Collection of platelets and peripheral progenitor cells with the fresenius AS.TEC 204 blood cell separator.

The aim of the present study was to clinically evaluate the blood cell separator AS.TEC 204. One hundred fifteen platelet collections were carried out with the dual or single needle procedure. Platelet yield was 3.21 +/- 0.80 x 10(11) (mean +/- standard deviation) and 59.1% of the collections showed platelet counts > or = 3.0 x 10(11). Leukocyte contamination was 1.77 +/- 2.81 x 10(6) and 89.0% of the platelet concentrates had a white blood cell content < 5 x 10(6). Using a dual needle technique with an alternating interface adjustment, all of the products were contaminated with less than 1 x 10(6) leukocytes. Furthermore, 23 peripheral progenitor cell collections were performed in 12 patients and three allogeneic donors. Median numbers of harvested CD 34 antigen expressing cells/kg body weight were 0.78 (range 0-4.7) and 3.67 x 10(6) (range 2.2-5.23), respectively. We conclude that platelet and progenitor cell collections can be carried out with efficient results. The collections were well tolerated by the donors.

Evaluation of a new protocol for peripheral blood stem cell collection with the Fresenius AS 104 cell separator.

In this report we analyzed sixty leukapheresis procedures on 35 patients with a new protocol for the Fresenius AS 104. Yields and efficiencies for MNC, CD 34+ cells, and CFU-GM indicate that the new protocol is able to collect large quantities of hemopoietic progenitors. Procedures were performed processing 8.69 +/- 2.8 liters of whole blood per apheresis and modifying 3 parameters: spillover-volume 7 ml, buffy-coat volume 11.5 ml, centrifuge speed 1,500 rpm; blood flow rate was 50 ml/min and the anticoagulant ratio was 1:12. No side effects were observed during apheresis procedures except for transient paresthesia episodes promptly resolved with the administration of calcium gluconate. Yields show a high capacity of the new program to collect on average MNC 17.28 +/- 10.85 x 10(9), CD 34+ 471 +/- 553.5 x 10(6) and CFU-GM 1278.7 +/- 1346.3 x 10(4) per procedure. Separator collection efficiency on average was 49.91 +/- 23.28% for MNC, 55.1 +/- 35.66% for CFU-GM, and 62.97 +/- 23.09% for CD 34+ cells. Particularly interesting are results for MNC yields and CD 34+ efficiency; these results make the new program advantageous or similar to the most progressive blood cell separators and capable to collect a sufficient number of progenitor cells for a graft with a mean of 1.80 +/- 0.98 procedures per patient.

Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells.

BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.

Evaluation of the Fresenius cell separator AS 104 for harvesting peripheral blood stem cells in pediatric patients.

In a single institution trial we carried out 35 peripheral blood stem cell harvesting procedures in 12 children with advanced malignancies to evaluate the procedure's safety and the collection efficiency of the Fresenius blood cell separator AS 104 in a pediatric population. Despite a significant mean decrease of 21% (+/-8%) in systolic blood pressure after starting the procedure, all children tolerated leukapheresis without any adverse reaction. After termination of leukapheresis there was a significant decrease of all determined hematological parameters, as compared with pre-harvest values. The mean mononuclear cell recovery was 64% (+/-26%), and in 25/35 (71%) harvesting procedures the minimum progenitor number required for safe autografting could be obtained by one single leukapheresis. We conclude that the Fresenius AS 104 blood cell separator provides a high cell yield and is a safe device for leukapheresis in pediatric patients.

Automated bone marrow concentration using the Fresenius AS 104 blood cell separator.

