Continuous Flow Blood Cell Separator Research Articles

Cohn de Laval Award Lectureship: The Science Behind the Success Development of a Continuous Flow Blood Cell Separator

On behalf of Dr. Jeane Hester, I would like to thank everyone in the American Apheresis Society and World Apheresis Society involved with the nomination and presentation of this award. She is truly honored by the recognition of her peers and wishes that she could be here to thank each and every one in person. The reason I am standing here goes back 43 years to July of 1971 when a new ‘crop’ of oncology fellows came to MD Anderson Cancer Center and the Department of Developmental Therapeutics. The chairmen of the department were Emil Frei and Emil J Frereich, with Ken McCredie as the head of the then fledgling apheresis center and I was the tech working in Ken’s research lab. Back then it was somewhat rare to have a female physician in the mix, so word traveled quickly about the tall attractive ‘fellow’ who was winning everyone over with her personality, commitment to her profession and, above all, dedication to her patients. As a fellow, part of the responsibility was to choose a research project and work in the lab in their free time. Several months later this here-to-for phantom Dr. Hester appeared in my lab to discuss where she could work on her project regarding Leukoagglutins in Transfusion. So we began sharing the lab, I used it during the day and she used it at night and occasionally we shared! To paraphrase the last line from Casablanca as Bogart and Claude Rains walk into the night: “This was the beginning of a beautiful friendship”. From that time forward, we worked together on many projects until her retirement from MD Anderson in 1995. The most notable achievement was the development of the first blood cell separators to use disposable channels and all the associated outcomes – the calcium citrate binding incorporated into the software, calcium replacement therapy used during collection, bag parameters for the storage and cryopreservation of cells and optimization of the software for the various procedures. The initial development was accomplished through collaboration with the Biomedical Division of IBM and moving forward she collaborated with GAMBRO COBE and until her 2nd retirement this year with TerumoBCT. The very first 2997 manufactured was called the ‘Genie’ and was donated to Dr. Hester’s Apheresis Unit. It was marketed as the IBM 2997, followed by the next generation – the COBE Spectra. Fast forward 30 years and today, this same blood cell separator is the work horse of apheresis, performing stem cell collections, plasma depletions, plateletpheresis, WBC reductions, and RBC depletions. Like all of us, the Spectra will be retired to make room for a newer model, but the scientific principals established during the development of the continuous flow separators (2990 – 2997 – Spectra) serve as the foundation for the future in apheresis. On a personal note, this award has been a time-traveling experience for Jeane and me. We are back 30+ years where she is drafting the speech and I am preparing the slides of our data. Only back then, it was truly hand-produced 35 mm slides and at least now I have the modern technology of PowerPoint. However, several of the slides and photos presented are digitized from the original 35 mm slide. The speech I will be giving has been written by Dr. Hester as if she were here presenting it herself. Many of you who know her will be able to pick up on several of the ‘mantras’ that she often presented and challenged at meetings, such as. . .‘Showme the data’. She is fiercely protective and loving, steadfast when right and can be stubbornly tenacious and I am proud to call her mentor and friend. – April G. Durett, MSc, Manager, Flow Cytometry Facility, Center for Cell & Gene Therapy I will now channel Dr. Hester in Houston, TX, and present her talk. International Forum: from the WAA Congress in San Francisco, 2014. * Corresponding author. Canadian Apheresis Group. E-mail address: cag@cagcanada.ca (G. Rock).

Leukocyte Transfusion and the Development of the Continuous-Flow Blood Cell Separator

The treatment of anemia and thrombocytopenia with allogeneic cell transfusions is an effective and well-developed technology. However, leukocyte replacement transfusion has been frustrated by the physiology of the leukocytes. To achieve effective leukocyte replacement, the continuous-flow centrifugal blood cell separator was developed, and it soon proved to be an important instrument for separation, collection, and transfusion of all the components of the blood. Thus, the continuous-flow centrifugal blood cell separator has become an important instrument in the science of blood collection and transfusion.

L’érythraphérèse thérapeutique : technique et applications cliniques

Purpose Therapeutic erythrocytapheresis is a selective red cell-depletion aphaeresis technique. Current knowledge and key point Using a discontinuous or continuous flow blood cell separator, the technique safely allows quickly and automatically collecting a large volume of red cells. The main obstacle of implementation is a poor peripheral venous access. The procedure is well-tolerated. Polycythemia vera, hereditary hemochromatosis, complicated sickle-cell-disease and retinal venous occlusion are the main clinical applications. Prospect and project Therapeutic erythrocytapheresis is a more modern and more effective method than the classic patient-bleeding. It has to be preferred in the early treatment of patients with polycythemia vera and hereditary hemochromatosis.

