Quality of red blood cells washed using a second wash sequence on an automated cell processor.

BACKGROUND: Washed red blood cells (RBCs) are indicated for immunoglobulin (Ig)A-deficient recipients when RBCs from IgA-deficient donors are not available. Canadian Blood Services recently began using the automated ACP 215 cell processor (Haemonetics Corporation) for RBC washing, and its suitability to produce IgA-deficient RBCs was investigated. STUDY DESIGN AND METHODS: RBCs produced from whole blood donations by the buffy coat (BC) and whole blood filtration (WBF) methods were washed using the ACP 215 or the COBE 2991 cell processors and IgA and total protein levels were assessed. A double-wash procedure using the ACP 215 was developed, tested, and validated by assessing hemolysis, hematocrit, recovery, and other in vitro quality variables in RBCs stored after washing, with and without irradiation. RESULTS: A single wash using the ACP 215 did not meet Canadian Standards Association recommendations for washing with more than 2 L of solution and could not consistently reduce IgA to levels suitable for IgA-deficient recipients (24/26 BC RBCs and 0/9 WBF RBCs had IgA levels < 0.05 mg/dL). Using a second wash sequence, all BC and WBF units were washed with more than 2 L and had levels of IgA of less than 0.05 mg/dL. During 7 days' postwash storage, with and without irradiation, double-washed RBCs met quality control criteria, except for the failure of one RBC unit for inadequate (69%) postwash recovery. CONCLUSION: Using the ACP 215, a double-wash procedure for the production of components for IgA-deficient recipients from either BC or WBF RBCs was developed and validated.

Plasma and cryoprecipitate manufactured from whole blood held overnight at room temperature meet quality standards.

BACKGROUND: With buffy coat (BC) processing of whole blood (WB) donations, the preparation of plasma occurs within 24 hours rather than 8 hours of collection. The effect of this change on coagulation factor function in plasma and cryoprecipitate was evaluated during the validation of this production method and with routine production. STUDY DESIGN AND METHODS: Plasma frozen after an overnight hold of WB was prepared via BC or whole blood filtration (WBF) methods and quality control (QC) variables were measured. Additionally, plasma prepared with the BC method was compared to plasma produced using the platelet-rich plasma (PRP) method with an extended plasma factor analysis. Selected plasma factor levels were also measured in both cryoprecipitate and cryosupernatant plasma prepared using the WBF method from plasma frozen on the day of collection or after an overnight hold of WB. RESULTS: When comparing BC plasma to PRP plasma, coagulation factors (F)II, VII, VIII, IX, X, and XI had somewhat lower levels, and fibrinogen and antithrombin levels were elevated. As expected the most sensitive to the prolongation of production time was FVIII with 72 and 78% of the activity of PRP plasma and cryoprecipitate, respectively. However, both still met QC standards. Similarly, products made in routine production show acceptable levels of FVIII. CONCLUSION: Plasma and cryoprecipitate products, prepared using methods in which the plasma is frozen close to 24 hours after collection, meet current quality standards. The longer WB storage time has been implemented into general use in Canada.

[Indications of urgent plasma exchange and cytapheresis therapies--a review based on literature data and personal experience]. / A sürgosségi plazmacsere és cytapheresis kezelések indikációja--irodalmi és tapasztalati összefoglaló.

Emergency plasma exchange therapy is life saving in many cases. Therefore, clinicians must be aware of the indications at which any delay in initiating therapy may prove to be fatal. Different hematological (Moschkowitz-, hyperviscosity- and catastrophic antiphospholipid syndrome; massive haemolysis [e.g Wilson's disease]), neurological (myasthenic), endocrine (thyrotoxicosis) and nephrological (rapidly progressive glomerulonephritis) crisis situations and for prevention of them; certain poisonings, fulminant liver failure, severe pancreatitis due to chylomicronaemia, meningococcus sepsis and iatrogenic or suicidal drug-overdose. In this latter, it is of fundamental importance that the protein binding of the drug should be high (>80%), whereas the volume of its distribution should be relatively low (<0,2 l/kg body weight) and the endogenous clearance of it should be less, than 500 ml/min. Urgent leukocytapheresis should be performed above 50.000 blasts/microl, in acute or chronic myeloid leukemia if symptoms of leukostasis are present (if blasts are above 100.000/microl, cytoreduction is mandatory even without symptoms). Similarly, urgent thrombocytapheresis should be administered above platelet numbers 1000 G/l, when there is concomitant thrombophilia or clinical symptoms of thrombostasis are present.

Automated red cell collection--quality and value.

