Chimaerism of FACS-sorted leucocyte subsets (CD14 þ , CD15 þ , CD3À/56 þ , CD3 þ /4 þ , CD3 þ /8 þ , CD19 þ ) was monitored prospectively between days þ 14 and þ 100 in 39 children undergoing allogeneic stem cell transplantation with reduced intensity-conditioning regimens. Cell subsets exceeding 1% of nucleated cells were subject to cell sorting. Chimaerism was analysed by dual-colour FISH and/or by short tandem repeat-polymerase chain reaction. The chimaerism pattern on day þ 28 was evaluated with regard to its correlation with graft rejection. Of 39 patients, nine patients had donor chimaerism (DC) in all subsets. Mixed/recipient chimaerism (MC/RC) was detectable within T cells in 62%, within NK cells in 39% and within monocytes and granulocytes in 38% of the patients. The correlation of secondary graft rejection with the chimaerism pattern on day þ 28 revealed the strongest association between RC in NK-cells (Po0.0001), followed by T cells (P ¼ 0.001), and granulocytes and monocytes (P ¼ 0.034). Notably, patients with RC in T cells rejected their graft only if MC or RC was also present in the NK-cell subset. By contrast, none of the children with DC in NK cells experienced a graft rejection. These observations suggest that, in the presence of recipient T-cell chimaerism, the chimaerism status in NK-cells on day þ 28 might be able to identify patients at high risk for late graft rejection.
There is increasing demand for mononuclear cell (MNC) harvests not only for PBPC but also for immune therapies using dendritic cells and donor lymphocytes. We determined the collection efficiencies (CE) of various MNC subpopulations during CD34+ cell harvests using a Fenwal CS 3000 Plus Omnix system in small children and adolescents. The cell content of 140 leukapheresis products (LP) was prospectively evaluated in 45 pretreated patients with solid tumors and hematological malignancies. The median age was 12 years (range 0.8-22), and the median body weight (BW) 43 kg (range 9-92). Depending upon the BW of the patients, the media used for priming were saline (SP) in 86, human albumin (HA, HAP) in 10, and packed red blood cells (BP) in 44 apheresis procedures. The major nucleated cell (NC) fractions collected were monocytes (52% of NC) and CD3+ T cells (26%). The median cell yield for monocytes was 174 * 10(6)/kg (range 24-613) representing a CE of 55%. The median number of CD3(+) T cells was 84 * 10(6)/kg (range 5.6-380; CE = 74%). CD34+ cells represented a very small cell fraction of the LP (1.3% of NC), with a median yield of 4.2 * 10(6)/kg (range 0.2-87) and a CE of 63%. The cell yield of various MNCs was significantly correlated with the cell count in the peripheral blood (PB) and with the blood volume processed (ANOVA, P < 0.0001). No influence on the CE was observed for the priming procedure, the patients' age or sex, or the other adaptations used in the harvesting protocol. In conclusion, the Fenwal CS 3000 Plus OMNIX system with the CD34+ cell program and the described adaptations, is also predictably useful for harvest of monocytes or lymphocytes in pediatric patients. We present regression equations that predict the cell yield of various MNC subpopulations in apheresis products.
Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30 -500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components. Cytometry (Clin. Cytometry) 50:231-237, 2002.
The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.
Background: Methods for clinical-scale selection of CD34-positive hematopoietic stem and progenitor cells have facilitated allogeneic transplants using HLA-mismatched healthy donors. We examined different approaches to purify mobilized CD34+ cells, focusing on yield, purity, and viability of the selected cells and T-cell depletion levels. Methods: Sixty-seven CD34-positive selections were performed for a total of 37 allogeneic transplantations, 23 of which from HLA-haploidentical donors. The selection devices were the Isolex® 300i (v. 1.12) used alone (n =13) or with the SuperMACS (n = 29); the CliniMACS (n = 3); and the Isolex 300i (v. 2.0b1). The latter was used for CD34-positive selection (n = 7) and combined CD34+/CD4 8 19-negative selections (n =15). DNAse was included to reduce cell clumping. Results: With the Isolex 300i (v. 1.12), the median CD34+-cell recovery increased from 51% (without DNAse) to 61% (15 mg DNAse) and 70% (7.5 mg). DNAse (5 mg) was used for 22 selections with the Isolex (v. 2.0b1) without cell clumping. CD34-positive cell purity, yield, and viability, as well as the degree of CD3 depletion varied with the selection device and procedure used. Conclusion: With regard to all of the above-mentioned parameters, the best results were obtained with the Isolex 300i (v. 2.0b1). Values achieved for CD34-positive cells were 98% for purity, 50–60% for yield, and > 96% for cell viability; T-cell depletion was 4.5 to > 5 log. The automated and closed system provides target cells that are free of both magnetic particles and murine monoclonal antibody.
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