CD4+CD28+ T Lymphoctye Subsets Decreased in Healthy Donors after G-CSF Mobilization.

Objective Allogeneic transplantation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) -mobilized peripheral blood stem cells (PBSCs) is now being increasingly performed, but safety considerations for hematologically normal PBSC donors have not been fully addressed. The effection of G-CSF on donors' lymphocytes have not been defined clearly. Our study was to detect and analyze the phenotypical and functional properties of lymphocytes from allo-PBSC donors treated with recombinant human G-CSF by flow cytometry. Methods Thirty-four HLA-identical sibling donors (13male, 21female; median age, 35 years) were treated by subcutaneous injection with rhG-CSF at a dose of 5 μg/kg twice daily for 4–6 consecutive days to mobilize HSCs to the peripheral blood. Leukapheresis was performed using a continuous flow blood cell separator (COBE Spectra, Lakewood, CO) on 1 to 2 consecutive days beginning on day 4 of rhG-CSF administration. Donor blood samples, which were obtained before the first administration of G-CSF (pre-G), on day 4 of G-CSF administration (post-G), and one week after the last rhG-CSF were analyzed by 3-color flow cytometry. Monoclonal antibodies included: CD3, CD4, CD8, CD20, CD16, CD56, CD25, CD69, CD45RA, CD45RO, CD28, CD95. Results Absolute counts of lymphocytes, B cells, T cells, CD4+ and CD8+cells post-G were significantly elevated two more times than pre-G (P<0.05), but these changes recovered when the last G-CSF administrated one week later. The percentage of CD3+, CD4+, CD8+ and the ratio of CD4 and CD8 T cells had no significantly difference between pre-G and post-G. The percentage of CD4+CD25+, CD4+CD45RO+ and CD4+CD28+ T cells were significantly decreased post-G (45.26±9.68% to 37.34±11.12%; 65.53±13.74% to 52.32±13.47%; 94.75±4.02% to 91.74±8.72%, P=0.00329, 0.0003 and 0.0947, respectively). We found that CD4+CD28+ T cells(91.77 ± 6.15, P=0.00218) were still lower than pre-G when stopped administrate G-CSF one week later, while others changes have recovered to the Pre-G level. Conclusion: The study shows that CD4+CD28+ T lymphoctye subsets were still lower than pre-G, although most of other change recover one week later after mobilization

No Significant Benefit of Marrow and Blood HLA-Haploidentical Stem Cell Transplantations Given by Mothers with Long-Term Fetomaternal Microchimerism.

Non-T-cell-depleted maternal HLA-haploidentical hematopoietic stem cell transplantations(SCT) have been reported feasible based on the hypothesis that long-term fetomaternal microchimerism(FMC) is linked with hyporesponsiveness to inherited paternal antigens(IPAS),but the hypothesis is still in debate due to lack of direct evidence. To determine whether the existence of such microchimerism affect the outcome of maternal HLA-haploidentical SCT, we investigated the outcomes of 18 patients (median age 19) with hematologic malignancies (ALL 8, AML 6, CML 3, NHL 1) who underwent haploidentical SCT from mothers with the same regime from July 2003 to January 2005. Donor was treated with granulocyte colony stimulating factor subcutaneously at a dose of 300μg per day for 5 days before bone marrow harvesting. Leukaphereses were performed using a continuous-flow blood cell separator if CD34+ cells in the bone marrow harvest were less than 4.0×106 cells/kg. Patients with CML or AML in CR received 7.5Gy TBI based standard-intensity regimen combined with cyclophosphamide plus cytarabine. The remaining received non-TBI standard-intensity regimen consisting of cytarabine, busulfan and cyclophosphamide. Antithymocyte globulin(ATG) at a dosage of 2.5mg/kg/day on days −5 to −3 was given as an additional immunosuppressive measure in all patients. To prevent GVHD, patients also received cyclosporin at a dose of 3mg/kg/day as a continuous infusion from day −10, 30mg/kg/day mycophenolate mofetil starting on day −10, and short-term methotrexate administered on days 1, 3, 6, 11 at doses of 15, 10, 10, and 10mg/m2. Among all these patients, microchimerism were confirmed with nested polymerase chain reaction using sequence-specific primers(PCR-SSP) typing for HLA in 10 donor-recipient pairs. In the 2 categories as to whether the microchimeric status of the donor is positive or negative, they had similar characteristics in the background factors such as recipient sex, type of conditioning regime, stem cell source, and disparity of HLA except recipients of positive group had more CML. Engraftment was obtained in all patients. As of august 1, 2005, 5 patients died (1 of relapse, 4 of complication), other 13 patients were alive and free of disease. The 2-year overall survival for the whole cohort was 58%. Although there was a survival advantage for pairs with microchimerism over pairs without it, the influence on outcome was not statistically significant (P=0.9, log-rank test). The cumulative incidences of grade Ò/Ô acute graft-versus-host disease (GVHD) were 39% (95%CI, 17%–64%). Also no significant difference was observed between the two groups. Extensive chronic GVHD developed in 5 of 13 patients who could be evaluated. These results indicate maternal HLA-haploidentical hematopoietic SCT is a acceptable option for patients who lack immediate access to a conventional stem cell source, but the presence of fetomaternal microchimerism may not correlate to the existence of immunological tolerance between mother and offspring.