Automated red cell collection was initially used largely for therapeutic purposes. New technology has rendered the procedure safer for donors and easier for machine operators. Optimal additive solution can be automatically added and the red cells filtered to provide a leucodepleted product. Two units of red cells may be collected during a single procedure from individuals who have a high enough red cell mass, whilst a single unit of red cells plus platelets or plasma can be collected from smaller donors. In vitro studies suggested that red cells collected by automated methods would be of better quality than those collected by gravity. This was not confirmed in vivo, but red cells collected by automated methods have the major advantage of consistency in terms of haemoglobin content, volume and haematocrit, compared with red cells collected by gravity. This standardised product is of particular value for transfusion dependent patients as the patient's haemoglobin can be maintained within narrow limits. The use of a double dose red cell product for transfusion to a single patient also confers benefit in terms of reduction in donor exposure.

Quality of packed red blood cells and platelet concentrates collected by multicomponent collection using the MCS plus device.

The demand for blood components is constantly increasing, while the exclusion criteria for donors are strengthened in order to reach maximal safety for donors and patients. To counterbalance reduced availability of volunteers, multicomponent collections (MCC) is an attractive approach to produce more than one component during a single apheresis procedure from one donor, such as packed red blood cells (PRBCs) and platelet concentrates (PCs). Further, the exposures of patients to a limited number of donors reduces the possibility of alloimmunization and transfusion-related diseases. We measured the quality of PRBCs and PCs obtained by MCC, using the MCS+ device with the LDPRBC program, Revision B, and compared them with the quality of manually collected PRBCs and PCs collected with the Revision C2 of the MCS+. We found higher pH levels and lower hemolysis assessed by means of fHb and K+ in the supernatant of PRBCs over the whole storage period of 42 days in MCC-derived PRBCs. The functional metabolism assessed by intracellular ATP was higher in PRBCs collected by MCC than in manually collected units. Furthermore, PCs obtained during MCC showed an increase in p-selectin expression on day 5 of storage compared to PCs collected with the Revision C2 of the MCS+. The p-selectin expression on MCC platelets was within the range of p-selectin expression found in PCs obtained by other apheresis devices. These results indicate less storage lesion in MCC-derived PRBCs compared to manually collected units and no compromise in the quality of MCC PCs obtained in the same apheresis procedure.

A phase II trial evaluating the safety and effectiveness of the AastromReplicell system for augmentation of low-dose blood stem cell transplantation.

To reduce the number of apheresis procedures and maintain the usual rate of hematopoietic recovery in patients treated with high-dose chemotherapy, we studied the effect of adding a small volume of ex vivo expanded bone marrow to low doses of CD34(+) blood stem cells. Thirty-four patients with breast cancer received G-CSF (10 microg/kg/day) priming followed by a limited volume (50-100 ml) bone marrow aspiration and standard 10-liter aphereses. Marrow was expanded ex vivo using the AastromReplicell system and infused along with low doses of blood-derived CD34(+) cells, collected in one apheresis. Thirty-one evaluable patients received a median CD34(+) blood stem cell dose of 0.7 x 10(6)/kg (range, 0.2-2.5) and 4.7 x 10(7) nucleated cells/kg (range, 1.98-8.7) of ex vivo expanded marrow. All patients recovered with normal blood counts and engrafted 500 neutrophils/microl and 20 000 platelets/microl in a median of 10 and 13 days, respectively. Multivariate analysis revealed that, in addition to CD34(+) lineage negative cell quantity, the quantity of stromal progenitors contained in the ex vivo expanded product correlated with engraftment outcome (r = 0.551, P = 0.004). Our results indicate that ex vivo expanded bone marrow is capable of facilitating engraftment when combined with low doses of mobilized blood derived CD34(+) cells.

Studies on the improvement of leucodepletion performance of the Haemonetics MCS+ for production of leucodepleted platelet concentrate.

With the implementation of universal leucodepletion, an in-line, negatively charged LRF6H leucodepleting filter became an essential part of the Haemonetics MCS+ plateletpheresis system. A larger-scale (968) study using the standard protocol revealed a 2.79% leucodepletion failure rate (standard < 5 x 10(6) leucocytes per adult therapeutic dose). Factors influencing the efficacy of the filter were investigated. The pH of the filtrate was 7.0, the temperature 28 degrees C and filtration rate 80 ml/min. Reduction of the filtration rate to 30 ml/min (784 doses) reduced leucodepletion failure to 0.38%. Measurement of the leucocyte count, pre- and post-filtration of the platelet products, revealed that donations from 1% of donors contained substantially larger numbers of leucocytes in pre-filter samples (300-1500/microl) than in control samples (35-70/microl). This number tends to increase progressively with subsequent donations in these individuals, leading to leucodepletion failure, whilst peripheral leucocyte counts remain normal. The new continuous filtration protocol (version C) using a less impact filter LRF-XL and a lower (7 ml/min) head pressure was also effective but failure still occurred twice on one of the donors who persistently showed high pre-filter count. We conclude that leucodepletion failures in the Haemonetics system are related to both donor leucocyte (i.e., being light and non-adherent) and operational/filter performance.

