Colony stimulating activity and continuous flow centrifugation leukapheresis induced proteinuria.

Urinary granulocyte colony stimulating activity (CSA) was studied in normal individulas donating granulocytes. Donors were given corticosteroids 2 h prior to a 4-h leukapheresis using an Aminco celltrifuge in which hydroxyethyl starch was introduced into the donor line. Urine was collected 12-24 h prior to the procedure and 12-24 h beginning at the time of administration of corticosteroids. Colony stimulating activity was measured using mouse marrow cells grown in soft agar. After leukapheresis a significant increase in protein excretion was noted (139.94 +/- 28.1 to 288.69 +/- 63.8 mg per gram of creatinine) and the bulk of the protein was albumin. CSA isolated from G-75 Sephadex columns was increased in five donors, decreased in five donors and undetectable in nine donors. No CSA inhibitors were detectable. There was a significant correlation between the quantity of protein recovered from G-75 Sephadex column and CSA.

Leukocyte donor and recipient reactions with filtration leukapheresis: their character, frequency, and management.

Neutrophil collection by filtration leukapheresis can be recommended chiefly on the basis of simplicity, low cost and efficiency. The disadvantages of the FL technique include a slightly increased incidence of adverse donor reactions, an increased risk of donor bleeding complications due to the large doses of heparin employed and a marked increase in the incidence recipient reactions, chiefly in the form of fever and chills. Because of these disadvantages, alternative methods of neutrophil collection should be considered when possible. The recently described technique of gravity leukapheresis is one such alternative which does not require investment in expensive new equipment and could be undertaken in any center familiar with plasmapheresis procedures (5).

Mobilization of peripheral blood progenitor cells for human immunodeficiency virus-infected individuals.

Gene therapy is becoming one of the most promising modalities for the treatment of acquired immunodeficiency syndrome. The purpose of this study was to investigate the mobilization and collection of peripheral blood progenitor cells from human immunodeficiency virus (HIV)-infected individuals using granulocyte colony-stimulating factor (G-CSF). A total of 10 patients (9 male, 1 female; median age 36.5 years) with varying circulating CD4+ cell counts (13.9-1467/microL) were administered 10 microg/kg G-CSF daily for 6 days. Peripheral white blood cells (WBCs), CD34+ cell counts, lymphocyte subsets, and plasma viremia were monitored before each G-CSF injection. An average sixfold increase in WBCs was observed, which stabilized on day 4 or thereafter. The level of CD34+ cells was increased by 20-fold, and did not differ between days 5 and 6. Smaller increases in CD4+, CD8+, and CD4+CD8+ cells were observed. HIV viral load, as measured by RNA copy number in plasma, was not significantly altered by G-CSF administration. The leukapheresis product (LP), collected on day 7, contained an average of 6.25+/-4.52 (mean +/- standard deviation) x 10(10) WBCs and 3.08+/-2.98 x 10(6) CD34+ cells/kg. The levels of different CD34+ cell subsets were similar to those in the LPs of G-CSF-mobilized healthy individuals from an earlier study. Primitive hematopoietic cells (CD38- and CD38-HLA-DR+ cells) were detected in LPs (1.19+/-0.46% and 0.87+/-0.23%, respectively, of CD34+ cells). All parameters (WBC counts, lymphocyte populations, CD34+ cells, and HIV-1 RNA copies) measured 3 weeks after leukapheresis returned to baseline values. The administration of G-CSF was well tolerated by the HIV patients; side effects included bone pain, headache, flulike symptoms, and fatigue. There were no correlations between baseline CD4+ cell count and the WBCs, mononuclear cells, or CD34+ cells collected in the LP. Similarly, no correlation existed between baseline CD4+ and CD34+ cells, peak CD34+ cells, or days to achieve peak CD34+ cell counts after G-CSF mobilization. Our results showed that: (1) maximal mobilization can be achieved after 4 days of G-CSF administration; (2) therapeutic quantities of hematopoietic cells can be collected and used for gene therapy; and (3) G-CSF administration is well tolerated and does not cause a clinically significant increase in viremia.

Large-scale collection of circulating haematopoietic progenitors in cancer patients treated with high-dose cyclophosphamide and recombinant human GM-CSF.

