bluuebunny/crossref_metadata_2025 huggingface dataset:
Pressure cycling technology:a novel approach to virus inactivation in plasma
Authors: Bradley D.W. , Hess R.A. , Tao F. , Sciaba‐Lentz L. , Remaley A.T. , Laugharn J.A. , Manak M.
Published: March 2003

BACKGROUND: Hydrostatic‐pressure virus inactivation is a novel approach to the inactivation of pathogens in plasma and blood‐derived components, that retains the therapeutic properties of these products.

STUDY DESIGN AND METHODS: A custom‐built apparatus was used to pressurize human plasma samples spiked with lambda phage. Phage titer and plasma protein activities were monitored after pressure treatment.

RESULTS: Pressure‐mediated inactivation of lambda phage was found to be an effective means for virus inactivation, particularly when performed at near‐zero (0°C) temperatures, rather than at temperatures above 20°C and below –40°C. The efficiency of inactivation was improved by an increase in applied pressure and repeated cycling from atmospheric to high pressure. In contrast, activities of plasma proteins alkaline phosphatase and total amylase did not vary with temperature and remained within 29 percent and 6 percent, respectively, of starting values after the same pressure treatments. By combining cycling, near‐zero temperatures, and high pressure, phage titers in serum were reduced approximately 6 log after 10 to 20 minutes of treatment. Activities of plasma proteins IgG, IgM, and factor X were at 104 percent, 89 percent, and 80 percent, respectively, of starting values after 20 minutes of the same temperature and pressure treatment.

CONCLUSION: High‐pressure procedures may be useful for the inactivation of viruses in blood and other protein‐containing components.
Amino acid substitutions in human erythroid protein band 3 account for the low‐incidence antigens NFLD and BOW
Authors: McManus K. , Pongoski J. , Coghlan G. , Zelinski T.
Published: March 2003

BACKGROUND: The low‐incidence red cell antigens NFLD (700.37) and BOW (700.46) were first described in 1984 and 1988, respectively. Recent investigations showed that antigens of the Diego blood group system (including a number of low‐incidence antigens) are coded by SLC4A1 (solute carrier family 4, anion exchanger member 1 gene). Among these newly characterized Diego system antigens is Wu (designated DI9). Because a serologic relationship among Wu, NFLD, and BOW has been established, a series of genetic and molecular investigations of SLC4A1 in relation to NFLD and BOW were undertaken.

STUDY DESIGN AND METHODS: By the use of exon‐specific primers, single‐strand conformational polymorphism (SSCP) analysis of SLC4A1 was performed on DNA isolated from an NFLD+ person from Japan, from the members of a Canadian kindred segregating for NFLD, and from two unrelated BOW+ persons. Exons displaying SSCPs were subjected to genetic linkage analysis (for NFLD only) and DNA sequencing.

RESULTS: SSCPs in DNA amplified from exons 12 and 14 of SLC4A1 were observed for all NFLD+ subjects. Linkage between each of these polymorphisms and NFLD was established with peak lods = 4.82 at θ = 0.00 for combined paternal and maternal meiosis. DNA sequencing of exons 12 and 14 of SLC4A1 from NFLD+ persons identified A→T and C→G mutations that underlie Glu429Asp and Pro561Ala substitutions in human erythroid band 3 protein (band 3). DNA from the two unrelated BOW+ persons only exhibited an SSCP in exon 14 of SLC4A1. Subsequent DNA sequencing revealed a C→T mutation that accounts for a Pro561Ser substitution in band 3.

CONCLUSION: SLC4A1 codes for the low‐incidence red cell antigens NFLD and BOW. In light of these findings, both antigens have been assigned to the Diego blood group system.
Incidence and clinical presentation of posttransfusion TT virus infection in prospectively followed transfusion recipients: emphasis on its relevance to hepatitis
Authors: Wang J.T. , Lee C.Z. , Kao J.H. , Sheu J.C. , Wang T.H. , Chen D.S.
Published: March 2003

BACKGROUND: A novel transfusion‐transmissible human DNA virus, TT virus (TTV), has been discovered recently. An attempt was made to determine the incidence and clinical outcome of TTV infection in recipients of blood transfusion.

STUDY DESIGN AND METHODS: Serial serum samples collected as part of a prospective study of posttransfusion hepatitis were examined for TTV DNA by a nested PCR assay.

RESULTS: Among 150 adults undergoing cardiac surgery, posttransfusion specimens from 59 individuals were positive for TTV DNA. Pretransfusion sera were found to be positive in 13 of these individuals. Therefore, 46 (33.6%) of the 137 previously uninfected patients developed new TTV viremia after transfusion. Among the 46 patients, 3 were coinfected with HCV, 5 were coinfected with HGV, and 38 were infected with TTV alone. No apparent symptoms or signs were noted in the 38 patients infected by TTV alone or the 5 infected with HGV plus TTV. The average peak serum ALT activity was 31 IU per L, with persistently normal levels in 34 of the 38 patients with TTV infection alone. In 8 other patients who subsequently developed well‐documented non‐A‐G hepatitis, 3 were positive for TTV (3/8 vs. 46/137, p = 0.8). In 12 patients followed for more than 1 year, TTV viremia persisted in every case.

