Leukocyte reduction filters as an alternative source of peripheral blood leukocytes for research

ABSTRACT Introduction: Peripheral blood leukocytes are a suitable cell model for science research. However, blood samples from healthy volunteers are limited in volume and difficult to obtain due to the complexity of volunteer recruitment. Objective: Therefore, it is urgent to find an alternative source of peripheral blood leukocytes. Method: One of the possibilities is the use of leukocyte reduction filters (LRFs) in blood banks that is used for preparation of leukoreduced blood products. More than 90% of the leukocytes are trapped in the leukofilters allowing the desired blood product to pass through. Results: It has been reported that the biological function of leukocytes collected from the filters are no different from those isolated from buffy coats, leukapheresis products and whole blood (WB) cells. Moreover, LRFs are waste products that are discarded after leukoreduction. Conclusion: Thus, leukofilters represent an economic source of human cell populations that can be used for a variety of investigative purposes, with no cost. In the present study, we reviewed the different usage of LRFs in the research, clinical and commercial applications.

Effects of irradiation and leukoreduction on down-regulation of CXCL-8 and storage lesion in stored canine whole blood

White blood cells (WBCs) and storage period are the main factors of transfusion reactions. In the present study, cytokine/chemokine concentrations after leukoreduction (LR) and irradiation (IR) in stored canine whole blood were measured. Red blood cell storage lesion caused by IR and LR were also compared. Blood samples from 10 healthy Beagles were divided into four groups (no treatment, LR-, IR-, and LR + IR-treated). Leukocytes were removed by filtration in the LR group and gamma radiation (25 Gy) was applied in the IR group. Immunologic factors (WBCs, interleukin-6 [IL-6], C-X-C motif chemokine ligand 8 [CXCL-8], and tumor necrosis factor-alpha) and storage lesion factors (blood pH, potassium, and hemolysis) were evaluated on storage days 0, 7, 14, 21, and 28. Compared to the treated groups, IL-6 and CXCL-8 concentrations during storage were significantly higher in the control (no treatment) group. LR did not show changes in cytokine/chemokine concentrations, and storage lesion presence was relatively mild. IR significantly increased CXCL-8 after 14 days of storage, but IR of leukoreduced blood did not increase CXCL-8 during 28 days of storage. Storage lesions such as hemolysis, increased potassium, and low pH were observed 7 days after IR and storage of blood, regardless of LR. IR of leukoreduced blood is beneficial to avoid immune reactions; however, storage lesions should be considered upon storage.

Blood transfusion-related immunomodulation

The phenomenon of transfusion-related immunomodulation (TRIM) has been studied since the observation of a higher kidney allograft survival in patients who had received a higher number of transfusions. Conversely, it has been suggested as one of the possible causes related to the development of infections in patients with multiple blood transfusions and/or after a major surgery, and has been also associated with a decreased function of natural killer cells (NK) and antigen-presenting cells (APCs), reduced cell-mediated immunity, and increased regulatory T cells (Tregs). This review aimed to conceptualize TRIM and discuss some aspects related to its mechanisms and the prevention of immunomodulatory events.

[Comparison of the side effects of blood transfusion between pre-storage filtration and post-storage filtration methods in thalassemia patients]

Application of WBC-reduced blood products can decrease side effects related to white blood cells transfer, such as febrile reactions, CMV infection and .. Advances in biotechnology have led to production and development of filters that are capable of reducing WBC; hence, diminishing these side effects. The aim of this study was to determine and compare side effects of blood transfusion between pre-storage filtration and post-storage filtration methods in thalassemia patients. This study was a randomized clinical trial with crossover design and included adult thalassemia patients referring to hospital from 1388 to 1389 and receiving alternately red blood cell concentrates by using pre-storage and post-storage filtration methods. Side effects of blood transfusion including hypotension, fever, hemoglobin drop and blood transfusion rate were recorded and compared between the two methods. Data were introduced in to SPSS software and analyzed by paired t-test, X[2], t-test and P>0.05 was considered significant. Comparison of the incidences of hypotension, FNHTR, allergic reactions and hemoglobin drop between pre-storage and post-storage filtration methods showed no significant differences. A significant difference was observed in the transfusion rates between the two methods which indicated a lower transfusion rate in the pre-storage method [p<0.001]. Pre-storage filtration method is not superior to post-storage filtration method for blood transfusion in thalassemia patients.

