Reduction of transmissible spongiform encephalopathy infectivity from human red blood cells with prion protein affinity ligands

BACKGROUND: There is a demonstrated risk of infection by transmissible spongiform encephalopathies (TSEs) through transfusion from asymptomatic donors. Currently, blood-borne TSE infectivity cannot be detected with a diagnostic test, nor is it likely to be amenable to inactivation; however, its depletion with specific adsorp-tive ligand resins is possible. STUDY DESIGN AND METHODS: Six ligands that bind the prion protein, PrP, were selected by screening large solid-phase combinatorial chemical libraries. The selected resins were placed in columns and challenged with a unit of leukoreduced human red blood cells (RBCs) spiked with hamster brain-derived scrapie infectivity. The performance of each ligand was assessed by comparing the TSE infectivity titer in the RBCs before and after passage through each of five resin columns in series. RESULTS: Four resins were able to reduce infectivity titer by 3 to more than 4 log ID(50) per mL. The reduction was not due to nonspecific matrix interactions since a chemical modification of the most effective ligand completely abolished its ability to bind infectivity (negative control). A small subfraction of the infectivity, 0.01 percent, could not be removed, even upon repeated passage through successive columns. CONCLUSION: If endogenous TSE infectivity in RBCs binds to the ligands in the same proportion as brain-derived infectivity spiked into RBCs, the four most effective ligands would remove 3 to 4 log ID(50) per mL. A follow-up experiment is in progress to test whether endogenous blood-borne infectivity is also reduced.     

Removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter

BACKGROUND: Two recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In the absence of a preclinical screening test, removal of the infectious agent by processing is the only means by which risk to recipients of blood from donors with inapparent vCJD infections can be eliminated. STUDY DESIGN AND METHODS: In the endogenous infectivity study, a pool of 500 mL of whole blood was collected into CP2D anticoagulant from 263K-strain scrapie-infected hamsters, processed into 300 mL of red cells (RBCs), and then passed through a prion removal filter. Pre- and postfiltration samples were tested for PrP(sc) by Western blot and for infectivity by inoculation of healthy hamsters. In the exogenous (spiking) infectivity study, 30 mL of 10 percent (wt/vol) scrapie-infected brain homogenates was added to 270 mL of human RBCs and then filtered. Levels of PrP(sc) and infectivity were determined by Western blot and bioassay. RESULTS: In the endogenous infectivity study, the prefiltered RBCs transmitted disease to 6 of 43 animals, whereas the postfiltered RBCs did not transmit disease to any of 35 animals, and a barely visible prefiltration PrP(sc) Western blot signal was reduced below the level of detection in the postfiltration sample. In the exogenous (spike) study, infectivity was reduced by 3.7 log LD50 per mL, from 9.2 to 5.5 log LD50 per mL. CONCLUSION: The new filter was effective in removing both infectivity and PrP(sc) from RBCs. The use of this type of filter should reduce the risk of vCJD transmission through blood transfusion.     

Prestorage removal of Yersinia enterocolitica from red cells with white cell-reduction filters

Prestorage removal of phagocytic white cells (WBCs) may increase the survivability of contaminating bacteria in units of stored red cells. Fourteen units of whole blood were inoculated with 65 colony-forming units per mL of Yersinia enterocolitica (serotype O:3) and processed into AS-3-preserved red cells. Five red cell units were filtered with a prototype third-generation filter and five red cell units with a second generation filter. WBC reduction was performed on the day of collection. Four red cell units were not filtered. Three noninoculated whole blood units served as negative controls; two were filtered (one with each type of WBC-reduction filter) and one remained unfiltered. All red cell units were then stored at 4 degrees C for 42 days. One of the five filtered red cell units (20%) in each filter group supported growth of Y. enterocolitica. In contrast, 4 (100%) of 4 unfiltered inoculated red cell units had growth (p = 0.04). Overall, 2 (20%) of 10 units of WBC-reduced red cells supported the growth of Y. enterocolitica, as compared to 100 percent of unfiltered red cell units after inoculation (p = 0.015). Bacterial contamination was not detected in any of the three noninoculated units. It can be concluded that prestorage WBC filtration significantly reduces the potential for growth of Y. enterocolitica in red cells stored at 4 degrees C for 42 days.     