Early total white blood cell recovery is a predictor of low number of apheresis and good CD34 + cell yield

Objective. We analysed peripheral blood progenitor cell (PBPC) mobilisation and collection in order to assess the main factors related to CD34 + cell yields in patients affected by haematological malignancies. Patients and Methods. The features of CD34 + cell mobilisation of patients with haematological malignancies that underwent autologous bone marrow transplantation were examined. Mobilisation chemotherapy consisted mainly of cyclophosphamide (CY) 4 or 7 g/m 2 followed by growth factors. Leukapheresis was started when the WBC counts reached 1.0 × 10 9/l with the aim to collect at least 5 × 10 6 CD34 + cells/kg body weight. The aphereses were performed on continuous-flow blood cell separators. The analysed variables were: age, diagnosis, CT mobilisation regimen, type of growth factor, number of previous CT lines, prior radiotherapy, days for WBC recovery and number of aphereses procedures to achieve the target of CD34 + cells. Results. There were 41 consecutive patients (26 M/15 F): 21 non-Hodgkin’s lymphoma (NHL), 15 Hodgkin’s disease (HD), two chronic myeloid leukaemia (CML) and three multiple myeloma (MM). Eleven patients could not collect the proposed threshold of CD34 + cells. CY 4 mobilised patients recovered WBC counts in less days ( P=0.03). By ANOVA, the days to WBC recovery had a linear function of the predictors “number of aphereses” and “type of mobilisation CT” (coefficients: 0.86 and 0.95, respectively). For the number of aphereses and WBC recovery after CT mobilisation, we obtained a correlation coefficient of 0.36 ( P=0.02). Conclusion. This study shows that it is feasible to mobilise and collect PBPC in patients previously treated with CT with or without RT. There was a linear correlation between the days for WBC recovery and the number of aphereses needed to collect the target number of CD34 + cells. The study suggests that early WBC recovery, using mainly CY 4 mobilisation chemotherapy, is an important predictor of a low number of aphereses to achieve a good CD34 + yield.

Lymphocyte Subset Proportions in Guillain-Barré Syndrome Patients Treated with Plasmapheresis

We examined the changes in the proportions of certain T cell subsets in 18 Guillain-Barré syndrome patients treated with plasmapheresis (PP). PP was performed within 8 days after the onset using a continuous-flow blood cell separator or immunoadsorption. The proportions of CD3+, CD4+ and CD4+CD45RA+ cells at the onset were decreased and that of CD19+ was increased compared with those of 25 normal controls. However, the proportions of CD4+CD45R–, CD4+HLADR+, CD4+CD25+ cells and the CD8+ subpopulations were similar in patients and controls. After PP, the altered subsets tended to normalize, particularly in the group that improved after PP.

Peripheral Blood Progenitor Cell Collection by Large-Volume Leukapheresis in Low-Weight Children

