bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel, automated method of temperature cycling to produce cryoprecipitate
Authors: Rock Gail , Berger Rachel , Lange John , Tokessy Melanie , Palmer Douglas S. , Giulivi Antonio
Published: March 2003

BACKGROUND: Cryoprecipitate continues to find wide application in transfusion practice. Current AABB standards call for a minimum of 80 units (U) of factor VIII and 150 mg of fibrinogen per bag of cryoprecipitate. However, individual cryoprecipitates can vary greatly in content, with as many as 20 different factors known to affect the yield.

STUDY DESIGN AND METHODS: Plasma was processed in a new, rapid, automated device (CryoSeal, Thermogenesis) with computer‐controlled temperature cycling to produce cryoprecipitate.

RESULTS: In repeat runs (n = 20), the automated procedure yielded a product containing 184 mg of fibrinogen and 158 U of factor VIII in 55 minutes. Additional studies using plasma pools to compare the quality of the machine‐generated products to those of traditionally prepared cryoprecipitate showed comparative recoveries of 182 and 187 mg of fibrinogen and 172.1 and 129.7 U of factor VIII and no significant difference in the levels of plasminogen, protein C, or protein S.

CONCLUSION: The new system offers an automated method of cryoprecipitate production in which the steps involved in temperature cycling are initiated sequentially, producing within 1 hour a preparation that is equivalent to standard cryoprecipitate.
Patients with neoplastic and nonneoplastic hematologic diseases acquire CTLA‐4 antibodies after blood transfusion
Authors: Pistillo Maria Pia , Tazzari Pier Luigi , Gaudiano Carlo , Cilla Vito , Kato Tomohiro , Matsui Toshihiro , Nishioka Kusuki , Capanni Paolo , Conte Roberto , Ferrara Giovanni Battista
Published: March 2003

BACKGROUND: The presence of antibodies to CTLA‐4, a negative regulator of T‐cell activation, was investigated in multiply transfused patients with malignant and non‐ malignant hematologic diseases. A previous study showed that, in multiply transfused patients, an immune response against nuclear matrix proteins can be induced by WBCs undergoing apoptosis during RBC unit storage. This study evaluated whether the same phenomenon could be involved in the induction of CTLA‐4 antibodies in the patients analyzed.

STUDY DESIGN AND METHODS: Patient sera were tested for binding to the recombinant full‐length CTLA‐4 β‐galactosidase fusion protein by an ELISA. Immuno‐fluorescence stainings were performed to analyze the CTLA‐4 epitopes recognized by the antibodies and to detect such epitopes in the apoptotic cells present in the RBC units.

RESULTS: CTLA‐4 antibodies were found in multiply transfused patients with β‐thalassemia (40%) and with other hemolytic diseases (33%) including leukemias (42%). A higher incidence of CTLA‐4 antibodies was found in patients receiving non‐WBC‐reduced blood (88%) than in those receiving WBC‐reduced blood (26%). Immunofluorescence staining showed that WBCs undergoing apoptosis in the RBC unit expressed CTLA‐4 epitopes.

CONCLUSIONS: The apoptotic WBCs present in the RBC units, after cold storage, express CTLA‐4 epitopes. These epitopes can be released and induce formation of CTLA‐4 antibodies with profound implications in the development of autoimmune disorders and in facilitating tumor dissemination and metastasis.
Evaluation of a whole‐blood WBC‐reduction filter that saves platelets: in vitro studies
Authors: Larsson Stella , Gulliksson Hans , Paunovic Dragica
Published: March 2003

BACKGROUND: In this study, a new WBC‐reduction in‐line filter that removes WBCs but not platelets was evaluated. Three WBC‐reduced blood components were prepared: RBCs, plasma, and platelet concentrates (PCs).

STUDY DESIGN AND METHODS: Whole‐blood components (n = 30) were filtered within 2 to 4 hours after collection and then were centrifuged and separated into RBCs, plasma, and WBC‐reduced buffy coat. Saline‐adenine‐glucose‐mannitol solution was added to the RBCs. The WBC‐reduced buffy coats were stored overnight; on the following day, PCs were prepared from pooled WBC‐reduced buffy coats and stored in a medium composed of approximately 35 percent CPD plasma and 65 percent platelet additive solution (T‐Sol, Baxter). The WBC‐reduction capacity of the filter, the recovery of cells after filtration, and the in vitro storage of RBCs (n = 10) and platelets (n = 6) were evaluated.