Removal of Plasmodium falciparum–infected red blood cells from whole blood by leukoreduction filters

BACKGROUND: There has been an unexplained decrease in the incidence of transfusion‐transmitted malaria in recent years. The decrease in incidence has paralleled the increasing use of leukoreduction filters. Malaria‐infected red blood cells (RBCs) share surface characteristics of hemoglobin S–containing cells. Because units collected from donors with sickle trait do not filter optimally due to adherence of RBCs to the filters, the possibility that malaria‐infected RBCs may also adhere to filters was investigated. STUDY DESIGN AND METHODS: Malaria‐infected whole blood or calcium ionophore (A25187)‐treated and control RBCs were filtered with leukoreduction filters. Quantitation of malaria‐infected RBCs before and after filtration was performed by flow cytometry to determine the presence of DNA within RBCs, indicating malaria infection. Annexin V binding was also determined before and after filtration of RBCs treated with A25187. Immediately after filtration, filters were fixed and examined by scanning and transmission electron microscopy. RESULTS: There were at least three configurations of adherence of malaria‐infected RBCs demonstrated within the filters. The first was direct adherence of infected RBCs to filter fibers; the second involved adherence of malaria‐infected RBCs to platelets, which were adherent to filter fibers; and the third was adherence of infected RBCs to other RBCs. Filtration also resulted in preferential removal of phosphatidylserine (PS)‐expressing cells as seen by the reduction of annexin V binding after filtration. This was further confirmed by electron micrographic examination of the filters in which untreated RBCs sit within the filter resting on top of filter fibers; however, calcium ionophore–treated RBCs are seen to cling tightly to the fibers. CONCLUSIONS: PS expression by RBCs leads to their adherence within leukoreduction filters. Malaria‐infected RBCs are retained via more than one mechanism. The efficiency of removal requires further study.     

Investigation of a new in-line leukocyte reduction filter for packed red blood cells

Background and objectivesOccasionally there are adverse transfusion reactions in the therapeutic use of packed red blood cells. Some of those reactions are caused by the presence of white blood cells (WBCs). Both immunogenic and infectious transfusion reactions are significantly influenced by the level of white blood cell contamination.Materials and methodsThe flexible in-line red cell filtration system Leucoflex LCR Diamond (Macopharma) was investigated. According to manufacturer information the system has a smaller filter surface (46 cm²) than the previous filter LCR-5 (53 cm²). Main difference with the previous model is the rhomboid design. The filter tube connections are not at the level of the centre edge, but at two opposite corners. Eighteen red cell concentrates were produced under Good Manufacturing Practice conditions in routine operation. To ensure the quality of the filter system every 7 days metabolic parametres such as WBC count, haemoglobin content, haemolysis rate, potassium load, pH and ATP content were analysed over a storage period of 49 days.ResultsThe mean product volume was 260.7 mL after filtration. Average haemoglobin content was 51.8 g per unit and WBC contamination was 0.02 × 10⁶ per unit. Haemolysis rate was 0.05% directly after filtration and 0.20% at the end of storage. Immediately after filtration the potassium concentration was 1.3 mmol/L and the pH was 7.37. During whole storage time the ATP level was maintained above 2.0 μmol per g haemoglobin.ConclusionThe tested filtration system is suitable for quality-assured production of red blood cell concentrates meeting national and international guidelines.     

Leukocyte-poor red blood cells prepared by the addition and removal of glycerol from red blood cell concentrates stores at 4 C

Glycerol was added to and removed from red blood cells to prepare red blood cells free of white blood cells, platelets, and plasma protein. The red blood cells were stored in the primary polyvinylchloride (PVC) plastic collection bag for up to eight days. Red blood cell concentrates not treated with glycerol were washed either within four to six hours of collection or after seven days of storage at 4 C. Red blood cells mixed with glycerol were either washed immediately after addition or were equilibrated for 15 minutes at room temperature before washing. Leukocyte removal was influenced by the length of storage of the red blood cell concentrate at 4 C after collection and by the equilibration of the red blood cell-glycerol mixture prior to washing. The greatest number of leukocyte was removed when red blood cell concentrates were stored at 4 C for at least five days, mixed with glycerol solution, and the red blood cell-glycerol mixture equilibrated for 15 minutes before washing the Haemonetics Blood Processor 115. Leukocyte-poor red blood cells prepared by this procedure have been shown to be safe and effective in eliminating febrile transfusion reactions.     