Circulating haematopoietic progenitors from 36 cancer patients were collected by continuous-flow leukapheresis during the phase of rapid haematopoietic recovery after pancytopenia induced by high-dose cyclophosphamide and then cryopreserved for autologous transplantation. 20 of the patients also received intravenous infusion of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) for 7, 10 or 14 days after cyclophosphamide. 106 leukapheresis procedures were done for 2-5 consecutive days. Leukapheresis was started significantly earlier in patients receiving rhGM-CSF. In these patients, yields of peripheral blood elements (leucocytes, mononuclear cells, haematopoietic progenitors and platelets) were significantly higher than in controls treated with cyclophosphamide only. In particular, the mean number of granulocyte-monocyte colony-forming cells was 43.88 X 10(4) vs. 6.16 X 10(4) per kg patient body weight per leukapheresis. Side-effects of leukapheresis were limited to central venous catheter occlusion and fever in 4% and 2% of all procedures, respectively.

Collection of large quantities of mononuclear cells from individual donors by continuous flow centrifugation leukapheresis.

Continuous flow centrifugation leukapheresis allows the continuous removal of leukocytes from the peripheral blood of individual donors. With the IBM 2997 cell separator, leukapheresis of granulocytes has been well established. On the other hand, no standard procedure for the collection of lymphocytes and/or monocytes has yet been described. Here, we investigated the conditions necessary for collection of large amounts of mononuclear cells with highest possible purity. The average yield from more than 100 healthy donors was 8 X 10(9) leukocytes per donor containing 80-100% mononuclear cells (MNC). Of the MNC, 10-55% were monocytes and 45-90% lymphocytes, depending on the donor. The rotor speed for maximal MNC collection was between 800 and 1000 rpm. Highest yields of monocytes were obtained at 900 rpm. No sedimenting agents were added to the blood. With more than 150 donors no rebound leukocytosis or lasting depression of blood cell counts was observed after leukapheresis. One patient with the Sézary syndrome who was subjected to leukapheresis for 26 months at 3-6 week intervals showed no pathological changes in his blood cell count to date. The disease was stabilized and no abnormalities in resistance to infection became apparent.

Collection of peripheral blood stem cells in pediatric patients: a concise review on technical aspects.

Peripheral blood stem cells (PBSC) are now routinely collected for use as hematopoietic support after high-dose chemotherapy for various malignancies. Nevertheless, few data are still available on PBSC collection in pediatric patients, owing to technical problems associated with the leukapheresis procedure in children. This paper briefly summarizes current knowledge about some technical aspects of pediatric leukapheresis for PBSC collection, according to the review of the literature and our personal experience on 60 procedures performed in 36 children affected with various malignancies. Technical issues include venous access, risk of volume shift due to exceeding extracorporeal circulation, and anticoagulation, that can induce severe side-effects. Moreover, criteria for optimizing the PBSC harvesting procedure in children, in particular the correct timing of leukapheresis, are discussed.

Comparison of anxiety, pain and discomfort in two procedures of hematopoietic stem cell collection: leukacytapheresis and bone marrow harvest.

The aim of this study was to compare anxiety, pain and discomfort of cancer patients submitted to either peripheral blood progenitor cell collection (PBPCC) or bone marrow harvest (BMH). Patients, randomized (7/1993-2/1994), in view of autograft, to receive the first procedure or the second one, completed self-administered questionnaires. Anxiety was assessed by the State Trait Anxiety Inventory and pain using visual analogical scale (VAS) and McGill Pain questionnaire. Before the procedure, BMH patients (n = 25) experienced more anxiety (P < 0.01) and more trouble or inconvenience for having to come and stay at the hospital (P < 0.0001) than PBPCC patients (n = 40). Pain due to BMH is significantly higher than pain induced by PBPCC (P < 0.001 for VAS and total McGill score). However, patients submitted to PBPCC with a femoral catheter (n = 19) had significantly higher total McGill scores and sensory sub-scores than patients without it (n = 21). At discharge from the hospital, PBPCC patients expressed more positive judgements towards the collection procedure than BMH patients. These results suggest that a better patient acceptability of high-dose chemotherapy followed by autograft may be obtained by substituting PBPCC for BMH for stem cell collection.