Evaluation and comparison of washed RBCs by closed and open systems

Leukocyte filters are effective for WBC reduction but they cannot inhibit passing plasma proteins and as a result repeated protein entry may produce allergic transfusion reactions. To deal with this problem, washed RBC method is used. The traditional wash method is an open system through which waste products are carried away in sewers with the risk of environmental pollution. Newly introduced approach for washed RBCs consists of a closed system whereby waste products enter into a bag. In this study, the two methods were compared. The two open and closed wash methods were compared in terms of health system, leukoreduction, risk of transmission of infection and quality control. In each method, 100 bags were washed, coded and then transmitted to different units of blood culture, flowcytometry as well as quality control. The data were collected and analyzed by SPSS 14. 200 bags [100 for each method] were studied. Microbiologically, there were no positive results for any of the methods. In quality control also there was not any significant difference in the two methods. In flowcytometry, we didn't observe any significant correlation in leukocyte count in the two methods before washing [p=0.072], however. The correlation between them after washing [p<0.0001], demonstrating that the new method was better for leukoreduction. The new washing system method was a superior way because it involves a closed system where waste products are discharged into a side bag and disposed as hospital waste. Meanwhile, this approach is more convenient for leukoreduction. In our country, since we still need a washing system for some transfusions, this method is deemed to be a decent and practical one because it impedes environmental pollution

Protein Profile Changes in Platelet Concentrates According to Storage and Leukoreduction- Analysis Using Proteomics Technology / 대한진단검사의학회지

BACKGROUND: Knowing how the protein profile of platelet products changes with storage or leukoreduction may give us greater insight into cell physiology and the cause of transfusion reactions other than cytokines and chemokines. METHODS: We filtered four packs of platelet concentrates (PC) within 24 hr of blood collection and after 120 hrs of storage. Four aliquots of each supernatant in PC were obtained: pre-storage+prefiltration, pre-storage+post-filtration, post-storage+pre-filtration and post-storage+post-filtration. Routine chemistry tests and a two-dimensional electrophoresis (2-DE) were performed. The stained images were analyzed and the significant spots were identified using a peptide mass finger printing (PMF) with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis after trypsin digestion. RESULTS: The protein spots increased with storage and decreased after filtration (P<0.05, prestorage+post-filtration). The spot density of various proteins, including macrophage inflammatory protein-2 alpha, megakaryocyte colony stimulating factor and interleukin-22 changed with storage and leukoreduction. CONCLUSIONS: The database of identified protein spots and their changes produced in this study is a useful basic tool for future studies on the mechanism of transfusion reactions. Further studies should validate the significance of each protein spot.

Efecto de los leucocitos sobre la lesión por almacenamiento en plaquetas de aferesis / Effect of leukocytes on the storage lesion in apheresis platelet

En el Banco de Sangre , las plaquetas sufren una serie de cambios físicos, metabólicos y fisiológicos que se denominan "lesión por almacenamiento" (LA), que depende de varios factores: métodos de preparación del concentrado, tipo de bolsa utilizada, concentración de plaquetas, número de leucocitos presentes en la unidad y acumulación de citoquinas. Todos ellos podrían producir activación plaquetaria y así afectar la calidad del producto, lo cual se reflejaría en una menor sobrevida de las plaquetas transfundidas. Basándose en lo anterior, se plantea que la remoción precoz de leucocitos aminoraría la LA en los concentrados plaquetarios (CPs) obtenidos por aféresis. Se estudiaron veinte CPs obtenidos mediante dos métodos de aféresis que difieren en el número de leucocitos residuales que permanecen en el producto final; un CP leucoreducido (Cobe Spectra) y otro estándar (Baxter CS 3000 plus). Las determinaciones se realizaron el día cero (prealmacenamiento) y al quinto día de almacenamiento. La evaluación de la LA incluyó marcadores de menbrana plaquetaria: p-selectina (CD62-P), glicoproteína Ib (CD42b), fosfatidilserina (Ax V.), factor tisular (CD142), formación de micropartículas (MPs), los cuales se analizaron por citometría de flujo, y citoquinas liberadas por los leucocitos y/o plaquetas activadas (IL-1. IL-6,FNT y RANTES), las cuales se analizaron por ELISA. El principal marcador de activación de las plaquetas (p-selectina) se encontró significativamente aumentado en los CP leucorreducidos (P: 0.001) y en los obtenidos en forma estándar (p: 0.02). La expresión de GPIb disminuyó significativamente solo en las plaquetas no leucorreducidas (p: 0.01). En relación a la actividad procoagulante de las plaquetas, se observó un aumento significativo en la expresión de fosfatidilserina sobre la cara externa de la membrana (p: 0.019) y de MPs plasmáticas (p: 0.025) solo en las plaquetas leucorreducidas y un muy leve aumento de la expresión de factor tisular...

