Apheresis Protocol Research Articles

Selective red blood cell depletion by apheresis in sheep causes severe normovolemic anemia

Background: The setting of normovolemic anemia is required for a variety of research applications, such as testing of novel medication for anemia treatment. Unfortunately, large animal models using full blood draw and replenishment with balanced electrolyte solution (BES) lead to bleeding complications, as coagulation factors and platelets are also drawn. We therefore aimed to establish a model of selective red blood cell (RBC) depletion to the main endpoint of hemoglobin (Hgb) levels of 4–6 g dL−1 using apheresis in sheep. Methods: In vitro experiments were performed first to establish the apheresis protocol. In vivo, anesthetized ewes underwent a sham protocol without apheresis (n = 5) or apheresis (n = 4). Both groups were observed for the following six hours at a defined starting point (BE0) to compare Hgb, hematocrit (Hct), coagulation and clinical parameters. For statistical analysis, unpaired t-test with Welch`s correction was used. Results: Hgb levels were effectively decreased by 51 % to mean Hgb of 4.4 g dL−1 in the apheresis group compared to 9.1 g dL−1 in sham (*p < 0.0001). Hct (11.2% vs 25.1 %, *p = 0.01) and RBCs (3.7 vs 8.2 × 106/µl, *p = 0.003) also decreased. The relative number of platelets compared to baseline was different (55.6 ± 10.6% vs. 100 ± 0 %, *p = 0.004), but no hemorrhage was observed. White blood cells (WBCs), lactate, prothrombin ratio and activated partial thromboplastin time (aPTT) remained within similar ranges. Conclusions: Critical normovolemic anemia without bleeding complications was successfully reached by selective RBC depletion in sheep. Investigations of physiological adaptations to severe anemia and pharmaceutical testing can be performed in large animals with depleted RBCs.

Efficacy of plasmapheresis and semi-selective immunoadsorption for removal of anti-HLA antibodies.

In organ transplantation, apheresis is frequently used for removal of anti-HLA antibodies. However, it is unclear whether plasmapheresis (PP) or semi-selective immunoadsorption (IA) should be employed, and the optimal number of apheresis sessions required to reach post-treatment objectives is also unknown. We enrolled 43 patients from Bordeaux University Hospital who were treated with PP (n=29) or IA (n=14) for antibody-mediated rejection or pre-transplant desensitization. Using Luminex single-antigen flow beads, we assessed the initial mean fluorescence intensity (MFI) of 1416 positive beads with MFIs obtained after 7 to 8 apheresis sessions (extended protocol) and, if a serum was available, after the first four sessions (short protocol). MFI reduction after extended apheresis protocol was stronger with IA [87% (61%-100%)] than with PP [73% (22%-100%)] (P < .001). Indeed, 59% of the beads had a final MFI < 2000 with IA, whereas only 38% with PP (P < .001). The efficacy of removal depended on initial MFI but not on HLA specificity. A short protocol of apheresis showed excellent results without superiority of IA over PP for antibodies with an initial MFI < 3000. For antibodies showing MFI ≥2000 after four sessions, the residual MFI predicted the effectiveness of four additional sessions. Monitoring the MFI of anti-HLA antibodies before and during apheresis protocol can guide physicians in the selection of apheresis technique and the number of sessions to be performed.

Therapeutic plasma exchange: Experience in a third level hospital, 2013-2016, Lima (Peru).