Cell salvage and leucodepletion.

Cell salvage has been used as a method of blood conservation for more than three decades. Although the principles and development of the Latham bowl had occurred in the 1960s, it was not until the early 1970s that washing of the concentrated red cells was introduced and a product that was universally acceptable was obtained. The last 25 years have seen little in the way of development of cell salvage, although significant refinement has taken place. Although the simple picture of cell salvage involves removal of the buffy coat, including platelets and leucocytes, in practice there are reports of great variation in the removal of these cells. Most recent studies suggest that there is very little removal of leucocytes by cell salvage. The leucocytes that remain in the red cell suspension following cell salvage have undergone significant morphological changes and the surface expression of leucocyte adhesion receptors increases dramatically during the process. There is little evidence that removal of these activated leucocytes has any significant clinical benefit. Although leucofiltration of blood before storage has been shown to be an extremely safe process, 'bedside leucofiltration', including leucofiltration of cell salvage blood, may not be without problems. Reports of hypotensive events while receiving blood products through a bedside leucocyte reduction filter have emerged during the last few years. This may be due to bradykinin production following platelet exposure to negatively charged leucocyte filters.

Quality of processed blood for autotransfusion.

Centrifugal red blood cell washers for intraoperative autotransfusion process shed blood during surgery. In this study, the quality of processed fresh, human bank blood was assessed in a standardized laboratory setting during standard, medium, and high flow processing. Red cell recovery rates and plasma washout efficiencies were compared using three different devices. The accurate parameters measuring effectiveness and product quality were red cell mass (RCM) flow rate and the plasma washout efficiency. Cobe BRAT 2, a system with discontinuous flow (DF) and a cylindrical centrifuge bowl, permitted processing in standard and medium flow of 26 and 35 mL RCM/min, respectively, with washout of residual plasma albumin of 93.2 and 91.2%. The Medtronic Sequestra 1000, a DF system with a conical centrifuge bowl processed blood at 15 and 23 mL RCM/min and eliminated plasma albumin with 98.4 and 96.8% washout during standard and medium flow, respectively, with significant red cell loss occurring during medium flow. The respective speeds of high-flow programs with BRAT 2 and Sequestra 1000 were 15 and 22 mL RCM/min, related to a hematocrit in the holding bag, less than that of the incoming blood from the reservoir. Washout was 58.2 and 58.3%, respectively. Fresenius CATS, a continuous flow (CF) device, produced flow rates of 19, 24, and 43 mL RCM/min and plasma albumin elimination of 97.8, 94.4, and 93.3% in standard, medium, and high-flow, respectively. Holding bag hematocrits with CF exceeded that of DF. Standard, medium, and high-flow programs of CATS may be used without restriction.

Granulocyte colony-stimulating factor versus granulocyte-macrophage colony-stimulating factor for collection of peripheral blood progenitor cells from healthy donors.

The harvesting of peripheral blood progenitor cells (PBPCs) after granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor stimulation instead of bone marrow in healthy donors has become increasingly popular. Donors, given the choice between bone marrow and PBPC donation, often prefer cytapheresis because of the easier access, no necessity for general anesthesia, and no multiple bone marrow punctures. In addition, accelerated engraftment and immunomodulation by granulocyte colony-stimulating factor-mobilized PBPCs are advantageous for the recipient. However, because of donor inconvenience and poor mobilization, there is a need to develop improved procedures. Aspects such as durability of hematopoietic engraftment, characterization of the earliest stem cell, and composition of PBPCs are not yet well defined, and international donor registration and follow-up must be considered when evaluating long-term safety profiles in healthy donors. This review concentrates on the most significant developments on mobilization of PBPCs published during the past year.

Selection and use of chemotherapy with hematopoietic growth factors for mobilization of peripheral blood progenitor cells.