CONCLUSION: In this population, TTV is transmitted by transfusion to approximately 30 percent of patients who undergo cardiac surgery. Most of the infections appear to become persistent. Despite the high prevalence rate, TTV does not appear to cause hepatitis on its own.
Heparin‐induced coagulopathy associated with staphylococcal protein A immunoadsorption treatment columns: an in vitro and in vivo analysis
Authors: Kunkel J.F. , Sarode R. , Verba M. , Yomtovian R.
Published: March 2003

BACKGROUND: The staphylococcal protein A (SPA) column used to treat refractory autoimmune and alloimmune thrombocytopenia and rheumatoid arthritis patients is primed with heparin to prevent possible fibrin clot formation when the patient's plasma is passed through the column. A BMT patient with refractory alloimmune thrombocytopenia had prolonged activated partial thromboplastin times (aPTTs) at the end of SPA column treatments. This observation led to in vivo and in vitro analysis of the kinetics of heparin elution from the SPA column.

STUDY DESIGN AND METHODS: Two patients with refractory rheumatoid arthritis, who were treated on five occasions with the SPA column (as a part of a national trial) primed with 5000 U of heparin, were monitored for aPTT and heparin in their plasma. In addition, two in vitro analyses were performed with FFP for heparin elution from the SPA column.

RESULTS: The in vivo studies showed the presence of 0.3 to 1.5 U per mL of heparin in patients' plasma at the end of the SPA column treatments that corresponded with the prolonged aPTTs. The in vitro studies showed that 82 to 85 percent heparin (approx. 4400 U) was eluted from the SPA column during rather than before the procedure.

CONCLUSION: Patients undergoing SPA column treatments, especially those with thrombocytopenia, may be at increased risk of bleeding as a result of the presence of a significant amount of heparin in their circulation during the entire period of SPA column treatment.
V(D)J germline gene repertoire analysis of monoclonal D antibodies and the implications for D epitope specificity
Authors: Perera W.S. , Moss M.T. , Urbaniak S.J.
Published: March 2003

BACKGROUND: The D antigen is a highly immunogenic human RBC antigen. Alloimmunization against the D antigen produces high‐affinity antibodies that cause hemolytic transfusion reactions and HDN.

STUDY DESIGN AND METHODS: Cloning and subsequent sequence analysis of 11 new samples of monoclonal anti‐D was performed in an attempt to identify V(D)J germline gene usage. Sequences were compared and analyzed with 37 previously published samples of anti‐D for identification of VH and VL pairings, canonical structures, and conformation of restricted germline gene usage.

RESULTS: The VH and VL pairings used by the new D MoAbs resulted in seven canonical combinations, three of which had not been described previously. Preferential usage of gene segments from the VH3 and VH4 families and of D3, D6, JH6, and DPK9 germline gene segments was also determined. Three samples of anti‐D from different donors were found to use similar VH and Vκ germline genes, despite the fact that two of the antibodies recognized epD6/7 and the third recognized epD1. From the cumulative analysis of the anti‐D IgG, 24 VH and VL gene pairings were identified, resulting in only 10 canonical structures.

CONCLUSIONS: Despite the potential for diversity, only a minority of VH and VL germline genes are used by anti‐D. Consequently, VH and VL pairings and the resulting canonical structures are similarly restricted.
Correlation of in vivo and in vitro functions of fresh and stored human platelets
Authors: Rothwell S.W. , Maglasang P. , Reid T.J. , Gorogias M. , Krishnamurti C.
Published: March 2003

BACKGROUND: Long‐term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model.

STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 μM cytochalasin B and 80 μM EGTA/AM at 4°C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet‐induced clot retraction and by platelet aggregation.

RESULTS: Platelets stored at either 4°C or ‐145°C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4°C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO‐treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO‐treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet‐induced clot retraction. With thrombin as the agonist, only frozen DMSO‐treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets.

CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.
Separation of dendritic cells from highly purified human monocytes by counterflow centrifugation induces tissue factor expression
Authors: Broussas Matthieu , Cornillet‐Lefebvre Pascale , Bernard Jacky , Adjizian Jean‐C. , Potron Gérard , Nguyên Philippe
Published: March 2003

BACKGROUND: In vitro generation of dendritic cells (DCs) from human monocytes represents a promising tool in immunotherapy. However, it is not known whether the separation of DCs from monocytes induces tissue factor expression and therefore may trigger coagulation in patients receiving these DC preparations. The aim of this study is thus to analyze tissue factor expression on monocyte‐derived DCs and to compare their ability to trigger thrombin generation to that of macrophages obtained from the same monocytes.

STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis and washed by using counterflow centrifugation in sterile, endotoxin‐free conditions. Macrophages are grown from human monocytes in the presence of GM–CSF alone and immature DCs are grown in the presence of GM–CSF plus IL‐4 for 5 days with fetal calf serum (IDC‐FCS). Immature DCs are also grown from human monocytes for 7 days in the presence of GM–CSF plus IL‐4 with human group AB serum (IDC‐HS). The addition of prostaglandin E2 and TNFα in this culture medium at Day 5 leads to mature DCs (MDC‐HS). Tissue factor mRNA expression is studied by RT‐PCR analysis. Tissue factor antigen is measured by ELISA in cell lysates and by direct flow cytometry. The procoagulant activity of intact cells is assessed by using an amidolytic assay or a chronometric assay.

RESULTS: IDC‐FCS express tissue factor mRNA and antigen and trigger thrombin generation. Procoagulant activity of IDC‐FCS is dependent on both tissue factor expression and exposure to anionic phospholipid. Monocyte‐derived macrophages cultured for 5 days with GM–CSF alone express lower levels of tissue factor mRNA, tissue factor antigen, and procoagulant activity than IDC‐FCS. IDC‐HS and MDC‐HS also express high levels of tissue factor mRNA and antigen and support procoagulant activity.