Therapeutic plasma exchange (TPE) is an extracorporeal procedure which consists of removing the patient's plasma and replacing it with an appropriate replacement fluid. Plasma and blood cells are separated by a centrifugation process. Our department has used TPE for several years, and in 2013 we introduced an institutional apheresis protocol. The main objective of this report is to describe the TPE procedures performed between 2013 and 2016 in the Peruvian population. We analyzed the technical and clinical aspects of 864 centrifugal TPE procedures as well as the associated complications. We evaluated 230 patients treated in our institution. The therapeutic indications included 16 different diseases: 89.5% (N = 206/230) neurological, 7.3% (N = 17/230) hematological, 1.7% (N = 4/230) rheumatologic, 0.8% (N = 2/230) dermatological and 0.4% (N = 1/230) nephrological. 70.4% (N = 142/230) of patients were diagnosed with Guillain-Barré syndrome. Albumin 5% solution was the most frequent replacement solution, used in 65.8% (N = 569/864) of the procedures. The mean plasma volume (PV) replaced was 2451.73 ml, corresponding to 1.0 PV in all procedures. Complications occurred in 10.9% (N = 95/864) of the sessions. Allergic reactions were the most common events and cardiopulmonary arrests were recorded in two patients. This is the first report of TPE performed in the Peruvian population. The use of an institutional apheresis protocol was beneficial to improve registries in our service and our professional health attention. This study reports a low rate of complications, suggesting that TPE is safe. There is a need to create a multicenter Peruvian apheresis registry to assess the benefits and risks of TPE in Peru.

A safe therapeutic apheresis protocol in paediatric patients weighing 11 to 25kg.

Erythrocytapheresis and leukapheresis (LPE) of small children are logistically complex and many centres are reluctant to perform these procedures. In children, both sickle cell and leukaemic emergencies demand prompt action to prevent additional morbidity but detailed protocols for small children are lacking, and often are performed using guidelines shown to work in larger patients. We report a 3-year experience with children weighing 11-25 kg at a large academic medical centre. All patients were treated with the COBE® Spectra apheresis system; circuit was primed with blood not adjusted for haematocrit and anticoagulant citrate dextrose A was used as anticoagulation. Procedures were performed in the paediatric intensive care unit by apheresis nursing staff. Twenty-five apheresis procedures in 19 patients were performed; 17 of 19 patients presented with sickle cell-related acute complications and two (2/19) with newly diagnosed acute leukaemia and hyperleucocytosis. None of the patients required medications during the procedures. Vital signs and clinical condition remained stable and did not worsen during or postapheresis. One patient had a delayed haemolytic transfusion reaction 1 week posterythrocytapheresis as he developed alloantibodies as a result of the procedure. All sickle cell patients achieved a target haematocrit of 21-30% and Haemoglobin A of ≥68%. Both leukaemia patients who underwent LPE had no further signs of leukostasis and achieved marked reductions in leucocyte counts. Apheresis of children weighing 11-25 kg can be safely performed without increased morbidity. We outline a protocol that can be used to perform apheresis with minimal complications.

Autologous haematopoietic stem cell mobilisation in multiple myeloma and lymphoma patients: a position statement from the European Group for Blood and Marrow Transplantation

Autologous haematopoietic SCT with PBSCs is regularly used to restore BM function in patients with multiple myeloma or lymphoma after myeloablative chemotherapy. Twenty-eight experts from the European Group for Blood and Marrow Transplantation developed a position statement on the best approaches to mobilising PBSCs and on possibilities of optimising graft yields in patients who mobilise poorly. Choosing the appropriate mobilisation regimen, based on patients' disease stage and condition, and optimising the apheresis protocol can improve mobilisation outcomes. Several factors may influence mobilisation outcomes, including older age, a more advanced disease stage, the type of prior chemotherapy (e.g., fludarabine or melphalan), prior irradiationor a higher number of prior treatment lines. The most robust predictive factor for poor PBSC collection is the CD34(+) cell count in PB before apheresis. Determination of the CD34(+) cell count in PB before apheresis helps to identify patients at risk of poor PBSC collection and allows pre-emptive intervention to rescue mobilisation in these patients. Such a proactive approach might help to overcome deficiencies in stem cell mobilisation and offers a rationale for the use of novel mobilisation agents.