Effect of peripheral blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy.

The haematopoietic growth factor (HGF), granulocyte colony stimulating factor (G-CSF; filgrastim) substantially shortens the period of severe neutropenia that follows high-dose chemotherapy and autologous bone marrow infusion by stimulating granulopoiesis. Filgrastim also increases numbers of circulating progenitor cells. We have studied the ability of filgrastim to mobilise peripheral blood progenitor cells (PBPC) and assessed their efficacy when infused after chemotherapy on recovery of neutrophil and platelet counts. Seventeen patients with non-myeloid malignant disorders received filgrastim (12 micrograms/kg daily for six days) by continuous subcutaneous infusion. Numbers of granulocyte-macrophage progenitors in peripheral blood increased a median of 58-fold over pretreatment values, and numbers of erythroid progenitors increased a median of 24-fold. Three leukapheresis procedures collected a mean total of 33 (SEM 5.7) x 10(4) granulocyte-macrophage progenitors per kg body weight. After high-dose chemotherapy in 14 of the patients (busulphan and cyclophosphamide), these cells were used to augment autologous bone marrow rescue and post-transplant filgrastim treatment. Platelet recovery was significantly faster in these patients than in controls who received the same treatment apart from the infusion of peripheral blood progenitors; the platelet count reached 50 x 10(9)/L a median of 15 days after infusion of haematopoietic cells in the study patients compared with 39 days in controls (p = 0.0006). The accelerated neutrophil recovery associated with filgrastim treatment after chemotherapy was maintained. Subsequently, 10 patients received filgrastim-mobilised PBPC without marrow after high-dose chemotherapy. The rate of platelet and neutrophil recovery in these patients was at least equal to that observed in the patients receiving PBPC and bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed effects of rhG-CSF mobilization treatment and apheresis on circulating CD34+ and CD34+ Thy-1dim CD38- progenitor cells, and lymphoid subsets in normal stem cell donors for allogeneic transplantation.

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) is emerging as a new stem cell transplant modality. Rather than undergoing general anesthesia for bone marrow harvest, normal blood stem cell donors are subjected to rhG-CSF mobilization treatment followed by single or multiple apheresis. Whereas the effects of cytokine treatment and apheresis on stem cell peripheralization and collection have been described, little is known about delayed effects of rhG-CSF treatment and apheresis on a normal hematopoietic system, and there are no long-term data that address safety issues. Ten normal, patient-related donors underwent a 3 or 4 day rhG-CSF (filgrastim) treatment (12 micrograms/kg/day) followed by single or tandem apheresis. We monitored peripheral blood (PB) cellularity including CD34+ and lymphoid subsets at baseline, during cytokine treatment, prior to apheresis, and at days 2, 4, 7, 30 and 100 post-apheresis. The PB progenitor cell concentration peak prior to apheresis was followed by a nadir by day 7 and normalized by day 30, with the exception of the most primitive CD34+ Thy-1dim CD38- progenitor subset that reached a nadir by day 30. Lymphoid subsets such as CD3, 4, 8, suppressor cells (CD3+ 4- 8- TCR+ alpha beta), and B cells (CD19+) showed a similar pattern with a nadir concentration by day 7, followed, except for B cells, by a rebound by day 30 and subnormal counts at day 100. The PB concentrations of hemoglobin and platelets dropped mainly due to the apheresis procedure itself, and normalized by day 30. With cytokine treatment, the PB alkaline phosphatase and lactate dehydrogenase concentrations increased 2.2- and 2.8-fold, respectively, over baseline, and returned to normal range by day 30. Based on the preliminary nature of this study, the clinical relevance of these findings is still unclear.

Current status of leukocyte and platelet administration in cancer therapy.