Under Blood Bank storage conditions, platelet undergo a series of physical, metabolic and physiological changes that are denominated "platelet storage lesion" (PSL). This condition depends on several factors: the platelets number and the methodology used for the preparations of platelet concentrates (PC), type of storage bag, the number of leukocytes present in the cell unit, cytokines release, among others. All these factors may produce platelet activation and thus affect the quality of the product, which would be reflected in a shorter survival of the transfused platelets. Based on the previous knowledge, we hypothesized that early removal of leukocytes from the apheresis concentrate will diminish platelets "activation/lesion" during storage. We studied twenty PC obtained by two methods of apheresis that differed in the number of residual leukoreduced PC (Cobe Spectra) and a standard PC (3000 Baxter CS extra). The determinations were made at day zero (pre-storage) and at the fifth day of storage. The evaluation included markers present in platelets membrane, such as, p-selectin (CD62-P), glycoprotein Ib (CD42b), phosphatydilserine expression (PS). Tissue Factor (CD142) and microparticles (MPs) generation, that were analyzed by flow cytometry. Cytokines released by leucocytes or activated platelet (IL-1). IL-6, TNF and RANTES), were analysed by the ELISA technique. The most important marker of platelets activation, CD62-P, was significantly more increased in leukoreduced CP (P: 0.001) than in the standard method (p: 0.02). The expression of GPIb diminished significantly only in non-leukoreduced platelets (p: 0.01). With regard to the procoagulant activity of platelets, a significant increase in the PS expression was observed on the external face of the platelet membranes (p: 0.019) and on MPs (p: 0.025) only in leukoreduced preparations, changes that were accompanied by a very slight increase of tissue factor expression (p: 0.055). The determinations...

The effect of two leukocyte depletion in-line filters on the efficiency of whole blood filtration / 生物医学工程学杂志

The aim of this study was to observe the difference in respect to the leukocyte reduction efficiency and quality of fresh-frozen plasma (FFP) from filtered whole blood between two types of in-line filters wherein only filter materials were surface modified by the two methods respectively. Whole blood was kept in refrigerator and filtered within 6 h of collection at ambient temperature. Samples were taken pre- and post filtration for analysis of WBC numbers, coagulation factors and complement activation (n = 8 for each type of filter). All filtered units contained < 2. 5 x 10(6) residual leucocytes. RBCs recovery was over 93%. No significant difference between group A and B was seen. But group B appeared to take longer time for filtration than did group A (9'29" vs. 8'01"). Neither group A nor group B showed statistically significant losses of total protein, album, IgG, IgM, fibrin, factors VIII, IX, vWF and C3 (P > 0.05). Factor V, XI and AT-III decreased significantly in two group filters. Group B showed more significant losses of IgA content and factor V activity than did group A, which appeared to be related to the difference in surface character between group A and group B filters. These two types of filters could remove leukocytes effectively, and no significant changes were observed in the quality of FFP from the filtered whole blood. It is presumed that the filter material with better bio-compatibility will give a high recovery of plasma protein and coagulation factors after filtration.

Calidad de hemocomponentes leucorreducidos: la validación del procedimiento de leucorreducción / Quality of WBC-reduced blood components: validation of leukoreduction process

Las Guías Internacionales y Nacionales regulan la práctica de leucorreducción para garantizar la calidad de los hemocomponentes leucorreducidos. La normativa local establece que el procedimiento deberá estar validado y que el nivel máximo de leucocitos residuales en productos leucorreducidos es 5 x 10 elevado a la 6. Para alcanzar dicho objetivo se analizan los factores críticos que influyen sobre el proceso de leucorreducción y se presentan métodos de recuento de leucocitos residuales, planes de muestreo y análisis estadístico.