CONCLUSION: Monocyte‐derived DCs express a high level of functional tissue factor and support procoagulant activity. This finding should be taken into account in clinical trials.
Posttransfusion survival (24‐hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4°C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions
Authors: Valeri C. Robert , Pivacek Linda E. , Cassidy George P. , Ragno Gina
Published: March 2003

BACKGROUND: Previously frozen human RBCs currently are glycerolized and deglycerolized by the use of open systems that limit storage of the deglycerolized RBCs at 4°C to only 24 hours.

STUDY DESIGN AND METHODS: Healthy male volunteers who met AABB requirements for blood donors (n = 38) were studied. A volume of 450 mL of blood was collected into CPDA‐1. The RBC concentrates were stored at 4°C for 3 to 6 days before being frozen with 40‐percent (wt/vol) glycerol and stored at –80°C. The RBCs were deglycerolized, resuspended in 0.9‐percent sodium chloride and 0.2‐percent glucose (SG) solution or SG solution supplemented with AS‐1, AS‐3, or AS‐5, and stored in the resuspension medium at 4°C for 14 days.

RESULTS: The mean ± SD freeze‐thaw‐wash process recovery was 90.0 ± 4.0 percent for all 38 units. The mean 24‐hour posttransfusion survival value was 79 percent for deglycerolized RBC stored at 4°C for 7 days in SG alone, SG plus AS‐3, or SG plus AS‐5. Deglycerolized RBC that were stored at 4 C for 14 days in SG supplemented with AS‐1, AS‐3, or AS‐5 had a mean 24‐hour posttransfusion survival of 74 percent. After 7 days of storage of deglycerolized RBCs in SG alone, the mean hemolysis was 3.7 percent. After 14 days of storage of deglycerolized RBCs in SG supplemented with AS‐1, AS‐3, or AS‐5, the mean hemolysis was 2.5 percent.

CONCLUSIONS: The levels of hemolysis did not correlate with the 24‐hour posttransfusion survival values.
Psoralen photochemical inactivation of Orientia tsutsugamushi in platelet concentrates
Authors: Belanger Kevin J. , Kelly Daryl J. , Mettille Frank C. , Hanson Carl V. , Lippert Lloyd E.
Published: March 2003

BACKGROUND: The risk of transfusion transmission of disease has been reduced by the combination of predonation questions and improved transfusion‐transmitted disease assays, but the risk is still present. This study was conducted to determine if psoralen photochemistry could inactivate an obligate intracellular bacterium, with documented potential for transfusion, in PCs to further improve safety.

STUDY DESIGN AND METHODS: PCs were inoculated with MNCs infected with Orientia tsutsugamushi. The concentrates were treated with amounts ranging from 0.86 to 138 μmol per L of 4′‐(aminomethyl)‐4,5′,8‐trimethylpsoralen hydrochloride (AMT) combined with a constant long‐wave UVA light (320‐400 nm) exposure of 5 J per cm2. The effects of photochemical treatment were analyzed by using a mouse infectivity assay along with in vitro testing by PCR, indirect fluorescence antibody, direct fluorescence antibody, and Giemsa staining.

RESULTS: AMT, at 0.86 μmol per L or more, combined with UVA light of 5 J per cm2, inactivated O. tsutsugamushi that contaminated PCs. The PCs that did not receive the combined treatment caused infection.

CONCLUSIONS: The psoralen AMT, in conjunction with UVA light exposure, effectively abolished the infectivity of PCs deliberately contaminated with the scrub typhus organism O. tsutsugamushi, as tested in a mouse infectivity assay.
Coagulase‐negative staphylococcal contamination of whole blood and its components:the effects of WBC reduction
Authors: Holden Fiona , Foley Marie , Devin Gerard , Kinsella Anthony , Murphy William G.
Published: March 2003

BACKGROUND: Most bacteria present in blood components are normal skin flora, particularly Staphylococcus epidermidis and other coagulase‐negative staphylococci. Growth patterns of these bacteria and the effects of different methods of component preparation may depend on variations in behavior between different isolates of the same species.

STUDY DESIGN AND METHODS: Whole‐blood units were inoculated with 19 different coagulase‐negative staphylococcus (CNS) isolates at 1 to 10 and 10 to 100 CFUs per mL. After overnight holding at 22°C, the units were processed into components. The components were cultured before inoculation and during processing, including before and after WBC reduction.

RESULTS: At low inoculum levels, CNS was detected in 15 (79%) of 19 whole‐blood units and in 12 (63%) of 19 RBCs after separation; after filtration, bacteria were detected in 3 (16%) of 19 (p = 0.0069). For platelet concentrates, 6 (32%) of 19 grew bacteria before filtration and 1 of 18 after filtration (difference not statistically significant). Three (16%) of 19 plasmas were positive before and after freezing. At high inoculum levels, 16 (89%) of 18 whole‐blood samples and RBCs were positive before filtration; 6 (33%) of 18 RBCs were positive after filtration (p = 0.0002); 8 (44%) of 18 platelets were positive before filtration; 3 (17%) of 18 were positive after filtration (difference not statistically significant), and 7 (37%) of 18 plasma samples were positive before and after freezing.