Large-volume leukapheresis (LVL), defined as the processing of at least three blood volumes in a single session for peripheral blood progenitor cell (PBPC) collection, was performed in 32 small children weighing < or = 25 kg, aged 10 months to 8 years, with a variety of malignancies. Harvesting of PBPC was started after 4 days of cytokine (G-CSF, 12 micrograms/kg s.c.) alone. Procedures were performed using a continuous flow blood cell separator (COBE Spectra). The automated program of lymphocytapheresis was modified to achieve a collection rate of 0.9 ml/min. The extracorporeal line was primed with a unit of a packed red blood cells before the procedure. Acid citrate dextrose (ACD) was used as anticoagulant with an ACD inlet ratio of 1:14 and an ACD infusion rate of 1.1 ml/min/L of total blood volume. The inlet flow ranged between 6 and 35 ml/min (median 20 ml/min). A total of 37 apheresis procedures were performed (median 1, range 1-3). In 84% of patients, a single apheresis yields the minimum number of PBPC cells required for transplantation. No consistent side effects were observed, and LVL was well tolerated by children. A median of 7.7 x 10(8) kg MNC, 5.4 x 10(6)/kg CD34+, and 6.2 x 10(4)/kg CFU-GM per apheresis were harvested. Patients with neuroblastoma had a significantly lower yield than other patients. To date, 27 patients have been transplanted after myeloablative treatment, and rapid and sustained engraftment was achieved in all cases. The number of CD34+ cells infused was highly correlated with engraftment kinetics. LVL can be safely and easily performed in small children, allowing adequate PBPC collection for transplantation with rapid hematologic recovery.

Hydroxyethylstarch sedimentation by gravity ex vivo for red cell reduction of granulocyte apheresis components

BACKGROUND When selecting only leukocyte compatible donors, the requirement of ABO compatibility limits the investigation and application of granulocyte transfusion therapy by reducing the pool of potential donors. Ex vivo hetastarch (HES) sedimentation was evaluated as a method of red blood cell (RBC) reduction of granulocyte components. The objective was to determine if this procedure consistently resulted in reduction of component packed RBC (PRBC) volume to < 5 ml, the range acceptable for infusion of ABO incompatible blood components based on guidelines set forth by the American Association of Blood Banks (AABB). STUDY DESIGN AND METHODS: HLA-matched, ABO-compatible sibling marrow donors were selected to donate granulocyte components, which were transfused into the allogeneic bone marrow transplant (BMT) recipient as prophylaxis against infection. Three granulocyte components were collected from each of 5 donors receiving G-CSF (daily × 5). Leukapheresis (LA) began 1 day after the first G-CSF dose (Day 1), and was repeated on Days 3 and 5. LA were performed using a continuous-flow blood cell separator, with 7L blood processed during each procedure. RBC sedimentation was facilitated by administration of a 6% HES solution to the donor line. The 5 granulocyte components collected on Day 1 were not manipulated after collection. The 10 components collected on Days 3 and 5 were manipulated by ex vivo gravity sedimentation for 60 minutes followed by transfer of the buffy coat (red cell poor [RCP] fraction) to a transfer bag with residual RBCs retained in the collection bag (red cell rich [RCR] fraction). The PRBC volume and cellular composition of the components and fractions were determined. RESULTS: When data for the 10 manipulated components were combined, the fraction of the components with < 5 ml PRBC was 0.4 in the RCP and 0.1 in the RCR fractions. All unmanipulated components contained > 5 ml PRBC. The mean PRBC volume (ml) of the RCP and RCR fractions were 6.3 and 16.4, respectively (P = .06). The mean number of RBC (×1011) in the RCP and the RCR fractions were .41 and 1.73, respectively (P = .03). The average proportion of cells in the manipulated components lost to the RCR fraction was 19.2% of granulocytes and 18.6% of platelets. CONCLUSION: Ex vivo HES sedimentation, as performed, significantly reduced the number of RBCs from granulocyte components, but did not consistently result in PRBC volumes in the RCP fraction within the range acceptable for infusion of ABO incompatible blood components based on the AABB guidelines. Moreover, significant numbers of granulocytes were lost to the RCR fraction. J. Clin. Apheresis 13:56–61, 1998. © 1998 Wiley-Liss, Inc.

Hydroxyethylstarch sedimentation by gravity ex vivo for red cell reduction of granulocyte apheresis components.