Leukapheresis and plateletpheresis are rather commonly performed in order to obtain single donor concentrates of granulocytes and platelets. These procedures, although relatively safe, present occasional risks to donors and recipients. Some of the occasional adverse problems experienced by donors include citrate toxicity or acute hypocalcemia, hypotension, hypervolemia, venospasm or vein occlusion, chills, anaphylactoid reactions, hemorrhage, abdominal pain or complications related to equipment failure and related technical problems. Potential risks to donors include those related to the receiving of six percent hydroxyethyl starch (HES), dextrans, or corticosteroids, lymphocyte depletion or immunosuppression, and effects on the complement system. Prophylactic granulocyte transfusions to prevent the occurrence of infections and associated complications in neutropenic patients have not proven to be efficacious; therapeutic granulocyte transfusions appear to be more effective. Indications for therapeutic granulocyte transfusions include those patients with known infections unresponsive to appropriately aggressive antibiotic chemotherapy over a two or three day period combined with findings of a peripheral granulocyte count less than 500 mm3 and especially those with counts below 100 mm3 and/or prolonged fever greater then 38 degrees C (100.4 degrees F) for 24 to 48 hours. In addition, the patient should have a reasonable chance for bone marrow recovery. Hazards or complications associated with granulocyte transfusions include: (a) immediate transfusion reactions, (b) hypersensitivity reactions, (c) pulmonary infiltrates, (d) alloimmunization, (e) transmission of infections, and (f) the possibility of Graft vs. Host (GVH) disease. The current best use of apheresis platelets is to provide therapeutic doses of single donor matched platelets for patients refractory to pooled random donor platelets. Alloimmunization represents the major complication of therapeutic platelet transfusion and is characterized clinically by the failure to achieve expected platelet count increments after transfusion. Future developments which might greatly improve the effectiveness of therapeutic and possibly prophylactic leukapheresis and plateletpheresis include the development of effective sedimenting agents with shorter biological half-lives, more efficient and less expensive methods of procurement of granulocytes and platelets, improved methods of cryopreservation of granulocytes and platelets, better methods for detecting and quantitating antigranulocyte and/or antiplatelet antibodies, and more efficient evaluation of possible synergism of granulocyte transfusions with antibiotic therapy and residual host defense. These improvements may be of great value in the effective utilization of granulocyte and platelet products and in determining which patients are most likely to receive the maximum benefit from granulocyte and platelet support.

Guidelines for efficient peripheral blood progenitor cell collection.

In recent years peripheral blood progenitor cells (PBPC) have increasingly been used to support hematological recovery after high-dose chemotherapy treatment. PBPC are collected by leukopheresis after mobilization by chemotherapy and/or hematopoietic growth factors. Efficient mobilization and correct timing of leukopheresis is essential to minimize the number of leukophereses required for collection of sufficient PBPC for transplantation. Mobilization efficiency is influenced by various factors and recruitment of cells can be assessed by cell assays and FACS analysis. Target values of cells required for rapid hematological reconstitution after high-dose chemotherapy have been reported, but threshold values for various conditions still need to be established. CD34+ selection of the leukopheresis is of value for tumor cell purging and may be important for reduction of relapse rate of solid tumors and hematological malignancies.

Cytokine production and protein adsorption in a stainless steel filter used for leukocyte reduction.

A new metallic filter made from a stainless steel fiber has been under development. To evaluate biocompatibility of this filter, the authors compared cytokine production with that of stainless steel fibers and polyester fibers by using a mononuclear cell culture. Furthermore, adsorbed proteins on each fiber were identified by using sodium-dodecyl sulfate (SDS)-polyacrlyamide gel electrophoresis (PAGE). The levels of tumor necrosis factor (TNF)-alpha in the cultured supernatant without fibers as the control, with polyester fibers, and with stainless steel fibers were 28.1 +/- 8.1, 39.3 +/- 2.6, and 29.1 +/- 6.7 pg/ml, respectively. The levels of interleukin (IL)-1 beta were 7.6 +/- 3.2, 8.9 +/- 1.5, and 8.9 +/- 2.1 pg/ml, respectively. The IL-4 levels were less than 0.25 pg/ml, and the interferon-tau levels were less than 7.8 pg/ml in all three conditions. The amount of adsorbed proteins was 3.39 +/- 0.27 microgram/cm2 for the polyester fibers and 2.72 +/- 0.23 microgram/ cm2 for the stainless steel fibers. The protein bands adsorbed to the polyester fibers by SDS-PAGE analysis were observed at approximately 180, 120, 90, 76, 67, 59, 56, and 28 kd molecular weight. In contrast, the protein bands adsorbed to the stainless steel fibers were observed at 90, 76, 67, 62, 56, 28, and 12 kd molecular weight. Thus, the proteins adsorbed to the stainless steel fibers differed from those on the polyester fibers. By western blot analysis, the amounts of albumin, IgG tau chain, and fibronectin adsorbed on the stainless steel fibers were smaller than those on the polyester fibers. The results of this study suggest that the stainless steel fibers do not stimulate monocytes, Th1, and Th2 cells. In addition, lesser adsorption of IgG tau chain and fibronectin may indicate that the stainless steel is a superior material for anti thrombogenicity compared to polyester.