CONCLUSION: The growth characteristics of CNS in blood components vary with differences either in the subtype of bacteria or in the donor blood. Filtration reduces but does not eradicate contamination of RBCs and platelets by CNS. Plasma may act as a reservoir for CNS infection.
Influence of ABO blood groups on primary hemostasis
Authors: Moeller Ansgar , Weippert‐Kretschmer Monika , Prinz Helge , Kretschmer Volker
Published: March 2003

BACKGROUND: Recently, the in vitro bleeding time test (IVBT) was proved to be a very sensitive screening method for the detection of vWD, showing rather good correlation between the closure time and the level of vWF. The vWF levels have been found to be significantly lower in healthy humans who are group O than in those who belong to the other ABO blood groups (non‐group O). The aim of this study was to detect whether these differences in vWF levels in normal persons correspond to differences in nonvascular primary hemostasis when investigated by the IVBT.

MATERIAL AND METHODS: Healthy blood donors (n = 162) without evidence of hemostatic disorders, without ingestion of drugs for at least 2 weeks, and with normal in vivo bleeding time endpoints, normal factor VIII clotting activity levels, normal structure of vWF multimers, and normal ristocetin‐induced platelet aggregation were examined by IVBT. IVBT was performed with two automated systems (Thrombostat 4000, VDG [TST]; and a platelet function analyzer (PFA‐100, Dade Behring [PFA]). CaCl2 and ADP were used as aggregants for the two TST tests (TST‐CaCl2 and TST‐ADP), and ADP‐ or epinephrine (Epi)‐coated membranes were used with the two PFA tests (PFA‐ADP and PFA‐Epi).

RESULTS: Closure time in the IVBT significantly correlated with the blood groups, but in reverse order (as did blood volume; data not shown): TST‐ADP (mean ± SD): group O, 89 ± 14.6 seconds versus non‐group O, 82 ± 13 seconds (p<0.01); TST‐CaCl2: group O, 154 ± 28.9 seconds versus non‐group O, 140 ± 31.3 seconds (p<0.01); PFA‐ADP: group O, 91 ± 13.4 seconds versus non‐group O, 86 ± 11.9 seconds (p<0.05); PFA‐Epi: group O, 112 ± 15.4 seconds versus non‐group O, 104 ± 16.7 seconds (p<0.05). Donors with vWF ≤77.5 % had longer closure time than those with vWF >77.5 % (p<0.05).

CONCLUSION: Significant ABO‐group‐specific differences in nonvascular primary hemostasis could be found by IVBT. The differences are small, however, and lie within the normal range. Whether these differences have any biologic relevance can only be speculated.
Comparison of a new WBC‐reduction system and the standard plateletpheresis protocol in the same donors
Authors: Zingsem Jürgen , Zimmermann Robert , Weisbach Volker , Glaser Anke , Bunkens Helena , Eckstein Reinhold
Published: March 2003

BACKGROUND: A cell separator (Spectra, Gambro BCT) with an integrated leukoreduction system (LRS) for producing WBC‐reduced single‐donor platelet concentrates has been shown to result in a slightly reduced collection efficiency as compared to the former Spectra system without LRS. A novel modified system for improved collection efficiencies (LRS Turbo, Gambro BCT) was evaluated.

STUDY DESIGN AND METHODS: Each of 37 donors underwent plateletpheresis using the LRS Turbo (LRS‐T) and the standard LRS (LRS) of the Spectra cell separator. The collection efficiency and WBC contamination of the different techniques were compared. Platelets were counted automatically and WBCs were counted by using one or two full grids of a Nageotte chamber.

RESULTS: The preseparation and postseparation numbers of RBCs, WBCs, and platelets, as well as the number of collected platelets, did not differ for the two techniques. In the LRS‐T separations, the collection efficiency was 112 percent of that in the LRS procedures. Median residual WBCs in the platelet components were 0.0256 × 106 per LRS‐T procedure and 0.0253 × 106 per LRS procedure. The purity of the LRS‐T components was not less than that of the standard LRS components, whereas the collection efficiency of the LRS‐T was significantly greater, 44.9 percent versus 40.7 percent.

CONCLUSIONS: The LRS‐T procedures produced platelet concentrates with WBC‐reduction capacity that is comparable to that obtained with the standard LRS procedures, which have previously been described as satisfying the most stringent criteria for WBC‐reduced platelets. The new technique significantly improved the collection efficiency of the plateletpheresis procedure.
Evaluation of oxygen extraction ratio as a physiologic transfusion trigger in coronary artery bypass graft surgery patients
Authors: Sehgal Lakshman R. , Zebala Luke P. , Takagi Ippei , Curran Ronald D. , Votapka Timothy V. , Caprini Joseph A.
Published: March 2003

BACKGROUND: Approximately 20 percent of all allogeneic blood transfusions are administered in connection with coronary artery bypass graft (CABG) operations. Transfusion practices vary across the country. The whole‐body oxygen extraction ratio (O2 ER) reflects the adequacy of the patient's response to acute normovo‐lemic anemia with an O2 ER of approximately 50 percent being shown to be an appropriate transfusion trigger. The present study monitored the O2 ER in patients undergoing CABG and determined if transfusion practices would have been different if an O2 ER ≥45 percent were used as a transfusion trigger.

STUDY DESIGN AND METHODS: Seventy patients with a postoperative Hct ≤25 percent were the test subjects. Arterial and mixed venous contents were determined before the operation, in the intensive care unit after the operation, and 12 hours after the operation.