When selecting only leukocyte compatible donors, the requirement of ABO compatibility limits the investigation and application of granulocyte transfusion therapy by reducing the pool of potential donors. Ex vivo hetastarch (HES) sedimentation was evaluated as a method of red blood cell (RBC) reduction of granulocyte components. The objective was to determine if this procedure consistently resulted in reduction of component packed RBC (PRBC) volume to < 5 ml, the range acceptable for infusion of ABO incompatible blood components based on guidelines set forth by the American Association of Blood Banks (AABB). HLA-matched, ABO-compatible sibling marrow donors were selected to donate granulocyte components, which were transfused into the allogeneic bone marrow transplant (BMT) recipient as prophylaxis against infection. Three granulocyte components were collected from each of 5 donors receiving G-CSF (daily x 5). Leukapheresis (LA) began 1 day after the first G-CSF dose (Day 1), and was repeated on Days 3 and 5. LA were performed using a continuous-flow blood cell separator, with 7L blood processed during each procedure. RBC sedimentation was facilitated by administration of a 6% HES solution to the donor line. The 5 granulocyte components collected on Day 1 were not manipulated after collection. The 10 components collected on Days 3 and 5 were manipulated by ex vivo gravity sedimentation for 60 minutes followed by transfer of the buffy coat (red cell poor [RCP] fraction) to a transfer bag with residual RBCs retained in the collection bag (red cell rich [RCR] fraction). The PRBC volume and cellular composition of the components and fractions were determined. When data for the 10 manipulated components were combined, the fraction of the components with < 5 ml PRBC was 0.4 in the RCP and 0.1 in the RCR fractions. All unmanipulated components contained > 5 ml PRBC. The mean PRBC volume (ml) of the RCP and RCR fractions were 6.3 and 16.4, respectively (P = .06). The mean number of RBC (x10(11)) in the RCP and the RCR fractions were .41 and 1.73, respectively (P = .03). The average proportion of cells in the manipulated components lost to the RCR fraction was 19.2% of granulocytes and 18.6% of platelets. Ex vivo HES sedimentation, as performed, significantly reduced the number of RBCs from granulocyte components, but did not consistently result in PRBC volumes in the RCP fraction within the range acceptable for infusion of ABO incompatible blood components based on the AABB guidelines. Moreover, significant numbers of granulocytes were lost to the RCR fraction.

Non-tunneled catheters for the collection and transplantation of peripheral blood stem cells in children.

We analyzed the use of non-tunneled (polyurethane, double lumen) central venous catheters (CVCs) for the collection, conditioning, transplantation and immediate post-transplantation periods in 56 children with various malignant diseases. A total of 71 leukaphereses were performed, with a mean of 1.2 apheresis per patient, following administration of granulocyte colony-stimulating factor (G-CSF) using a continuous flow blood cell separator (Cobe Spectra). The mean TBV (total blood volume) processed was 4.5 +/- 1.2 s.d. (range 2.4-7). The mean flow rate was 30.6 ml/min and the duration of a single apheresis was 327 +/- 84 s.d. (range 175-511 min). The mean purities and efficacies of collections were 77.38 +/- s.d. (range 42-100) and 42.78 +/- s.d. (range 24-80), respectively. The mean numbers of mononuclear cells (MNC) and CD34+ cells collected were 9.3 +/- 6.9 s.d. x 10(8)/kg (range 2-49) and 6.2 +/- 7.2 s.d. x 10(6)/kg (range 1-42), respectively. We observed the following complications during catheter insertion for collection: pneumothorax (1.7%), mechanical dysfunction (3.5%) that resolved with thrombolytic therapy. Complications during conditioning, transplantation and immediate post-transplantation periods were entry site infection in five patients (8.92%), catheter-related infection in two (3.57%) and catheter-related sepsis in three (5.35%). Our results indicate that the collection of PBSC with non-tunneled catheters is safe, effective and dis associated with a low incidence of complications.

Stem cell mobilization in normal donors.

We studied peripheral blood and apheresis samples from 39 consecutive normal donors who were parents or siblings of patients who received matched or mismatched bone marrow transplants using a combination of rhG-CSF-mobilized peripheral blood stem cells (PBSCs) and bone marrow (BM). BM was harvested from donors 1-7 days before starting rhG-CSF treatment: 12 micrograms/kg/day rhG-CSF was administered by continuous s.c. infusion for 4-7 days. Peripheral blood progenitor cells were harvested by leukapheresis using an automated continuous-flow blood cell separator, beginning on day 4 of rhG/CSF, for 1-4 consecutive days. Peak peripheral blood CD34+ cell and CFU-GM levels were reached simultaneously on day 5 or 6 of rhG-CSF administration. Median peak levels were 1.65% for CD34+ cells (range 0.34%-4.7%) and 142 CFU-GM/10(5) plated cells (range 16-700). The greatest numbers of CD34+ cells and CFU-GM, expressed per liter of blood volume processed, were harvested during the second and third leukapheresis: CD34+ cells 37.77 +/- 25.48 x 10(6) and CFU-GM 3.32 +/- 2.51 x 10(6) during the second leukapheresis, and CD34+ cells 37.01 +/- 16.33 x 10(6) and CFU-GM 3.82 +/- 4.36 x 10(6) during the third. The number of CD34+ cells and CFU-GM did not correlate with the sex, age, or body weight of the donors. This study indicates that this protocol for administration of rhG-CSF mobilizes large numbers of hematopoietic progenitor cells into the peripheral blood and that bone marrow harvesting before G-CSF administration does not impair stem cell mobilization.