The relationship of platelet yield, donor’s characteristic and apheresis instruments in China

Platelet yield was associated with donor's characteristic and property of apheresis instruments. Here, we have analyzed the relationship of platelet yield, physiologic parameters of donors for different apheresis instruments in China. Data were consecutively retrieved from plateletapheresis donors during March 1, 2007 and March 1, 2012. Three different apheresis instruments MCS+, Amicus, Trima system were used for plateletapheresis and defined as group 1, 2 and 3 respectively. Totally 77,091 Plateletapheresis donations were performed in this study. 17 donations were finally aborted because of vasovagal reaction with syncope. 5861, 37,036, 34,177 donations were performed in group 1, 2 and 3 respectively. Hct and platelet values before donations were similar, but platelet yield and collection rate were showed significantly difference (p<0.05) among the three groups. The values of platelet and Hct in the males before donations were higher than those in the females, and the platelet yield and collection rate were showed significantly difference between the male group and female group (p<0.05). The overall reaction rate was 1.56%. Most donors were chosen the group 2 (51.6%) for next donation, followed by group 3 (33%) and group 1 (15.4%). We concluded that the platelet yield and collection rate in the male group were higher than those in the female group and the efficiency of plateletapheresis was associated with the kind of apheresis instruments and donor's characteristic. These data will help to work out suitable apheresis protocol based on the Chinese donor's characteristic.

Comparison of source plasma quality in three different apheresis protocols

Comparison of source plasma quality in three different apheresis protocols

Therapeutic Apheresis in Children: Special Considerations

The provision of therapeutic apheresis to children is a technically challenging procedure, requiring trained personnel and an understanding of the disease processes that leads to the need for apheresis. Most apheresis protocols are derived from studies in adult patients, even though most studies are of limited sample size. The focus of this review is to highlight the disease processes commonly treated with therapeutic apheresis in children, and to address the technical considerations pertinent to the provision of safe and effective apheresis in the pediatric setting.

Stem cell harvesting protocol research in autologous transplantation setting: Large volume vs. conventional cytapheresis

The use of peripheral blood as a source of hematopoietic stem cells (SCs) is progressively increasing and has nearly supplanted bone marrow transplantation. Interpatient variability in the degree and kinetics of SC mobilization into peripheral blood is an expected event after conventional chemotherapy-based treatment, followed by sequential administration of recombinant granulocyte-colony-stimulating factor (rHu-CSF). In this study, specific factors associated with the application of two different SC-harvesting approaches, including the use of large volume leukapheresis (LVL) vs. repetitive conventional apheresis (RCA), were analyzed. The basic goal of the study was to evaluate the influence of apheresis protocol (collection timing, processed blood volume and cell yield) upon the clinical outcome of transplantation. Results obtained by LVL (76 pts) and RCA (20 pts--control group) were compared. The SC mobilizing regimen used was cyclophosphamide (4-7 g/m2) or polychemotherapy and rHuG-CSF 10-16 microg/kg of body mess (bm) per day. Cell harvesting was performed using Caridian-BCT Spectra (Gambro, USA). The volume of processed blood in LVL setting was > or = 3.5-fold of the patient's circulating blood quantity (ranged from 12.7 to 37.8 l). All patients tolerated well the use of intensive treatment, without any side or adverse effects. Our original controlled-rate cryopreservation was carried out with 10% dimethyl sulfoxide (DMSO) using Planer R203/200R or Planer 560-16 equipments (Planer Products Ltd, UK). Total nucleated cell (NC) and mononuclear cell (MNC) counts were examined by flow cytometry (Advia-2120 Bayer, Germany; Technicon H-3 System, USA). The CD34+ cell surface antigen was investigated by the EPICS XL-MCL device (Coulter, Germany). Performing LVL-apheresis, high-level MNC and CD34+ cell yields (7.6 +/- 4.6 x 10(8)/kg bm and 11.8 +/- 6.5 x 10(6)/kg bm, respectively) were obtained. As a result, rapid hematopoietic reconstitution ("graft-healing")--on the 9.4th and 12.4th day for granulocytes and platelets, respectively was achieved. Using repetitive conventional apheresis (2-3 procedures), the total MNC count was high (8.2 +/- 7.0 x 10(8)/kg bm), but the total CD34+ yield was lower 10.8 +/- 9.9 due to inferior CD34+ vs. MNC ratio. The results obtained suggest that well-timed LVL-apheresis increased SC-yield in cell harvest, resulting in faster bone marrow repopulation and hematological reconstitution, as well as better overall clinical outcome of transplantation. These results necessitate additional examinations of CD34+ subsets ratio in cell harvest.