RESULTS: There were no deaths. Forty‐one patients received allogeneic transfusion. These patients were older, weighed less, and had a preoperative Hct lower than the nontransfused patients. There were no significant differences between transfused and nontransfused patients with respect to postoperative Hct (21.0 ± 0.4 vs. 22.2 ± 0.4), cardiac index (2.5 ± 0.1 vs. 2.7 ± 0.1), O2 delivery (6.4 ± 0.3 vs. 6.7 ± 0.3), O2 consumption (2.5 ± 0.1 vs. 2.5 ± 0.1), and O2 ER (38.3 ± 1.7 vs. 37.5 ± 1.5). In the transfusion group, 7 of 21 patients had a postoperative O2 ER ≥45 percent, while 3 of 35 in the nontransfused group had that result.

CONCLUSION: The use of O2 ER as a transfusion trigger as part of a transfusion algorithm could lead to a reduction in allogeneic blood transfusion.
Determination of the degree of bacterialcontamination of whole‐blood collections using anautomated microbe‐detection system
Authors: De Korte Dirk , Marcelis Jan H. , Soeterboek A.M.
Published: March 2003

BACKGROUND : The prevalence of bacterial contamination in whole‐blood collections, either with immediate sampling or sampling after overnight storage as whole blood at 20°C, is determined.

STUDY DESIGN AND METHODS : Whole blood was collected under blood bank conditions in special five‐bag systems, allowing sampling in a closed system for culture bottles. Samples were taken within 2 hours after collection (Group 1) or after overnight storage of the whole blood at 20°C (Group 2). Culture bottles were incubated for 7 days, and positive samples were entered on agar plates for confirmation and determination.

RESULTS : In Group 1, 9219 units were tested; 27 units were positive with positive subculture, that is, 0.29 percent with a 95% CI of 0.19 to 0.42 percent. In Group 2, 9038 units were tested; 36 units were positive with positive subculture, that is, 0.39 percent with a 95% CI of 0.28 to 0.55 percent. No significant difference could be found between the two test groups. The majority of bacteria were either Staphylococcus (all coagulase‐negative) or Propionibacterium species.

CONCLUSION: For a total of 18,257 units, 0.34 percent (CI, 0.25‐0.44) of whole‐blood collections appeared to have bacterial contamination (mainly skin‐derived). Overnight storage of whole blood at 20°C did not have a significant effect on the prevalence of bacterial contamination.
In vitro immunosuppressive activity of soluble HLA class I and Fas ligand molecules:do they play a role in autologous blood transfusion?
Authors: Ghio Massimo , Contini Paola , Mazzei Clemente , Merlo Andrea , Filaci Gilberto , Setti Maurizio , Indiveri Francesco , Puppo Francesco
Published: March 2003

BACKGROUND: The immunomodulatory effects of allogeneic blood transfusion may contribute to a poor prognosis in patients with cancer who are undergoing surgery, and clinical trials have been carried out to investigate whether these patients would benefit from autologous blood donation. As the immunomodulatory effects of allogeneic blood transfusion have been related to soluble molecules released from residual WBCs during storage, the in vitro immunomodulatory activity of soluble molecules detected in supernatants from stored autologous blood was evaluated.

STUDY DESIGN AND METHODS: Blood was donated by four healthy volunteers. Packed WBC‐reduced RBCs were obtained and stored for 30 days, and supernatants were collected. FFP and serum were also obtained. The concentration of soluble molecules was determined by immunoenzymatic assays. The in vitro immunomodulatory activity of undiluted blood component supernatant was assessed by antigen‐specific cytotoxic T‐cell activity and mixed lymphocyte reactions in autologous combinations and by apoptosis induction in Fas+ cells.

RESULTS: The concentrations of soluble Fas‐ligand and HLA class I molecules were higher in packed RBCs than in WBC‐reduced RBCs, FFP, and serum. Undiluted supernatants of packed RBCs strongly inhibited functional assays and induced apoptosis in Fas+ cells. The immunomodulatory effects were correlated with the amount of soluble Fas ligand and HLA class I molecules.

CONCLUSION: The results of the present study are comparable with those already reported in allogeneic blood components, and they indicate that undiluted supernatants of autologous blood components may exert immunosuppressive effects in vitro.
A multivariable model for predicting the need for blood transfusion in patients undergoing first‐time elective coronary bypass graft surgery
Authors: Karkouti Keyvan , Cohen Marsha M. , McCluskey Stuart A. , Sher Graham D.
Published: March 2003

BACKGROUND: The incidence of blood transfusion in coronary artery bypass graft (CABG) surgery remains high. Preoperative identification of those at high risk for requiring blood will allow for the cost‐effective use of some blood conservation modalities. Multivariable analysis techniques were used in this study to develop a prediction rule for such a purpose.

STUDY DESIGN AND METHODS: Data were prospectively collected for all patients undergoing elective first‐time CABG surgery from January 1997 to September 1998 at a tertiary‐care teaching hospital (n = 1007). The prediction rule was developed on the first two‐thirds of the sample by using logistic regression methods to examine the relationship of patient demographics, comorbidities, and preoperative Hb with perioperative blood transfusion. The remaining one‐third of the sample was used to validate the rule.