Peripheral blood progenitor cells (PBPCs) have become the preferred means of stem cell support for high-dose chemotherapy in recent years. The biology of PBPC mobilization is complex and may be influenced by several variables. Signals from both stromal and hemopoietic cells may induce downregulation of adhesion molecules and upregulate the expression of metalloproteinases. Cytokines alone can mobilize PBPCs but a synergistic effect has been shown when they are used in conjunction with chemotherapy. Disease-specific mobilization strategies appear to have the advantage of less toxicity, greater stem cell yield, and enhanced antitumor activity. Studies have demonstrated that the number of peripheral blood CD34+ cells can be used as a predictor for the timing of apheresis and for estimating PBPC yield. Similarly the CD34+ cell dose is the strongest predictor of hematologic recovery after PBPC transplant. Age, prior radiotherapy, marrow involvement, and prior chemotherapy (especially with alkylating agents) are important factors influencing the yield of stem cells.

[Leukodepletion of labile blood products]. / Déleucocytation des produits sanguins labiles.

On the first of April 1998, French authorities decided on the systematic leukodepletion of two blood products: Red blood cell concentrates and platelet concentrates coming from homologous donors. This measure is based on the following principle arguments: Purification of the therapeutic product, as white cells are just passenger cells. White cells interfere with the red cells and the platelets during storage, deteriorating the overall quality of the blood product. There are multiple effects on the recipient of allogeneic leucocytes: CMH II structures stimulation of the host immune system; graft versus host reaction; cytokines secretion and mediators release; class I anti-HLA allo-immunisation; in general, immunomodulation (immunosuppression); intraleukocyte infectious agent transmission. Several techniques make leukodepletion in blood products possible: 1. For the red cells, just one technique is basically used today: filtration either when the red cell concentrate is being prepared (filtering the whole blood), or after having obtained the concentrate, filtering that. The filters used today are from a third generation and their efficiency is above 99.9%. The final product contains thus less than 1.10(6) per unit, whereas the initial product contained 1.10(9) per cent. 2. For the platelets, apheresis technology makes it possible to obtain platelet concentrates directly leucoreduced (1.10(5)). However, there are still two questions: is this process also useful for plasma? and what about for autologous blood products?

Treatment of graft-versus-host disease by extracorporeal photochemotherapy: a pilot study.

BACKGROUND: Graft-versus-host disease (GVHD) is a major complication after bone marrow transplantation, which may be refractory to immunosuppressive drugs. As preliminary case reports suggested that extracorporeal photochemotherapy (ECP) using a Therakos device might be beneficial, we conducted a pilot study to assess the efficacy and safety of a new ECP method that does not require administration of 8-methoxypsoralen (8-MOP) to the patient. METHODS: ECP was performed three times a week for 3 weeks and then tapered according to the patient's course. Soluble 8-MOP was added ex vivo to an enriched mononuclear cell suspension obtained by a cell separator. This cellular suspension was then ultraviolet A irradiated and reinfused into the patient. Evaluation was performed using specific objective tests depending on clinical conditions. RESULTS: The two patients in the study with acute GVHD and severe liver dysfunction resistant to steroid pulse showed no improvement with ECP treatment. The five patients with chronic GVHD (c-GVHD) had the following clinical features: three patients had myositis and two patients had severe cutaneous c-GVHD, including one patient with sclerodermoid lesions, one with bronchiolitis obliterans, one with bronchitis, and one with liver involvement. Immunosuppressive drugs were either prohibited or ineffective. The number of procedures for each patient ranged from 13 to 30. Cytapheresis required the use of a double-lumen catheter (4/5) or an arteriovenous fistula (1/5). No side effects were related to 8-MOP or ultraviolet A irradiation. Four of five patients improved after ECP; one patient with bronchiolitis obliterans, a fibrotic condition, remained stable. CONCLUSIONS: ECP treatment may be helpful for the treatment of severe c-GVHD and the avoidance of increased immunosuppression.

[Flow cytometry quality control in stem cell separation]. / Durchflusszytometrische Qualitätskontrolle bei der Stammzellseparation.

Peripheral blood-derived haematopoietic stem cells (PBSC) are used as an alternative to bone marrow stem cells for autologous transplantation. One of the most important prerequisites for successful PBSC separation is the precise determination of the optimal separation days. As we previously demonstrated that neither PLT counts nor WBC counts in the peripheral blood (PB) are of predictive value for the amount of colony-forming cells (CFC) in the patients' PB, we started a daily monitoring of CD-34-positive cells to determine the beginning of the separation series. In addition to routine cell counts and CFU testing we assessed the number of CD 34+ in the PBSC concentrates to ascertain the number of separations needed for each patient. Due to the strong correlation of CD 34+ cells to CFC it is possible to predict the number of CFC collected within 2 h after finishing the PBSC separation and to calculate the efficiency of the separation for quality control.