Cytokine generation in stored platelet concentrate: comparison of two methods of preparation.

BACKGROUND & OBJECTIVES: Contaminating white blood cells (WBCs) in stored platelet concentrates (PC) are the main source of pro-inflammatory cytokines including interleukin-6 (IL-6), interleukin- 8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) that are implicated in transfusion reactions. We compared the levels of these cytokines in stored platelet preparations prepared by two methods. Effect of pre-storage leucofiltration on these cytokine levels was also studied. METHODS: Twelve units of pooled PCs were prepared by platelet rich plasma (PRP) method and buffy-coat (BC) method each and stored for 5 days. IL-6, IL-8 and TNF-alpha levels were measured in platelet supernatants on day 0, 1, 3 and 5 of the storage using commercially available immunoassays. Pre-storage leucofiltration was done in 4-pooled units of PRP-PC and cytokine levels compared with unfiltered PCs. RESULTS: Median IL-6 levels increased from day 0 to day 5 in both PRP-PC and BC-PC. In PRP-PC, IL-8 increased from <3 pg/ml on day 0 to 817 pg/ml on day 5, while in BC-PC the corresponding levels were 10 and 346.5 pg/ml, respectively. No significant increase in levels of TNF-alpha was observed in BC-PC during storage period, while levels increased significantly in PRP-PC on day 1 only. There was no significant change in the levels of all three cytokines in leucofiltered PCs over 5 days of storage. INTERPRETATION & CONCLUSION: Findings of our study showed that method of preparation and WBC content are the critical factors in determining the cytokine levels in stored PCs.

Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis.

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.

Evaluation of two methods for counting residual white blood cells in thrombocytaphoresis concentrates / 中国实验血液学杂志

To evaluate flow-cytometry and Nageotte method for counting residual WBC in thrombocytaphoresis concentrates, their accuracies were determined by dilution studies separately; the repeatability was determined by measuring the interassay coefficient of variation for 14 replicates of a sample with known leukocyte concentration. 102 samples of leukocyte-depleted thrombocytaphoresis concentrates were detected by the above mentioned two methods, and the results were compared each other. The results showed that no difference was observed between two methods over a range of leukocyte concentrations from 0.8 to 10 WBC/microl (P > 0.05). In conclusion, flow-cytometry and Nageotte methods can be used for quality control of WBC-reduced blood components.

Effects of leukocyte depletion filter on canine kidney during cardiopulmonary bypass / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of a reforming leukocyte depletion filter (LDF-1) on the functional and pathologic changes of canine kidney during cardiopulmonary bypass (CPB).@*METHODS@#Twelve Mongolian dogs were randomly allocated into a control group (no LDF-1, n = 6) or a leukocyte-depleted filter group (LDF-1 placed in venous line, n = 6). CPB of the dogs anestheitized with sodium pentobarbitone at 25 mg/kg was set up. After 10 min of CPB, aorta was clamped and St. Thomas cardioplegic solution at 20 mg/kg was immediately injected into the root of aorta. The aortic cross-clamp was released and CPB was closed at 70 min. Dogs were observed for 2 h after weaning from CPB. LDF-1 was opened at 2 min and stoped at 7 min during initially running CPB in the LDF-1 group. Circulating leukocytes, plasma L-selectin, and plasma IL-8 were respectively counted before CPB, at 10 minutes, 40 min, and 75 min during CPB, the end of CPB, and 2 h after CPB. The urine analysis and renal pathology, which were obtained before CPB and 2 h after CPB, were observed.@*RESULTS@#The number of leukocytes significantly decreased by 55% - 68% in the LDF-1 group compared with the baseline during CPB. The value at 10 min of CPB in the LDF-1 group was lower than that in the control group (P < 0.05). Plasma levels of L-selectin and IL-8 obviously increased in the 2 groups compared with the baseline during CPB, but both levels at 2 h after CPB in the LDF-1 group were lower than those in the control group (P <0. 05). No statistic difference was found in plasma levels of urea and creatinine, but hematuria was observed in the 2 groups at 2 hours after CPB. The pathologic changes of kidney, which was mainly renal tubule swelling accompanied partly with vacuolar degeneration, were similar under the light microscope in the 2 groups at 2 h after CPB. Obvious glomerular damage was not found.@*CONCLUSION@#LDF-1 can effectively decrease leukocyte counts and the inflammatory reaction, but it can not bring about excellent protective effect on kidney during CPB when used alone. Attention to should be paid the renal protection in the postoperative CPB.