RESULTS: The transfusion rate was 29.4 percent. The prediction rule included preoperative Hb (g/dL, OR 0.928, p<0.0001), weight (kg, OR 0.938, p<0.0001), age (years, OR 1.037, p<0.01), and sex (male/female, OR 0.493, p<0.01); receiver operating characteristic = 0.86. When externally validated, the rule had a sensitivity of 82.1 percent and a specificity of 63.6 percent (at a selected probability cutoff).

CONCLUSION: A simple and valid prediction rule is developed for predicting the risk of blood transfusion in patients undergoing first‐time elective CABG surgery.
Multiplex PCR for detection of Enterobacteriaceae in blood
Authors: Sen Keya , Asher David M.
Published: March 2003

BACKGROUND: Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5′‐nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood.

STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL‐1 detected Serratia, Klebsiella, and Enterobacter species, and YE‐3 detected Y. enterocolitica.

RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection with probe SL‐1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y. enterocolitica with probe YE‐3 was 2 CFUs. When total chromosomal DNA was extracted from whole‐blood samples spiked with different numbers of Y. enterocolitica, S. liquefaciens, E. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood.

CONCLUSION: The 5′‐nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.
Localization of Knops system antigens in the long homologous repeats of complement receptor 1
Authors: Tamasauskas Daniel , Powell Vivien , Schawalder Alissa , Yazdanbakhsh Karina
Published: March 2003

BACKGROUND: The Sla (Knops system) located on complement receptor 1 (CR1) has been associated with malarial rosetting, a process associated with severe malarial infections. Moreover, the long homologous repeats (LHRs) B and C of CR1 were implicated in rosette formation. As a step toward mapping the location of Knops system antigens, truncated CR1 proteins have been expressed and their ability to inhibit antibodies to the high‐incidence Knops system antigens was assessed.

STUDY DESIGN AND METHODS: Individual LHRs (A, B, C, and D) of CR1 of the common CR1*1 (F) allotype were expressed as secreted forms in 293T cells. Their abilities to specifically neutralize Knops system antibodies were tested by both hemagglutination and flow cytometry.

RESULTS: Three examples of anti‐Kna (n = 6) were almost completely inhibited by LHR‐C and three by LHR‐D. Two examples of anti‐McCa (n = 2) and seven examples of anti‐Sla (n = 8) were inhibited by LHR‐D. Both examples of anti‐Yka (n = 2) were partially inhibited by LHR‐D.

CONCLUSION: The high‐incidence Knops system antigens reside within LHR‐D and to a lesser extent within LHR‐C. Because of the role of Sla antigen in malaria rosetting, these results indicate that LHR‐D may represent an additional malaria interaction region in CR1.
Higher affinity human D MoAb prepared by light‐chain shuffling and selected by phage display
Authors: Proulx Chantal , Boyer Lucie , St‐Amour Isabelle , Bazin Reneé , Lemieux Réal
Published: March 2003

BACKGROUND: In blood banks, D MoAbs are routinely used to phenotype donors and patients. However, most D MoAbs do not agglutinate RBCs that weakly express D. The use of higher affinity MoAbs could overcome this problem. In this work, an attempt has been made to increase the affinity of the human clone 43F10, an IgG anti‐D, by light (L)‐chain shuffling followed by selection using phage display.

STUDY DESIGN AND METHODS: PBMNCs of three polyimmunized individuals were used to construct the κ L‐chain repertoire that was recombined with the 43F10 heavy chain in a phagemid vector system (pComb3H, Scripps Institute, La Jolla, CA). L‐chain‐shuffled 43F10‐F(ab) phages were selected on intact RBCs and characterized by ELISA, indirect agglutination, and sequence analysis.

RESULTS: L‐chain shuffling combined with phage display permitted the selection of a 43F10 MoAb variant (p3.17) with improved reactivity with weak D RBCs in agglutination assays. Nucleic acid sequence analysis showed that p3.17 and wild‐type (wt) 43F10 L chains are encoded by different VL segments of the Vk1 family and different J segments, thus showing a relatively low degree of homology (86.4%).

CONCLUSION: The use of a variant such as p3.17 could permit a further increase of the potency of existing anti‐D reagents. The low homology between p3.17 and wt 43F10 sequences further exemplifies the predominant role of the heavy chain in determining the specificity of the anti‐D.
Construction of dimeric F(ab) useful in blood group serology
Authors: Czerwinski Marcin , Krop‐Watorek Anna , Lisowska Elwira , Spitalnik Steven L.
Published: March 2003

BACKGROUND: When expressed in Escherichia coli, recombinant F(ab) contain a heavy‐chain Fd fragment and a complete light‐chain fragment. Because these F(ab) are monovalent, their avidity is significantly lower than that of a corresponding bivalent IgG antibody. In addition, when monovalent F(ab) are used in hemagglutination assays, antiglobulin reagents are required. Therefore, it would be useful to develop a system that expresses recombinant bivalent F(ab) in E. coli.

STUDY DESIGN AND METHODS: Three modified vectors were constructed. Each contained cDNA sequences encoding a peptide linked to the C terminus of a heavy‐chain CH1 region: an IgG1 hinge region (Hinge), a leucine zipper (Zip), or a peptide containing the Hinge and Zip sequences in tandem (HingeZip). The vectors were used to express two cloned F(ab) recognizing human antigens M and N: NNA7 (anti‐N) and 425/2B (anti‐M). The recombinant proteins were expressed in E. coli and were purified and evaluated by ELISA and hemagglutination.