Collection and transplantation of peripheral blood progenitor cells mobilized by G-CSF alone in children with malignancies.

Results of collection and transplantation of peripheral blood progenitor cells (PBPC) mobilized by G-CSF in 31 children with different malignancies were analysed. A total of 43 aphereses were performed, following administration of granulocyte colony-stimulating factor (G-CSF), using a continuous flow blood cell separator (Cobe Spectra) through a central venous catheter. For patients weighing <or= 25 kg the extracorporeal line was primed with red blood cells. The mean blood flow rate was 33.2 ml/min (range 12-56 ml/min). The mean number of mononuclear cells (MNC), granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells collected were 7.22 x 10(8)/kg body weight (b.w.), 15.9 x 10(4)/kg and 5.44 x 10(6)/kg respectively. The morbidity related to PBPC collection was low and the mean apheresis time was 302.7 min (range 115-492). The volume of processed blood for apheresis (per kilogram body weight) ranged from 117 to 539.6 (mean 309.13 ml/kg). 20 patients required only one apheresis to collect the minimum requirement of 5 - 7 x 10(8)/kg of MNC. All patients subsequently underwent autografting with PBPC after myeloablative therapy. Days to achieve an absolute count of neutrophils (ANC) > 0.5 x 10(9)/l and a platelet count of 20 x 10(9)/l without platelet support were 9.5 and 18, respectively. The number of CD34+ cells infused correlated highly with engraftment kinetics. The extra-medullary toxicity was low and manageable.

Principles of bone marrow processing and progenitor cell/mononuclear cell concentrate collection in a continuous flow blood cell separation system.

The application of continuous flow apheresis technology to processing bone marrow for collection of the mononuclear progenitor cell population appears to follow the same principles as collection of mononuclear cells from peripheral blood. Unlike peripheral blood, however, where mobilization of cells from extravascular sites during the procedures contributes significantly to the final cell yield, the entire quantity of progenitor cells available for recovery from marrow is present in the original marrow when it is pooled. The process then becomes one of attempting optimal recovery of the cells of interest while excluding contaminating erythrocytes and cells of the myeloid series. This study reports the development of a protocol for recovery of MNC, CD33+, CD34+, and CD34+/DR- cells from harvested marrow for autologous and allogeneic transplants using a continuous flow blood cell separator, the variables influencing the recovery of the cells of interest and the clinical response to infusion of the processed cells.

Comparison of different protocols in plateletpheresis with the haemonetics MCS 3p blood cell separator with respect to parameters of product quality.

In thrombocytapheresis, intermittent flow cell separators produce platelet concentrates (PC) with a higher leucocyte contamination than continuous flow blood cell separators. The new discontinuous flow cell separator Haemonetics MCS 3p offers a low-leucocyte solution for PC. The quality of PC obtained by the MCS 3p was investigated in this study. Prospective study. Haemapheresis Unit of a University Clinic. Healthy blood donors from the haemapheresis unit. Platelet (PLT) yield, separation efficiency and white blood cell (WBC) contamination were studied in three different protocols. Two protocols used a haemocalculator, which calculated the target volume based on the donor's physical characteristics and the desired PLT yield for the procedure. Protocol I used 3,000 ml as target process volume and protocol II 3.3 x 10(11) as desired PLT yield. Protocol III was used without haemocalculator. Glucose, lactate, lactate dehydrogenase (LDH), morphology score and pH value were analysed to investigate the quality of the PC. Platelet yield and separation efficiency were not statistically different in the three protocols. Leucocyte contamination was lowest in the protocol without haemocalculator (median: 3.15 x 10(6), range 0.4-20.8 x 10(6)). Glucose, lactate, LDH and pH were not statistically different in the three protocols. Morphology score was best in protocol III. PLT collection with the MCS 3p blood cell separator results in sufficient thrombocyte yields. Using the haemocalculator we were not able to achieve the desired platelet yield. For this reason, and because of the higher WBC contamination in protocol II we prefer PLT collection without the haemocalculator. The quality of the platelet concentrates was good with respect to the parameters glucose, lactate, LDH, morphology score and pH.

