 8-70), respectively. In 64 (85%) cases, ⩾1 infections were diagnosed after transplant. The probability of nonrelapse mortality by day +100 after transplantation was 10±4%. With a median follow-up of 22 months, the probability of relapse was 32±6% and disease-free survival was 52±6%. Haploidentical transplantation using CD3/CD19 depletion is associated with encouraging results especially in patients in early phase of disease. Killer-cell Ig-like receptor B haplotype donors confer a rapid natural killer cells expansion early after transplantation, resulting in lower probability of relapse and suggesting a GvL effect apart from graft-versus-host reactions. Donor infusion of high numbers of CD34+ cells is recommended in order to improve T-cell reconstitution.

TCR-alpha/beta and CD19 depletion and treosulfan-based conditioning regimen in unrelated and haploidentical transplantation in children with acute myeloid leukemia.

We evaluated the depletion of TCR-alpha/beta cells from the graft of children with high-risk AML, who received transplantation from unrelated (n=20) and haploidentical donors (n=13). The preparative regimen included treosulfan, melphalan, fludarabine and anti-thymocyte globulin. Grafts were PBSC engineered by TCR-alpha/beta and CD19 depletion. The graft contained a median of 9 × 10(6)/kg of CD34+ and 20 × 10(3)/kg of αß-T cells. Post-transplant immune suppression included tacrolimus till day +30 and Mtx in 21 patients, tacrolimus in 5, Mtx in 2 and no prophylaxis in 5 patients. Sixteen patients received native or TCR-alpha/beta-depleted donor lymphocytes at a median of 47 (40-204) days. Median follow-up is 1.76 years. Primary engraftment was achieved in 33 patients (100%). Cumulative incidence of acute GvHD (aGvHD) grade 2-3 was 39 (26-60)%, half of them had skin-only aGvHD. Cumulative incidence of chronic GvHD was 30(18-50)%. Transplant-related mortality is 10(4-26)%. Event-free survival (EFS) is 60(43-76)% and overall survival (OS) is 67(50-84)% at 2 years. In a subgroup of patients, who received transplantation in CR, EFS is 66(48-84)% and OS-72(53-90)% at 2 years. Our data suggest that TCR-alpha/beta and CD19 depletion is a robust method of graft manipulation, which can be used to engineer grafts for children with AML.

Consistent, multi-instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference.

Detecting changes in the expression levels of cell antigens could provide critical information for the diagnosis of many diseases, for example, leukemia, lymphoma, and immunodeficiency diseases, detecting minimal residual disease, monitoring immunotherapies and discovery of meaningful clinical disease markers. One of the most significant challenges in flow cytometry is how to best ensure measurement quality and generate consistent and reproducible inter-laboratory and intra-laboratory results across multiple cytometer platforms and locations longitudinally over time. In a previous study, we developed a procedure for instrument standardization across four different flow cytometer platforms from the same manufacturer. CD19 quantification was performed on three of the standardized instruments relative to CD4 expression on T lymphocytes with a known amount of antibody bound per cell (ABC) as a quantification standard. Consistent and reliable measures of CD19 expression were obtained independent of fluorochrome used demonstrating the utility of this approach. In the present investigation, quantification of CD20 relative to CD4 reference marker was implemented within a single tube containing both antibodies. Relative quantification of CD20 was performed using anti-CD20 antibody (clone L27) in three different fluorochromes relative to anti-CD4 antibody (clone SK3). Our results demonstrated that cell surface marker quantification can be performed robustly using the single tube assay format with novel gating strategies. The ABC values obtained for CD20 expression levels using PE, APC, or PerCP Cy5.5 are consistent over the five different instrument platforms for any given apparently healthy donor independent of the fluorochrome used.

Comparative evaluation of two purification methods of anti-CD19-c-myc-His6-Cys scFv.

Different chromatographic methods have been used to purify bacterially expressed single chain antibodies in soluble or insoluble form. Here, we compared two methods for purification of anti-CD19-c-myc-His6-Cys scFv expressed in Escherichia coli as soluble protein. The protein-L-agarose purification method is a one step purification method that yielded significant amounts of pure protein compared to the two-step Ni-NTA-agarose plus Resource 15S purification method. However, the protein-L purification method exhibited an additional lower molecular weight protein contaminant. Based on results from in vitro gel digestion, mass spectrometry and database search results, we confirmed that the lower molecular weight protein contaminant, which could not be purified by Ni-NTA-agarose and 15S column method, is a degraded product of the full length scFv construct.

CD19+ B lymphocytes are the major source of human antibody-secreting hybridomas generated by electrofusion.

Human monoclonal antibodies may be generated by electrofusion of human B lymphocytes with a human/mouse heteromyeloma line. In addition to a fusion protocol optimised for the fusion partners, the activation of B lymphocytes is crucial for fusion and hybrid efficiency. In this study, we initially treated peripheral blood mononuclear cells (PBMC) from normal blood donors with a large panel of known stimulants and determined the yield of human antibody-secreting hybridomas after electrofusion with the heteromyeloma cell line H73C11; 3- to 5-day incubation with phytohaemagglutinin L (PHA-L) resulted in the highest number of secreting hybrids. In a second set of experiments, PBMC were depleted from various cell populations, including CD14+ monocytes, CD8+ T lymphocytes, and CD2+ T cells, respectively. Undepleted PBMC stimulated with PHA-L were shown to give rise to the highest number of secreting hybridomas when subjected to electrofusion, whereas depletion of CD2+ T lymphocytes greatly reduced the yield. In a final set of experiments, CD19+ B lymphocytes were identified as the major source of secreting hybridomas. For optimal fusion efficiency, CD19+ B cells were shown to require direct physical contact with other cell populations, most probably T lymphocytes, during the stimulation process. Our data highlight the importance of an adequate stimulation prior to electrofusion and may be helpful to further facilitate the development of human monoclonal antibodies.

A three-step procedure for the purification of human basophils from buffy coat blood.

OBJECTIVE AND DESIGN: We report a method for basophil purification from buffy coats, which avoids positive selection of the cells and gives rise to good purity, yield and functional integrity of the cells. SUBJECTS: Buffy coat blood (concentrated leukocyte fraction derived from 450 ml venipuncture donations) obtained from healthy blood donors (n = 51). METHODS: Basophils were enriched by a three-step process starting with Ficoll density centrifugation (1.6 +/- 0.1% basophil purity) followed by counter current centrifugal elutriation (17.7 +/- 1.4% basophil purity). The final stage involved negative selection using Dynal immunomagnetic beads directed against CD2, CD14, CD16 and CD19 positive cell contaminants. Functional integrity of which was assessed by comparing the anti-IgE or calcium ionophore A23187 induced histamine release from basophils obtained from each enrichment step. Furthermore, basophil morphology was investigated using light and electron microscopy. RESULTS: The final mean basophil purity of 67.3 +/- 1.4% with a yield of 3.5 +/- 0.5 x 10(6) basophils and a recovery of 21.8 +/- 2.4% was achieved. Net histamine release from basophils stimulated with optimal concentrations of anti-human IgE was 39.1 +/- 6.5% after Ficoll centrifugation, 41.6 +/- 7.7% following elutriation and 35.7 +/- 6.8% from the final purified fraction. Additionally, basophils enriched with our method showed intact morphology by electron microscopy and were functionally active to non-immunological stimulation. CONCLUSIONS: These results compare favourably with previous studies, which have often required the use of positive selection via the Fc epsilon RI receptor, which may result in cell degranulation, or cell sorting, which cannot be applied to large cell numbers. Our method provides a reproducible technique for basophil enrichment when large numbers of functionally intact basophils are required.