Activation of plasma components by leukocyte removal filters.

The technique of leukocyte filtration has been introduced into cardiac surgery to reduce leukocyte mediated reperfusion injury. When autologous whole blood (WB) is used as filtrate, it is more likely to be activated by the filter material than are banked red blood cell concentrates (RCC), because of its richness in plasma components. This study was designed to compare the activation of plasma components during leukocyte filtration from WB (n = 10) taken from the heart-lung machine, with RCC (n = 10) obtained from the blood bank. Leukocyte filters made from either cellulose acetate or polyester were used. Blood samples were taken simultaneously from the inlet and outlet of the filter after filtration of either 700 ml of WB or 350 ml of RCC. Results indicated that the complement cascade was activated, as reflected by the increase of C3a and C5a during filtration of WB by filters made from cellulose acetate. In contrast, there was no significant increase of C3a and C5a during filtration of RCC. The clotting system, indicated by fibrinopeptide A, and the fibrinolytic system, indicated by fibrinogen degradation products, were not activated during leukocyte filtration. These data suggest that it is the WB taken from the heart-lung machine rather than the RCC from the blood bank that is being activated during leukocyte filtration. Thus, careful selection of filter material is important for leukocyte filtration of autologous whole blood during cardiac surgery.

[Activation of bradykinin formation cascade on receiving autologous blood transfusion through a white cell-reduction filter in a patient treated with an ACE inhibitor].

We have experienced a case of anaphylactoid reaction on receiving autologous blood transfusion through a WBC filter for packed red blood cell (PRBC). The patient was a 71-year-old man with a history of hypertension treated with oral antihypertensive drug; enalapril, an angiotensin converting enzyme (ACE) inhibitor, who received anesthesia for Y-graft replacement. Autologous blood was obtained after the induction of general anesthesia in the operating room. Upon starting to return the stored blood with an unintentional use of a WBC filter, arterial blood pressure (ABP) fell within the first minute of the transfusion. We obtained three blood samples; pre-filtered blood (PRE), postfiltered blood (POST) and arterial blood (CIRC) after the event, and analyzed concentrations of bradykinin (BK), high molecular weight kininogen (HMWK) and high molecular weight kininogen-light chain (HMWK-LC). BK was higher in POST than in PRE. HMWK was lower in POST than in PRE, while HMWK-LC was higher in POST than in PRE. HMWK in CIRC was lower than in PRE, and HMWK-LC was higher in CIRC than in PRE. HMWK and HMWK-LC changes after the event suggest that BK formation cascade in the patient was activated on receiving the transfusion. ACE inhibitors were reported to augment such activation. The WBC filter has the negatively charged surface on filteration material and may activate the cascade. While WBC filters can avoid transfusion related reactions, hemodynamic responses should be watched closely in patients treated with ACE inhibitors.

[Adverse effects of hemapheresis]. / Vedlejsí úcinky hemaferéz.

The authors performed 1252 haemaphereses, incl. 689 plasmaphereses. They divide the side-reactions as follows: Technical reaction: these do not threaten the donor directly; these reactions were recorded in 4.4%. Clinical reactions--total 6.8%. a) early, b) late. The authors recorded one severe reaction during plasmapheresis, there was however, no fatality. The most frequent reactions were: general symptoms, failure to withdraw blood because of poor state of the veins and hypotension. The authors analyze the importance of the observed reactions. They used continual separators where in general the incidence of reactions is smaller. They investigated also late reactions, in particular after development of infections in donors or in the staff working with the separator. Repeated donors were subjected to detailed immunological examination, no abnormalities were, however, recorded.