A Case Report of Long-term Remission of Ulcerative Colitis After Lymphocyto-plasmapheresis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of the colorectum, with mucosal infiltration by activated leukocytes, which are the result of complex interactions between lymphocytes, antigen, and dendritic cells (DCs). We carried out lymphocyto-plasmapheresis (LCPA) in a UC patient with the aim of removing lymphocytes, DCs and inflammatory cytokines (ICKs). A 42-year-old female with UC in moderate activity phase was submitted to 5 weekly LCPA treatments. Before and after LCPA we monitored: (i) the percentage of T, B, NK lymphocytes, monocytes, and peripheral blood lymphoid and myeloid DCs; (ii) the T lymphocyte subpopulations; (iii) the ICKs; and (iv) the immune complexes (IC). We achieved the interruption of all pharmacological therapies, and so far the clinical and histological remission has lasted for 24 months. The flow cytometric assessment of the leukocyte subpopulations did not show any relevant variation of their numbers after LCPA, while TNFalpha, IL-6, IL-12 and serum IgG-C1q ICs decreased. In the present case, the contemporary depletion of plasma, lymphocytes and DCs, allowed LCPA to emerge as an efficient alternative to UC pharmacological therapy without affecting the number of white blood cells, DCs and leukocyte subpopulations that were assessed. Further studies are needed both to address LCPA mechanism of action and optimal apheresis protocol, and to compare this form of therapy to a placebo control group.

Apheresis in donor and therapeutic settings: Recruitments vs. possibilities—a multicenter study

In our country, the first apheresis was performed in the late 1960s (by manual technique), and the first cell separator was used in 1979. The number of blood component collections performed from 1994 to 2004 was: 11,170 (total), i.e., 8540 (NBTI), 1180 (IT-MMA), 1050 (BTI of Novi Sad) and 400 (BTI Nis). The number of PBSC harvests during 1996–2004 was 386 for treatment of 272 patients. For treatment of myocardial infarction, “cell-therapy” by autologous stem cells was introduced in 2004 at the MMA. The results of PE treatments performed (7632 sessions) by our group for various immune-mediated and other disorders were generally beneficial, but the effect is not associated with bone marrow remission. TC procedures (total number = 1279) resulted in a significant fall in the blood cell counts and hemorheological improvement, as well as the removal and replacement of abnormal red blood cells. Greater standardization of different apheresis protocols is required.

Technical and safety aspects of blood and marrow transplantation using G-CSF mobilized family donors

An allogeneic transplantation programme using immunoselected blood progenitor and bone marrow CD34+ cells has been established. Thirteen healthy HLA-matched, MLC negative sibling donors received two doses of 5 micrograms kg-1 G-CSF (s.c. daily) for 5 days. On days 4 and 5, large-volume mononuclear cell aphereses were performed (COBE Spectra) and on day 5 one unit of autologous blood was obtained. Mononuclear cells were pooled and cryopreserved after CD34+ cell-immunoselection on day 5. Bone marrow (BM) of the same donors was procured under routine conditions 10-45 days later (median: 27 days). The final graft consisted of blood CD34+ cells with either complete BM (n = 5) or immunoselected BM CD34+ cells (n = 8). The present paper describes the progenitor cell mobilization and apheresis protocol and analyzes the cell loss by BM and peripheral blood progenitor cell (PBPC) donation. Considerably larger amounts of mononuclear cells (CD45+), T-lymphocytes (CD3+) and platelets were lost by the apheresis as compared to bone marrow without apparent immediate clinical consequences for the donors. Owing to cross-cellular contamination of the apheresis concentrate, blood platelet count (PC) significantly decreased (mean PC after the second apheresis 116 x 10 microL-1); furthermore on average 3.04 x 10(10) CD3+ cells were removed by two apheresis sessions. This loss did not lead to long-term total lymphocyte count changes (2370 microL-1 versus 1889 microL-1) as observed during the long-term follow-up of 7/13 donors (mean 290 days). Subjectively, the PBPC collections were better accepted than BM donations in all but one family donor.