RESULTS: By gel filtration chromatography, 35, 90, and 70 percent of the purified F(ab) expressing the Hinge, Zip, and HingeZip tails, respectively, were dimers. By ELISA, the avidity of F(ab) containing the Zip or HingeZip tails was six to eight times higher than that of the corresponding monovalent F(ab). In addition, the dimeric F(ab) directly agglutinated RBCs in concentrations similar to those of corresponding bivalent IgG antibodies.

CONCLUSIONS: An introduction of dimer‐inducing peptides allowed the isolation of bacterially produced, bivalent F(ab). This approach could be useful for obtaining inexpensive, serologic reagents that may replace or complement conventional MoAbs produced by mammalian tissue culture methods.
Predictors of transfusion for spinal surgery in Maryland, 1997 to 2000
Authors: Berenholtz Sean M. , Pronovost Peter J. , Mullany Daniel , Garrett Elizabeth , Ness Paul M. , Dorman Todd , Klag Michael J.
Published: March 2003

BACKGROUND: The purpose of this study was to identify preoperative patient, hospital, and surgeon characteristics associated with transfusion for spinal surgery.

STUDY DESIGN AND METHODS: Discharge data were obtained from 39 Maryland hospitals for adult patients (n = 3988) who had a primary procedure code for spinal surgery between July 1997 through June 2000, and with these codes, surgeons and hospitals were characterized by annual patient volume. Outcome variables included any allogeneic transfusion, any transfusion, RBCs, autologous blood, FFP, or platelet transfusion. Logistic regression was used for univariate and multivariate analyses.

RESULTS: Characteristics independently associated with an increased risk of receiving any allogeneic transfusion (n = 786) included age >54 (OR, 1.6; 95% CI, 1.3‐2.1), age >66 (OR, 2.7; 95% CI, 2.0‐3.5), female sex (OR, 1.6; 95% CI, 1.2‐2.0), diabetes with chronic complications (OR, 2.5; 95% CI, 1.3‐4.9), and metastatic tumor (OR, 4.9; 95% CI, 2.3‐10.5), emergency room admission (OR, 2.3; 95% CI, 1.4‐3.8), and greater hospital volume (OR, 4.0; 95% CI, 1.8‐8.6). Characteristics independently associated with increased autologous transfusions (n = 574) included white race (OR, 1.7; 95% CI, 1.2‐2.4), female sex (OR, 1.4; 95% CI, 1.1‐1.8), and greater surgeon volume (OR, 3.5; 95% CI, 1.4‐9.1).

DISCUSSION: This information can be used to provide informed risk‐benefit discussions with patients regarding the risk for blood transfusion as well as to target high‐risk patients and institutions for interventions to reduce the risk of exposure to blood components.
Characterization and functional analysis of granulocyte concentrates collected from donors after repeated G–CSF stimulation
Authors: Joos Katja , Herzog Regina , Einsele Hermann , Northoff Hinnak , Neumeister Birgid
Published: March 2003

BACKGROUND: Neutropenic patients often develop bacterial or fungal infections not responding to broad‐spectrum antibacterial or antifungal agents. Clinical efforts were made with transfusion of granulocyte concentrates; however, functions of granulocytes after multiple G–CSF stimulations and after apheresis are not yet investigated and described sufficiently.

STUDY DESIGN AND METHODS: The aim of this study was to characterize functional and immunologic variables of granulocytes in blood samples drawn from donors before and after each stimulation episode with G–CSF, in the resulting granulocyte concentrates and in the patients 8 hours after transfusion.

RESULTS: Chemotaxis was not influenced, neither by G–CSF application nor by apheresis. Multiple G–CSF stimulations enhanced oxidative burst and phagocytosis of Escherichia coli in donor granulocytes. These values returned to basal levels in granulocyte concentrates. Expression of granulocytic surface antigens was downregulated after application of G–CSF but returned to normal and in part enhanced values in concentrates. A clinically relevant increase of proinflammatory cytokines could not be detected. Leukotriene B4 production was reduced after the fourth G–CSF stimulation in the donor blood and enhanced in the granulocyte concentrate after apheresis. Results in recipients indicate that changes of granulocyte function noted in concentrates were only transient.

CONCLUSION: Stimulation of healthy donors with repeated G–CSF injections and subsequent granulocyte apheresis does not dramatically change decisive functions of granulocytes.
Behavior of Nonselective Cation Channels and Large‐Conductance Ca2+‐Activated K+ Channels Induced by Dynamic Changes in Membrane Stretch in Cultured Smooth Muscle Cells of Human Coronary Artery
Authors: WU SHENG‐NAN , LIN PEI‐HSUAN , HSIEH KAI‐SHENG , LIU YEN‐CHIN , CHIANG HUNG‐TING
Published: March 2003

Stretch‐Activated Ion Channels. Introduction: The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery.

Methods and Results: In the cell‐attached configuration, membrane stretch with negative pressure induced two types of stretch‐activated (SA) ion channels: a nonselective cation channel and a large‐conductance Ca2+‐activated K+ (BKCa) channel. The single‐channel conductances of SA cation and BKCa channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on‐cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BKCa channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BKCa channel activity was greatly enhanced, but the activity of SA cation channels disappeared.