新型冰冻红细胞洗涤机洗涤效果的研究

目的研制一种全自动、封闭式,体积小、重量轻,洗涤程序设计简单的新型冰冻红细胞洗涤机。方法全血采自健康献血者,ACD抗凝,离心获浓缩红细胞,加入甘油到终浓度为40%,-80℃保存。42℃条件下冻融红细胞,利用自行设计的透析式新型冰冻红细胞洗涤机,使用3种自动化洗涤程序洗涤红细胞,直至可输注。结果洗涤后红细胞形态正常,3种程序洗涤后红细胞的回收率(%)分别为69.73±8.36,85.09±6.03和88.43±4.72;红细胞血红蛋白的回收率(%)分别为45.30±2.66,70.80±3.76和81.26±5.55;洗后上清液中游离血红蛋白的含量(g/L)为0.20±0.08,0.14±0.03和0.25±0.13,采用单因素k水平方法对数据进行统计学分析,得出了最佳洗涤程序。结论该新型冰冻红细胞洗涤机采用优化的洗涤程序,洗涤后的冰冻红细胞达到了国家的质量标准。     

Elution of antibody from red blood cells using xylene—a superior method

Chan-Shu and Blair's findings that xylene produces a strongly reactive eluate from red blood cells have been confirmed and extended. Alloantibodies of 21 different blood group specificities and red blood cells sensitized in vivo, including ABO sensitization of babies' blood, were tested. The xylene method produced stronger reacting eluates than the modified ether method in 70 per cent of the tests. Ether, acid, and heat methods never produced significantly stronger eluates than did xylene. Ether eluates prepared from red blood cells sensitized with anti-S and anti-s were nonreactive, but the same cells yielded easily detectable anti-S and anti-s when xylene eluates were prepared. The xylene method is sensitive and simple. In addition, xylene is not subject to such stringent control of storage conditions by inspection agencies as is ether.     

Supernatant from stored red blood cell primes inflammatory cells: influence of prestorage white cell reduction

BACKGROUND: The contribution of RBC transfusion to adverse patient outcomes is controversial. There is conflicting clinical data and limited biologic data that provide an underpinning biologic rationale for any adverse impacts from RBC transfusion. This study used in-vitro measures of PMN stimulation to determine the ability of supernatant from RBCs to stimulate allogeneic WBCs and to determine the influence of residual donor WBCs and storage time on the proinflammatory potential of RBCs. STUDY DESIGN AND METHODS: Three types of RBCs were assessed: standard non-WBC-reduced RBCs (S-RBCs), buffy coat-poor RBCs (BCP-RBCs), and prestorage WBC-filtered RBC (LF-RBCs). Supernatant was collected weekly up to Day 42 of storage. PMN priming by supernatant from RBCs was determined by three methods: induction of CD11b expression on PMNs, induction of IL-8 release from PMNs, and the chemotactic effect of supernatant on PMNs. RESULTS: Supernatant from S-RBCs induced the expression of CD11b on PMNs, primed PMNs to release IL-8, and was chemotactic for PMNs. The magnitude of this PMN-priming progressively amplified with storage time. In contrast, supernatant from BCP-RBCs or LF-RBCs did not significantly prime PMNs. The PMN-priming effect of supernatant from RBCs correlated more closely with the level of MNCs in the RBCs than PMN content. CONCLUSION: Supernatant from stored S-RBCs prime unstimulated allogeneic PMNs in vitro. Prestorage buffy-coat WBC reduction was as effective as WBC depletion in abrogating this proinflammatory response elicited by supernatants from RBCs. The clinical consequences, if any, of these findings for transfusion recipients are unknown.     

A single assay for multiple storage‐sensitive red blood cell characteristics by means of infrared spectroscopy

