Anke Glaser

Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application.

Dendritic Cell (DC)-based vaccination approaches in man require a reproducible DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multiple blood drawings to generate DC. To this end we modified our previously described method to generate mature DC from CD14 + monocytes by a two step method (priming in GM-SF + IL-4 followed by maturation in monocyte conditioned medium) for use with leukapheresis products as a starting population. Several adaptations were necessary. We established, for example, a modified adherence step to reliably enrich CD14 + DC precursors from apheresis mononuclear cells. The addition of GM-CSF + IL-4 at the onset of culture proved disadvantageous and was, therefore, delayed for 24 h. DC development from apheresis cells occurred faster than from fresh blood or buffy coat, and was complete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensitizing cells with a characteristic phenotype such as 85% CD83 + , p55/fascin + , CD115/M-CSF-R - , CD86 + ) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absence of cytokines, and to be resistant to inhibitory effects of IL-10. Freezing conditions were established to generate DC from frozen aliquots of PBMC or to freeze mature DC themselves for later use. The approach yields large numbers of standardized DC (5-10 x 10(8) mature CD83 + DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other.

A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use.

Dendritic cells (DC) are increasingly used as a vaccine. Unfortunately, a satisfactory cryopreservation of DC in the absence of FCS is not yet available, so that laborious repeated generation of DC from fresh blood or frozen peripheral blood mononuclear cells for each vaccination has been required to date. We now aimed at developing an effective cryopreservation method, and by testing several variables found that it was crucial to combine the most advantageous maturation stimulus with an improved freezing procedure. We generated monocyte-derived DC from leukapheresis products by using GM-CSF and IL-4 and showed that amongst several known maturation stimuli the cocktail consisting of TNF-alpha+IL-1 beta+IL-6+PGE(2) achieved the highest survival of mature DC. We then systematically explored cryopreservation conditions, and found that freezing matured DC at 1 degrees C/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10x10(6) DC/ml gave the best results. Using this approach 85-100% of the frozen DC could be recovered in a viable state after thawing (Table 1). The morphology, phenotype, survival as well as functional properties (allogeneic mixed leukocyte reaction, induction of influenza matrix or melan A peptide-specific cytotoxic T cells) of these thawed DC were equivalent to freshly prepared ones. The addition of CD40L or TRANCE/RANKL further improved DC survival. Importantly, we demonstrate that DC can effectively be loaded with antigens (such as Tetanus Toxoid, influenza matrix and melan A peptides) before cryopreservation so that it is now possible to generate antigen-preloaded, frozen DC aliquots that after thawing can be used right away. This is an important advance as both the generation of a standardized DC vaccine under GMP conditions and the carrying out of clinical trials are greatly facilitated.

Comparison of COBE white cell-reduction and standard plateletpheresis protocols in the same donors

BACKGROUND: It is necessary to protect patients from white cell (WBC)‐ caused side effects of platelet transfusion by reducing the WBC contamination in single‐donor platelets. STUDY DESIGN AND METHODS: A new COBE Spectra WBC (leuko)‐reduction system (LRS) was compared to the COBE standard plateletpheresis (standard) procedure. Each of 62 donors underwent plateletpheresis under the two protocols (LRS and standard). The collection efficiency and WBC contamination in the components collected using the techniques were compared. Platelets were counted in a cell counter and WBCs were counted using two full grids of a Nageotte chamber. RESULTS: The preseparation and postseparation numbers for red cells, WBCs, and platelets as well as the number of collected platelets were not different in the two techniques. Collection efficiency in the LRS procedures was 96.2 +/− 13.0 percent of that in the standard procedures. Median WBC contamination in the platelet components was 10,160 per LRS procedure and 56,500 per standard procedure. The purity of the LRS components was significantly improved (p = 0.001), as seen in a comparison of the WBC numbers in components per procedure after log10 transformation (LRS: 0.096 +/− 0.195 × 10(6); standard: 0.390 +/− 1.075 × 10(6)). CONCLUSION: These data suggest that the LRS procedure produces platelet concentrates with a collection efficiency that is comparable to that obtained with the standard technique and with a residual WBC content that satisfies even the most stringent criteria for filtered platelets. As this purity can be achieved without platelet loss or alteration, conventional fiber filtration no longer seems necessary or useful in this type of single‐donor platelet component.

Oral or intravenous iron as an adjuvant to autologous blood donation in elective surgery: a randomized, controlled study

BACKGROUND: This study was performed to evaluate the capacity of oral and intravenous (IV) iron administration during autologous blood donation (ABD) to improve the efficacy of ABD and to prevent the need for allogeneic blood transfusion in patients without iron deficiency who are undergoing major elective surgery for which a minimum of 3 autologous units have been ordered.

Comparison of the performance of four microtube column agglutination systems in the detection of red cell alloantibodies.

BACKGROUND: The purpose of this study was to compare the performance of four currently available microtube column agglutination systems in the detection of red cell alloantibodies to that of the standard tube low‐ionic‐strength solution (LISS) indirect antiglobulin test (IAT) (tube LISS‐IAT).

Evaluation of a Platelet Apheresis Technique for the Preparation of Leukocyte-Reduced Platelet Concentrates

Objectives: Reduction of the white blood cell (WBC) contamination in platelet concentrates (PC) protects patients from the immunological and infectious side effects of platelet transfusion caused by WBC. This can be done either by filtration of the PC or by improved apheresis techniques that yield WBC-poor preparations. Methods: To evaluate an improved technique for platelet collection, we carried out 201 separations in 89 healthy cytapheresis donors using the new COBE Spectra leukoreduction system (LRS) and compared the results with those of standard dual-needle separations obtained with the same cell separator. Results: A small but statistically significant difference was found in platelet collection efficiency in comparison with the standard non-LRS software procedures (LRS: 52.6 vs. 56.3% for the reference). However, median WBC contamination was only 0.01 × 106 WBC per LRS product. This significant (p < 0.0005) improvement corresponds to a 10-fold reduction of WBC as compared with the standard dual-needle technique. Conclusions: The COBE Spectra LRS system produced PCs with a platelet collection efficiency nearly equal to previous techniques and with a residual WBC content satisfying even the most stringent criteria for WBC-depleted blood components. As this purity is achieved without important platelet loss, conventional fiber filtration no longer seems necessary in this kind of PC.

Collection of MNCs with two cell separators for adoptive immunotherapy in patients with stage IV melanoma

BACKGROUND: MNCs for adoptive immunotherapy may be collected by leukocytapheresis with a cell separator.

Comparison of a new WBC‐reduction system and the standard plateletpheresis protocol in the same donors

BACKGROUND: A cell separator (Spectra, Gambro BCT) with an integrated leukoreduction system (LRS) for producing WBC‐reduced single‐donor platelet concentrates has been shown to result in a slightly reduced collection efficiency as compared to the former Spectra system without LRS. A novel modified system for improved collection efficiencies (LRS Turbo, Gambro BCT) was evaluated.

Preparation of FFP as a by-product of plateletpheresis.

BACKGROUND: To reduce the production costs of single‐donor platelets (SDPs), a study was conducted to investigate whether plasma collected as a by‐product of plateletpheresis satisfies the quality requirements for FFP without impairing the quality of the SDP component.

Effect of gamma radiation on the in vitro aggregability of WBC‐reduced apheresis platelets

BACKGROUND: The effect of gamma radiation on single‐donor apheresis platelet concentrates (SDPs) has been elucidated only incompletely. The only existing report on the function of SDPs stored in the irradiated state found a deterioration in the in vitro aggregability at the end of shelf life in SDPs divided before irradiation with 1500 cGy.