We concentrated freshly taken bone marrow (BM) pooled from 21 patients from an original 1.265 ml (+/- 537 ml) down to 128 ml (+/- 36 ml) within 40-70 min with a modified version of the 'grancollect protocol' on the Fresenius AS 104 blood-cell separator using the P1-Y set. An average of 47% (+/- 21%) fo the initially present mononuclear cells and 68% (+/- 47%) of the colony-forming cells could be obtained in the concentrate. The erythrocyte concentration was reduced to 7% (+/- 4%) of the original amount. The technique described is very effective and makes is possible to obtain a BM cell population that is suitable for both immunomagnetic purging and cryopreservation or transplantation, e.g. in the case of blood group incompatibility.

Automated processing of human bone marrow grafts for transplantation.

Prior to purging or cryopreservation, we concentrated 21 bone marrow (BM) harvests using a modification of the 'grancollect-protocol' of the Fresenius AS 104 cell separator with the P1-Y set. Within 40-70 min, the initial marrow volume of 1,265 ml (+/- 537 ml) was processed two to three times. A mean of 47% (+/- 21%) of the initial mononuclear cells was recovered in a mean volume of 128 ml (+36 ml). The recovery of clonogenic cells, measured by CFU-GM assays, was 68% (+/- 47%). Red blood cells in the BM concentrates were reduced to 7% (+/- 4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation and transplantation. At this time, 10 of the 21 patients whose BM was processed using this technique have been transplanted. Seven of these 10 patients have been grafted using the BM alone. Three of the 10 patients showed reduced cell viability and colony growth in the thawed BM samples, and therefore obtained BM and peripheral blood-derived stem cells. All transplanted patients showed an evaluable engraftment, achieving 1,000 granulocytes per microliter of peripheral blood in a mean of 18 days.

Evaluation of a novel automated protocol for the collection of peripheral blood stem cells mobilized with chemotherapy or chemotherapy plus G-CSF using the Fresenius AS104 cell separator.

Forty-seven peripheral blood stem cell (PBSC) collections were carried out on patients mobilized with chemotherapy and 63 on patients mobilized with chemotherapy plus G-CSF (Filgrastim), using the Fresenius AS104 cell separator and a novel automated PBSC collection protocol. As expected, cell yields were significantly higher in the series mobilized using chemotherapy plus G-CSF. The low platelet and red blood cell contamination permitted freezing of the apheresis product without further manipulation, other than plasma removal in both series. In patients mobilized with chemotherapy we obtained a MNC and a hemopoietic progenitor (CFU-GM, BFU-e, and CD34+ cells) collection efficiency comparable or superior to those reported by Bender (1992) with the Baxter CS3000 Plus after mobilization with cyclophosphamide. A significant decrease in MNC, BFU-e, and CD34+ cell collection efficiency was found in patients mobilized with chemotherapy plus G-CSF compared to those obtained in patients mobilized with chemotherapy alone. Ten patients achieved a prompt and stable engraftment after high dose chemotherapy and the infusion of cryopreserved PBSC collected using this protocol. Studies are in progress in order to improve MNC and hemopoietic progenitor collection efficiency in patients mobilized with G-CSF to obtain a graft in no more than one or two procedures.

Evaluation of a monocyte enrichment procedure using the blood cell separator Fresenius AS 104.

The small number of circulating monocytes in peripheral blood makes it difficult to collect enough cells for studies on the metabolism and analysis of receptor functions of this cell type for diagnostic aims and treatment. Therefore, a new procedure to enrich monocytes from peripheral blood using the cell separator Fresenius AS 104 was developed and evaluated. The separation is done using the tubing set 'P1Y-Gran-collect', which is a closed system with a single-stage separation chamber. The procedure is running continuously, while the cells are harvested cyclically. After processing of 4.9 l whole blood an average yield of (2.47 +/- 2.05) x 10(9) monocytes could be collected with an efficiency of 46 +/- 20%. The monocyte enrichment procedure using the Fresenius AS 104 provides a sufficient number of cells for the next steps: purification and concentration of the cells for drug monitoring, cell culturing, receptor analysis and further in vitro diagnostics.

Leukocytapheresis: evaluation of a new procedure for the blood cell separator Fresenius AS 104.