Conclusion: This study is the first to demonstrate that the behavior of SA cation and BKCa channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension.
Bigeminy Pacing: A New Protocol to Unmask Atrial Vulnerability
Authors: ATTUEL PATRICK , LECLERCQ JEAN‐FRANCOIS , HALIMI FRANCK , FIORELLO PIERRE , STIUBEI MARCEL , SEING SAO
Published: March 2003

Bigeminy Pacing and Atrial Vulnerability. Introduction: One of the most exciting developments in our understanding of atrial fibrillation (AF) mechanisms has been the recognition that “AF begets AF” in a process termed atrial remodeling. Little information is available about the events that mediate short‐term remodeling. In a bigeminy atrial pacing protocol that produces a continuous extrasystole‐postextrasystole cycle length, we sought to evaluate the electrophysiologic consequences of irregular atrial pacing.

Methods and Results: This study included 22 consecutive patients with documented paroxysmal AF and 10 control subjects. After evaluating the effective refractory period (ERP) and functional refractory period (FRP), bigeminy atrial pacing was performed for 5 minutes. The S1‐S2 coupling interval during bigeminy pacing was programmed to a mean value of

275 ± 45 msec

, i.e., 45 msec longer than the basic ERP measured at 100 beats/min. During bigeminy pacing, AF that lasted longer than 1 minute occurred in 12 AF patients and in none of the control subjects (group I). Short salvos of AF occurred in 5 patients and 3 controls (group II). No arrhythmia occurred in 5 patients and 7 controls (group III). Sensitivity, specificity, and negative and positive predictive values of sustained AF induced by bigeminy pacing were 54%, 100%, 50%, and 100%, respectively. No differences were observed between different pacing rates during bigeminy, the premature coupling interval S1‐S2, or the conduction parameters S2‐A2 and A2. Group I had the shortest basic ERP

(222 ± 38 msec)

and group III the longest ERP
(242 ± 21 msec, P < 0.05)
; group II was intermediate. Atrial ERPs and FRPs measured immediately after termination of 5 minutes of bigeminy pacing were shorter than during baseline. The degree of shortening was similar in AF patients and in controls. The locoregional conduction delay A2 did not change after the bigeminy protocol.

Conclusion: This study demonstrates that atrial bigeminy pacing highly increases atrial vulnerability. This protocol appears interesting because its sensitivity and specificity are higher than those of the conventional extrastimulation test. This makes it attractive for routine diagnosis of undocumented paroxysmal AF. Because it may induce atrial arrhythmias independently of the classic mechanisms of wavelength shortening, this study emphasizes the need for new modalities in the prevention of atrial arrhythmias.
Paroxysmal Supraventricular Tachycardia with Persistent Ventriculoatrial Block
Authors: STROHMER BERNHARD , SCHERNTHANER CHRISTIANA , PICHLER MAXIMILIAN
Published: March 2003

Supraventricular Tachycardia with VA Conduction Block. We report the case of a 64‐year‐old patient with paroxysmal supraventricular tachycardia and persistent VA block. Induction and maintenance of tachycardia occurred without apparent activation of the atria. Diagnostic characteristics were most compatible with AV nodal reentrant tachycardia (AVNRT). Automatic junctional tachycardia and orthodromic nodoventricular or nodofascicular reentry tachycardia were considered in the differential diagnosis. Upper common pathway block during AVNRT may be explained by either intra‐atrial conduction block or purely intranodal confined AVNRT. The arrhythmia was cured by a typical posteroseptal ablation approach guided by slow pathway potentials.
Neovascular Perfusion in Lung Cancer: Qualitative and Quantitative Study using MR Perfusion Imaging
Authors: Jingtao Miao , Yuesong Yang , Zhengyu Li , Weiming Cai , Yonghao Tao , Sijens Paul E. , Xianqiao Jin
Published: March 2003

The value of phase‐tracking subtraction MR pulmonary perfusion technique in the evaluation of the neovascular perfusion of lung cancer was investigated in 25 patients with lung cancer. All patients were examined with a GE Signa Horizon LX 1.5 T MR system with 2D fast spoiled gradient‐recalled‐echo and first‐pass images after bolus injection of contrast agents were obtained. The first‐pass images of pulmonary artery (PA), pulmonary parenchyma (PP) and pulmonary vein (PV)/ aorta phases and their time‐signal intensity curves (TSC) were calculated, subtracted and analyzed on a GE Advantage workstation with Functool software. The signal intensity changes of tumors occurred simultaneous to, or slightly later than the signal intensity changes of the descending aorta, whereas the signal intensity changes of corresponding regions of normal lung were simultaneous with those of the PA. Thus the TSCs of tumors matched those of the descending aorta, whereas the TSCs of corresponding regions of normal lung matched those of the PA. These findings indicate that the blood supply of these lung cancers comes from the aorta. On the other hand, the maximum slope of the TSC of the tumors was larger than that of normal lung, indicating that the blood supply to the tumor was larger in quantity and that the tumor probably had a higher density of vessels compared to normal lung tissue. It is concluded that MR perfusion imaging of differential phase subtraction of the pulmonary circulation can help to evaluate the angiogenesis of lung cancer by showing the source of the blood supply, its quantity and its hemodynamics.
Environment effects on effective magnetic exchange integrals and local spectroscopy of extended strongly correlated systems
Authors: Lepetit Marie-Bernadette , Suaud Nicolas , Gelle Alain , Robert Vincent
Published: February 2003

The present work analyzes the importance of the different components of the environment effects on the local spectroscopy of extended, strongly-correlated systems. It has been found that the usual formal charge definition of the charge transfer and Madelung potential are far too crude for an accurate determination of the local excitation energies in embedded fragment calculations. A criterion for the validation of the embedding against the infinite system density of states has been proposed.