Improved oxygenation in patients with mild lung dysfunction by leucocyte depletion during cardiopulmonary bypass

Cardiopulmonary bypass [CPB] has been shown to induce a systemic inflammatory response. This leads to release of toxic substances, such as elastase, which cause endothelial damage and adversely affect the outcome. Leucocyte depleting arterial filters have not dramatically improved lung function after cardiopulmonary bypass, but patients with pre-existing lung function may benefit from their use. This study was done to evaluate whether postoperative lung function could be improved by use of leucocyte filter during cardiopulmonary bypass. 30 patients with lung dysfunction having elective first-time coronary revascularization were randomized to either a leucocyte depleting or a standard 40mm arterial line filter during CPB. The alveolar-arterial oxygenation index was calculated before and 5min after CPB then at 1,2,4,8 and 18hr after surgery. Time to extubation was recorded. Preoperative, immediate postoperative and 24hr postoperative chest x-rays were scored for extravascular lung water. Activated white cells were identified using nitroblue tetrazolium, then total and activated white cell numbers counted after staining with leucoplate. Postoperative alveolar-arterial oxygenation were better in patients who received leococyte depletion during CPB [1.7 +/- 0.97 vs 3 +/- 1.74 in the control group, p<0.05] and also leucocyte filter reduced the total white cell count and was capable of selectively removing activated white cells during CPB. The duration of postoperative mechanical ventilation was less in the leucocyte depleted group [4.6 +/- 2.1 vs 8.1 +/- 4.8 hr in the control group, p<0.05]. The extravasular lung water scores immediately postoperatively were 13.0 +/- 8.7 in the study group vs 19.7 +/- 10.6 in the control group [p = 0.04] and at 24 hr postoperatively 9.5 +/- 7.6 vs -14.9 +/- 10 for controls. For patients with mild lung dysfunction, a leucocyte-depleting arterial line filter improves postoperative oxygenation, reduces extravascular lung water accumulation and reduces time on artificial ventilation after CPB. Leucocyte depleting filters may be an economic argument for every patient during CPB. Pulmonary dysfunction - after cardiopulmonary bypass remains an important clinical problem despite refinements in techniques of extracorporeal circulation and improvements in postoperative intensive care. The pathogenesis of postoperative lung injury consists of several steps .The initial step is a complement activation and release of inflammatory mediators as a result of blood exposure to the surface of the cardiopulmonary bypass circuit[1-5]. This causes the up regulation of leucocyte adhesive receptors CD11b /CD18 [known as Mac-1 or CR3][6] Furthermore the inflammatory cytokines induce the synthesis of ligand intercecullar adhesion molecules on the endotheliun cells, which lead to enhanced adhesiveness of endothelial for neutrophils[7]. In the phase of lung reperfusion at the termination of cardiopulmonary bypass neutrophils adhere to the endothelial surface of the pulmonary circulation creating a microenvironment that permits high concentration of toxic agents [such as elastase and oxygen radicals] released by activated neutrophils[8,9]. The technique of leucocyte depletion was developed in response to this with early studies using either cell separator technology, or leucocyte depleting trasfusion filters incorporated into the extracorporeal circuit[10-13]. The key benefits of leucodepletion are improved cardiac and lung function, especially an increased postoperative PaO2[10,11]. The leukoguard LG-6 arterial line filter [pall Biomedical Portsmouth, UK] removes leucocytes from the circulation although the systemic neutrophil count may[11,14,15] or may not be reduced[16-18]. Leucocyte depletion during cardiopulmonary bypass [CPB] may improve oxygenation in the early postoperative period in comparison with controls[11,16,18,19] and the duration of postoperative mechanical ventilation may also be reduced[11,16]. The benefit of leucocyte depletion may be small if the duration of bypass is short[20]. The aim of this study was to determine whether mechanical filtration of leucocytes during cardiopulmonary bypass improves the postoperative pulmonary function in human beings and to compare sequential total and activated white cells during CPB, using either a leucocyte depleting or standard arterial line filter