Technical and safety aspects of blood and marrow transplantation using G-CSF mobilized family donors

An allogeneic transplantation programme using immunoselected blood progenitor and bone marrow CD34+ cells has been established. Thirteen healthy HLA-matched, MLC negative sibling donors received two doses of 5μg kg −1 G-CSF (s.c. daily) for 5 days. On days 4 and 5, large-volume mononuclear cell aphereses were performed (COBE Spectra®) and on day 5 one unit of autologous blood was obtained. Mononuclear cells were pooled and cryopreserved after CD34+ cell-immunoselection on day 5. Bone marrow (BM) of the same donors was procured under routine conditions 10–45 days later (median: 27 days). The final graft consisted of blood CD34+ cells with either complete BM ( n=5) or immunoselected BM CD34+ cells ( n=8). The present paper describes the progenitor cell mobilization and apheresis protocol and analyzes the cell loss by BM and peripheral blood progenitor cell (PBPC) donation. Considerably larger amounts of mononuclear cells (CD45+), T-lymphocytes (CD3+) and platelets were lost by the apheresis as compared to bone marrow without apparent immediate clinical consequences for the donors. Owing to cross-cellular contamination of the apheresis concentrate, blood platelet count (PC) significantly decreased (mean PC after the second apheresis 116×10 μL −1); furthermore on average 3.04 × 10 10 CD3+ cells were removed by two apheresis sessions. This loss did not lead to long-term total lymphocyte count changes (2370 μL −1 versus 1889 μL −1) as observed during the long-term follow-up of 7 13 donors (mean 290 days). Subjectively, the PBPC collections were better accepted than BM donations in all but one family donor.

High-dose therapy and peripheral blood progenitor cell transplantation: effects of recombinant human granulocyte-macrophage colony-stimulating factor on the autograft

Between June 1989 and June 1992, 144 patients participated in sequential clinical trials using peripheral blood progenitor cells (PBC) as their sole source of hematopoietic rescue following high-dose chemotherapy. All patients had received prior extensive combination chemotherapy and had marrow defects that precluded autologous bone marrow transplantation (ABMT). PBC were collected according to a single apheresis protocol. The initial 86 patients (group 1) had PBC collected without mobilization. Beginning in April 1991, PBC were mobilized solely with recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF). Thirty-four patients (group 2) received rHuGM-CSF at a dose of 125 micrograms/m2/d by continuous intravenous infusion, and 24 patients (group 3) received rHuGM-CSF at a dose of 250 micrograms/m2/d by continuous intravenous infusion. Patients underwent at least six aphereses and had a minimum of 6.5 x 10(8) mononuclear cells (MNC)/kg collected. Cytokines were not routinely administered immediately after transplantation. A median of nine aphereses were required to collect PBC in group 1 and seven aphereses for groups 2 and 3 (P = .03). The time required to recover 0.5 x 10(9)/L granulocytes after transplant was significantly shorter (P = .0004) for the mobilized groups; the median time to recovery was 26 days for group 1, 23 days for group 2, and 18 days for group 3. Transplantation of PBC mobilized with rHuGM-CSF resulted in a shorter time to platelet (P = .04) and red blood cell (P = .01) transfusion independence. Mobilization with rHuGM-CSF alone resulted in efficient collection of PBC, that provided rapid and sustained restoration of hematopoietic function following high-dose chemotherapy. Mobilization of PBC with rHuGM-CSF alone is an effective method for patients who have received prior chemotherapy and have bone marrow abnormalities.