BACKGROUND: To maintain a high quality of red blood cells (RBCs), RBC characteristics must be followed during storage under blood bank conditions. By means of infrared (IR) spectroscopy, several characteristics can be measured simultaneously. STUDY DESIGN AND METHODS: IR spectra were acquired for samples from RBCs that were collected and stored according to Dutch blood bank procedures for a period of up to 50 days. Spectra of the soluble cell components were acquired separately after hypotonic lysis of the cells, followed by centrifugation. Characteristic vibrational bands were analyzed with respect to storage time–dependent changes in peak position and in intensity. RESULTS: A decrease in corresponding peak intensities indicates that RBCs lose protein and lipid during storage. Changes in protein secondary structure during storage are largely confined to integral membrane proteins and membrane‐associated proteins. A concurrent decrease in lipid packing density probably reflects the gradual change in cellular shape from discoidal to globular. By integration over a narrow range, storage‐dependent changes in intracellular adenosine triphosphate (ATP) and glucose levels could be estimated. ATP levels decrease during storage, but stay above the required 75% of the initial level after 35 days of storage. Glucose concentrations stay well above 5 mmol/L over the entire storage period. CONCLUSION: IR spectroscopy is a promising technique to follow structural and metabolic changes in RBCs during storage under blood bank conditions. Several variables can be determined rapidly in a single measurement.     

Detection of Mycoplasma pneumoniae using a highly sensitive rapid diagnostic method with silver amplification technology

To prevent an increase in the frequency of antimicrobial resistance (AMR), the specific and rapid diagnosis of causative pathogens is important, as the results can be used to initiate appropriate antibiotics treatment. Recently, the highly sensitive rapid immunochromatographic assay of silver amplification technology was developed for the detection of Mycoplasma pneumoniae. We investigated the sensitivity and specificity of the silver amplification immunochromatographic assay in adolescent and adult patients. A total of 767 patients with respiratory tract infection (RTI) and 605 with pneumonia were assessed by the silver amplification assay and real-time polymerase chain reaction (PCR). M. pneumoniae was identified by PCR in 95 patients with RTI and in 30 patients with community-acquired pneumonia (CAP), but it was not identified in patients with nursing- and healthcare-associated pneumonia. Eighteen of the 95 RTI patients and 7 of the 30 CAP patients with PCR-positive M. pneumoniae were found to be infected with macrolide-resistant M. pneumoniae. When PCR was used as the control test, the sensitivity, specificity, and overall agreement with the silver amplification assay were 90.5%, 99.0%, and 97.9%, respectively, in RTI patients and 90.0%, 99.1%, and 98.7%, respectively, in pneumonia patients. Our results show that the silver amplification assay has excellent sensitivity and specificity compared with PCR despite being a rapid diagnostic method. The silver amplification assay may be helpful for initiating appropriate antibiotic treatment and preventing AMR.     

Obtaining of CD34+ cells from healthy blood donors: development of a rapid and efficient procedure using leukoreduction filters

BACKGROUND: Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1 × 10³‐4 × 10³/mL), but their number trapped in one LRF after filtration of 400 to 450 mL of blood is high (0.4 × 10⁶‐1.6 × 10⁶). STUDY DESIGN AND METHODS: To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500‐mL continuous or after sequential elution (50‐ or 20‐mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. RESULTS: The 20‐mL back‐flushing of LRFs allowed the most efficient WBC recovery. The next steps (110 × g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76 ± 10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4 × 10⁵ to 6 × 10⁵ cells containing approximately 85% of functional CD34+ cells were obtained. CONCLUSION: This procedure allows the easy, rapid (<5 hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.     

Blood donor white blood cell reduction filters as a source of human peripheral blood-derived endothelial progenitor cells

BACKGROUND: Neovascularization in tumors, wounds, and sites of ischemic injury occurs by both angiogenesis (proliferation from existing vessels) and by vasculogenesis (differentiation into endothelial cells from circulating endothelial progenitor cells [EPCs]). EPCs can be obtained from marrow, from cord blood, or by ex vivo expansion of human peripheral blood (PB). The ease of obtaining human PB EPCs has led many recent studies to utilize PB EPCs. The ability to obtain large numbers of PB EPCs would greatly facilitate characterization to further our understanding of EPC biology and their application in cellular gene therapy. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) from whole blood or from the material obtained from white blood cell (WBC) reduction filters were isolated. The cells from both sources were then cultured separately under defined conditions to quantify EPC yield from each source. RESULTS: The yield of EPCs per million PBMNCs plated was approximately 3.5-fold higher from fresh PB. Because greater numbers of PBMNCs were obtained from each filter, however, the mean yield of EPCs from one filter versus fresh blood was 5.4 million versus 0.4 million, respectively (approx. 14-fold increased yield). CONCLUSION: The use of WBC reduction filters provides a safe, inexpensive, and readily available source for large numbers of PBMNCs from which culture-expanded EPCs can be generated for further study.