For granulocyte separation from peripheral blood with the Fresenius blood cell separator AS 104 a new procedure and a new tubing set were developed and evaluated. Separation is done in cycles of 400 ml of whole blood processed, with continuous blood flow of 50-55 ml/min, speed 750 rpm. HES-450 is added for sedimentation acceleration together with citrate, ratio 1:10. At the end of each cycle granulocytes were pumped out of the separation chamber into the collection container. After processing 13 cycles of 400 ml whole blood/HES-citrate-volume a total yield of 9.20 +/- 2.80 x 10(9) white cells were collected in volumes of 125 ml. 47 +/- 9% of the leukocytes collected were granulocytes, this corresponds to collection of 1.05 +/- 0.09 x 10(9) granulocytes per liter of processed blood. The efficacy of granulocyte collection is 22.5 +/- 5.6%, viability is more than 95%. With this procedure it is possible to collect granulocytes with the blood cell separator Fresenius AS 104 in therapeutical doses.

Bone marrow processing with the Fresenius AS 104: initial results.

Prior to purging, cryopreservation, or ABO-incompatible bone marrow (BM) transplantation, we have concentrated 23 BM harvests using a modification of the "grancollect-protocol" and the recently available bone marrow stem cell (BMSC) protocol of the Fresenius AS 104 cell separator with the P1-Y set. Within 40-70 minutes, the initial marrow volume of 922 ml (+/- 408 ml) was processed two to three times. A mean of 59% (+/- 20%) of the initial mononuclear cells was recovered in a mean volume of 119 ml (+/- 31 ml). The recovery of clonogenic cells, measured by CFU-GM assays, was 98% (+/- 80%). Red blood cells in the BM concentrates were reduced to 8% (+/- 4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation, and transplantation. All transplanted patients showed fast and sustained engraftments after autologous or allogeneic BM transplantation.

Dynamic properties of the Ca2+/calmodulin-dependent protein kinase in Drosophila: identification of a synapsin I-like protein.

Visual adaptation with blue light induces a change in a special light/dark choice behavior in Drosophila. On the molecular level adaptation induces long-term modulation of the in vitro autophosphorylation capacity of a Ca2+/calmodulin-dependent protein kinase. Here I describe a Drosophila phosphoprotein that is a substrate of this protein kinase. The molecular mass and phosphopeptide composition of this protein are similar to those of rat synapsin I. Furthermore, the Drosophila protein shows immunological cross-reactivity with monoclonal antibodies against rat synapsin I. I conclude that this 86-kDa protein in Drosophila is homologous to the vertebrate synapsin I.

Long-term modulation of Ca2+-stimulated autophosphorylation and subcellular distribution of the Ca2+/calmodulin-dependent protein kinase in the brain of Drosophila.

After prolonged visual adaptation of Drosophila, dramatic long-term changes of in vitro phosphorylation of a 50-kDa brain protein that is identical to the Ca2+/calmodulin-dependent protein kinase (EC 2.7.1.37) can be measured in isolated heads. By selective receptor cell desensitization in blue light, subcellular distribution of the 50-kDa kinase in fly brain is modified, and Ca2+-stimulated in vitro phosphorylation is increased. Concomitantly the 50-kDa kinase is translocated by in vitro phosphorylation from the membrane-cytoskeleton complex into the cytoplasm. After adaptation, association of the enzyme to the membrane shows long-term modification. In yellow light, which reverts receptor cell adaptation within seconds, the changes in kinase activity and distribution remain for about 2 hr, corresponding to the duration of behavioral modification induced by blue light. Reducing protein synthesis with cycloheximide inhibits the induction of behavioral modification as well as the prolonged modulation of the 50-kDa kinase by blue light. From our simple assay to measure biochemical changes induced in the intact organism by sensory stimulation, we propose that Ca2+/calmodulin-dependent kinase II is involved in long-term modulation of synaptic transmission.