High-Dose Therapy and Peripheral Blood Progenitor Cell Transplantation: Effects of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor on the Autograft

AbstractBetween June 1989 and June 1992, 144 patients participated in sequential clinical trials using peripheral blood progenitor cells (PBC) as their sole source of hematopoietic rescue following high-dose chemotherapy. All patients had received prior extensive combination chemotherapy and had marrow defects that precluded autologous bone marrow transplantation (ABMT). PBC were collected according to a single apheresis protocol. The initial 86 patients (group 1) had PBC collected without mobilization. Beginning in April 1991, PBC were mobilized solely with recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF). Thirty-four patients (group 2) received rHuGM-CSF at a dose of 125 µg/m2/d by continuous intravenous infusion, and 24 patients (group 3) received rHuGM-CSF at a dose of 250 µg/m2/d by continuous intravenous infusion. Patients underwent at least six aphereses and had a minimum of 6.5 × 108 mononuclear cells (MNC)/kg collected. Cytokines were not routinely administered immediately after transplantation. A median of nine aphereses were required to collect PBC in group 1 and seven aphereses for groups 2 and 3 (P = .03). The time required to recover 0.5 × 109/L granulocytes after transplant was significantly shorter (P = .0004) for the mobilized groups; the median time to recovery was 26 days for group 1, 23 days for group 2, and 18 days for group 3. Transplantation of PBC mobilized with rHuGM-CSF resulted in a shorter time to platelet (P = .04) and red blood cell (P = .01) transfusion independence. Mobilization with rHuGM-CSF alone resulted in efficient collection of PBC, that provided rapid and sustained restoration of hematopoietic function following high-dose chemotherapy. Mobilization of PBC with rHuGM-CSF alone is an effective method for patients who have received prior chemotherapy and have bone marrow abnormalities.

The safety and utility of leukapheresis of normal donors for obtaining products for immunologic research

In a recent report in this journal, Bach et al. (1990) described a method of preparing large numbers of human eosinophils from leukapheresis products obtained from normal donors. We have found the availability of leukapheresis products has enhanced several of the immunology research programs at our institution. Leukapheresis of normal donors for the sole purpose of laboratory research ethically should be constrained by a high degree of safety for the donor. However, the medical literature contains limited information on the safety or risks of leukapheresis of normal donors (Buchholz et al., 1975; Grindon et al., 1982). We have performed over 200 leukapheresis procedures on normal donors without the occurrence of serious adverse reactions. In the process of monitoring the safety of our protocol, we also prospectively evaluated the effects of leukapheresis on hematology, coagulation, and blood chemistry parameters for 40 procedures. We wish to report our safety experience and outline the donor protection features of our apheresis protocol for the benefit of other investigators or human investigation review boards considering the use of this procedure for obtaining leukocytes from healthy volunteers for research purposes. Our protocol is approved and monitored by the institutional review board of the hospital in which

Lymphocytopenia in donors undergoing regular platelet apheresis with cell separators

The effect of regular platelet apheresis on lymphocyte count was investigated in 142 platelet donors who donated up to 113 times on Haemonetics Model 30 and Fenwal Model CS-3000 cell separators. Lymphocytopenia (lymphocyte count less than 0.9 X 10(9)/l) was found in 21 out of 113 (18.6%) donors who donated solely on the Haemonetics Model 30 compared with 2 out of 100 (2%) whole blood donors and none of 100 plasmapheresis donors (P less than 0.002). Lymphocyte count was inversely related to the total number of platelet donations (r = 0.51, P less than 0.001) and total estimated lymphocyte loss (r = 0.49, P less than 0.001) suggesting a cumulative effect. T4 and T8 lymphocyte subsets were found to be reduced in each of 13 lymphocytopenic platelet donors tested but T4/T8 ratios were similar to those of controls (2.51 +/- 0.79 and 2.33 +/- 0.58 respectively). No adverse effects on platelet donor health were observed but it is suggested that platelet apheresis protocols should be designed to minimize lymphocyte loss.