Peripheral blood stem cells (PBSCs) collected after recombinant granulocyte colony stimulating factor (rhG-CSF): an analysis of factors correlating with the tempo of engraftment after transplantation.

Factors affecting mobilization and engraftment were analysed in 54 patients undergoing transplant using autologous PBSCs mobilized with high-dose recombinant granulocyte stimulating factor (rhG-CSF). Patients received 5-7 d of rhG-CSF, 16 micrograms/kg/d, administered subcutaneously. PBSCs were harvested by leukapheresis using automated continuous-flow blood cell separators beginning on day 4 of rhG-CSF, processing 10 litres of whole blood, for 2-6 consecutive days. Transplants were performed for the following diseases: breast cancer (n = 22), non-Hodgkin's lymphoma (n = 18), multiple myeloma (n = 7) and other (n = 7). Engraftment was rapid with patients reaching a neutrophil count of 1 x 10(9)/l a median of 12 d (range 9-22) after transplant. Platelets > 20 x 10(9)/l independent of transfusion support were achieved a median of day 10 (range 7-60) after infusion. Multiple factors potentially influencing engraftment were examined using a Cox regression model. The number of CD34+ cells per kg was highly correlated with the time to achievement of granulocyte and platelet recovery (P < 0.012, 0.0001). The use of a post-infusion growth factor and a radiation preparative regimen was important for neutrophil recovery, and a diagnosis of breast cancer was important for platelet recovery. In an analysis by linear regression of the logarithm of CD34+ cells collected, lower age, marrow without disease, no prior radiation, and lower number of prior chemotherapy regimens, were important factors influencing larger numbers of CD34+ cells in collections.

Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor

Peripheral blood mononuclear cells (PBMC) were collected after the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and used as the sole source of hematopoietic stem cells after myeloablative therapy with busulfan (Bu) and cyclophosphamide (Cy). These studies were performed in 12 patients with malignancies (4 non-Hodgkin's lymphoma, 5 breast cancer, 1 testicular carcinoma, 1 Wilm's tumor, and 1 undifferentiated carcinoma) who had bone or bone marrow disease or had low marrow cellularity. rhG-CSF (16 micrograms/kg/d) was administered for 5 to 7 days by subcutaneous injection and PBMC were collected for 2 to 5 days beginning on day 4 after initiation of rhG-CSF, using continuous-flow blood-cell separators that processed 10 to 12 L of whole blood. From a median of three collections, a mean of 24.0 x 10(8) (+/- 10.5 SD) total nucleated cells/kg containing 12.6 x 10(8) (+/- 4.5 SD) mononuclear cells/kg, 7.3 x 10(6) (+/- 4.3 SD) CD34+ cells/kg and 20.5 x 10(4) (+/- 28.1 SD) granulocyte-macrophage colony-forming units (CFU-GM)/kg were harvested and cryopreserved. After the administration of Bu 14 to 17 mg/kg and Cy 120 to 150 mg/kg, PBMC were thawed and infused. One patient received rhG-CSF after the infusion of PBMC and the remaining 11 patients did not receive postinfusion growth factors. Mean days to recovery of neutrophil levels of 0.1, 0.5, and 1.0 x 10(9)/L were 11.4 (range, 9 to 13), 12.7 (range, 10 to 15), and 13.6 (range, 11 to 16) and the mean day to platelet transfusion independence was 13.3 (range, 7 to 49). Time to recovery of neutrophils to 0.5 and 1.0 x 10(9)/L and platelets to 20 x 10(9)/L was more rapid than in historical patients treated with Bu and Cy who received marrow alone or marrow followed by the posttransplant administration of rh-G or GM-CSF. No graft failures have been observed with a follow-up of 4 to 12 months. These results indicate that PBMC collected after rhG-CSF lead to rapid hematopoietic recovery after myeloablative chemotherapy.

Autologous Transplantation With Peripheral Blood Mononuclear Cells Collected After Administration of Recombinant Granulocyte Stimulating Factor

Abstract Peripheral blood mononuclear cells (PBMC) were collected after the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and used as the sole source of hematopoietic stem cells after myeloablative therapy with busulfan (Bu) and cyclophosphamide (Cy). These studies were performed in 12 patients with malignancies (4 non-Hodgkin's lymphoma, 5 breast cancer, 1 testicular carcinoma, 1 Wilm's tumor, and 1 undifferentiated carcinoma) who had bone or bone marrow disease or had low marrow cellularity. rhG-CSF (16 micrograms/kg/d) was administered for 5 to 7 days by subcutaneous injection and PBMC were collected for 2 to 5 days beginning on day 4 after initiation of rhG-CSF, using continuous-flow blood-cell separators that processed 10 to 12 L of whole blood. From a median of three collections, a mean of 24.0 x 10(8) (+/- 10.5 SD) total nucleated cells/kg containing 12.6 x 10(8) (+/- 4.5 SD) mononuclear cells/kg, 7.3 x 10(6) (+/- 4.3 SD) CD34+ cells/kg and 20.5 x 10(4) (+/- 28.1 SD) granulocyte-macrophage colony-forming units (CFU-GM)/kg were harvested and cryopreserved. After the administration of Bu 14 to 17 mg/kg and Cy 120 to 150 mg/kg, PBMC were thawed and infused. One patient received rhG-CSF after the infusion of PBMC and the remaining 11 patients did not receive postinfusion growth factors. Mean days to recovery of neutrophil levels of 0.1, 0.5, and 1.0 x 10(9)/L were 11.4 (range, 9 to 13), 12.7 (range, 10 to 15), and 13.6 (range, 11 to 16) and the mean day to platelet transfusion independence was 13.3 (range, 7 to 49). Time to recovery of neutrophils to 0.5 and 1.0 x 10(9)/L and platelets to 20 x 10(9)/L was more rapid than in historical patients treated with Bu and Cy who received marrow alone or marrow followed by the posttransplant administration of rh-G or GM-CSF. No graft failures have been observed with a follow-up of 4 to 12 months. These results indicate that PBMC collected after rhG-CSF lead to rapid hematopoietic recovery after myeloablative chemotherapy.

Application of a new LDL apheresis system using two dextran sulfate cellulose columns in combination with an automatic column‐regenerating unit and a blood cell separator

Extracorporeal procedures for selective removal of low-density lipoproteins have become a promising new approach for treatment of severe familial hypercholesterolemia. We tested efficacy and safety of a new LDL apheresis system by using two dextran sulfate cellulose adsorbents (Liposorber LA 15TM from Kanegafuchi) under the control of an automatic column-regenerating unit for continuous alternate adsorption and desorption. Plasma was taken from a continuous-flow blood cell separator (model IBM/Cobe 2997) allowing an extracorporeal circuit from one cubital vein to another. A 57-year-old male with drug-resistant heterozygous familial hypercholesterolemia accompanied by moderate hypertriglyceridemia and severe coronary artery disease has been treated every 2 weeks for 3 months so far. Treatment of 4-5 liters of plasma resulted in a mean decrease of total cholesterol from 355 to 111 mg/dl (9.20 to 2.88 mmol/l), of LDL cholesterol from 272 to 49 mg/dl (7.05 to 1.53 mmol/l), and of apolipoprotein B from 175 to 44 mg/dl. HDL cholesterol, apolipoprotein A-I, and other plasma proteins did not substantially change apart from hemodilution. No side effects were seen. This new technique of LDL apheresis represents a very effective and safe method for treatment of drug-resistant familial hypercholesterolemia without or with concomitant hypertriglyceridemia.

Collection and cryopreservation of human stem and progenitor cells for bone marrow transplantation

Bone marrow collection was undertaken from human organ donors (Group 1: n = 7) to develop a closed-system single-step technique for stem and progenitor cell enrichment, using the Cobe 2997 continuous-flow blood-cell separator. The effects of programmed freezing, storage in liquid nitrogen, and thawing were then defined using these grafts. Once standardised, this method was extended to autografting following cryopreservation of a comparable fraction (Group 2: n = 8) and then to allogeneic transplantation after ex vivo exposure to the lytic monoclonal antibody. Campath-1 IgM and human complement, but without cryopreservation (Group 3; n = 9). The median number of mononuclear cells harvested was 5.0 x 10(6)/mL (n = 24), and this was not significantly different in the three groups. The ex vivo graft, composing marrow rich anticoagulated whole blood, was recirculated in the separator at a flow rate of 60 mL/minute, with a centrifuge speed of 1,100 r.p.m., and the mononuclear cell fractions were collected at the rate of 1.5 mL/minute. The average procedure time from formation of the interface in the single disposable channel to achievement of the final volume was 90 minutes. The mean recovery of the mononuclear cells was 101.4% (SD 38.0) and the GM:CFUc was 91% (SD 43.86). These figures were not significantly influenced by subsequent cryopreservation (Group 1; n = 7 and Group 2; n = 8) or following exposure to the monoclonal antibody. Campath-1 IgM (Group 3; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)