RESULTS: Mean and maximum WBC counts after filtration were 0.08 × 106 and 0.3 × 106, respectively, per filtered whole‐blood unit. Recovery of RBCs (mean values) after filtration was 90 percent in whole‐blood components and 73 percent in RBCs. Recovery of platelets (mean values) was 81 percent after filtration and 66 percent in PCs. The in vitro storage study of RBCs showed results comparable with previously published data, except for a lower degree of hemolysis. In the in vitro platelet storage study, results were compared with those of standard preparations. In all essentials, similar results were found.

CONCLUSION: The results of the present study suggest that effective WBC reduction meets current standards and satisfactory recovery after filtration. The storage characteristics for RBCs and PCs are similar to those of standard preparations. Use of a whole‐blood in‐line filter to save platelets is a new option for whole‐blood processing, which may simplify WBC reduction and blood component preparation, as well as reduce costs in the future.
Influence of temperature, filter wettability, and timing of filtration on the removal of WBCs from RBC concentrates
Authors: Van Der Meer Pieter F. , Pietersz Ruby N.I. , Reesink Hendrik W.
Published: March 2003

BACKGROUND: The efficacy of the removal of WBCs from buffy coat‐reduced RBC concentrates by filtration is determined by many variables. The aim of this study was to investigate the influence of the filtration temperature, the wettability of the filter material, and the timing of the filtration after collection.

STUDY DESIGN AND METHODS: The investigation used commercially available filters: 3 dry “online” filters (Cellselect FR, Fresenius Hemocare; BioR‐01‐max, Fresenius; Leucoflex LCG1, MacoPharma) and one wet “inline” filter (Leucoflex LCR4, MacoPharma) that contained saline‐adenine‐glucose‐mannitol additive solution for RBCs and differed from the online version only in wettability. After buffy coat removal and suspension in saline‐adenine‐glucose‐mannitol, filtrations were performed immediately at room temperature (RT) and after 2 hours' storage of the RBC concentrates at 4°C, while the Leucoflex LCR4 was also tested after 24 hours' storage of the RBC concentrates at 4°C. Sets of 12 pooled experiments were performed to prevent donor‐dependent differences.

RESULTS: The Cellselect FR gave significantly better WBC removal from RBC concentrates at 4°C than at RT, with residual WBCs of 1.44 ± 0.58 × 106 and 2.78 ± 1.23 × 106, respectively (p<0.001). The BioR‐01‐max gave no significant difference: 0.62 ± 0.27 × 106 WBCs (at 4°C) versus 0.61 ± 0.25 × 106 WBCs (at RT). Filtration with the Leucoflex LCG1 resulted in 0.06 ± 0.03 × 106 and 0.07 ± 0.07 × 106 WBCs at 4°C and RT, respectively, which is not a significant difference. The Leucoflex LCR4, however, gave 2.08 ± 0.84 × 106 WBCs at RT, 0.52 ± 0.44 × 106 WBCs at 4°C after 2 hours' cooling, and 0.05 ± 0.10 × 106 WBCs at 4°C after 24 hours' cooling (all p<0.001).

CONCLUSION: Temperature, filter wettability, and timing of filtration after collection influence the efficacy of a filter for RBC concentrates. These variables need to be established, validated, and controlled before a filter can be selected for routine use.
IL‐10 increases the number of CFU–GM generatedby ex vivo expansion of unmanipulated human MNCsand selected CD34+ cells
Authors: Wagner Thomas , Fritsch Gerhard , Thalhammer Renate , Höcker Paul , Lanzer Gerhard , Lechner Klaus , Geissler Klaus
Published: March 2003

BACKGROUND: Ex vivo expansion strategies with different cytokine combinations are currently used by several groups as a means of increasing the number of HPCs for a variety of special clinical applications. Because there is little information on the potential role of IL‐10 in such ex vivo expansion models, the effect of this cytokine on the generation of myeloid progenitor cells in suspension cultures was investigated.

STUDY DESIGN AND METHODS: On the basis of data from the literature and from new experiments, the combination of SCF and IL‐3 at concentrations of 100 ng per mL and 100 U per mL, respectively, was chosen as the standard cocktail. The addition of IL‐10 to such cultures resulted in a marked and dose‐dependent potentiation of myeloid progenitor cell production.

RESULTS: Using unmanipulated leukapheresis components from 13 individuals (including lymphoma and cancer patients and normal donors), the expansion multiple of CFU–GM after 14 days as compared with pre‐expansion values was 9.54 ± 2.31 times by SCF/IL‐3 and 46.38 ± 7.37 times by the combination of SCF/IL‐3 and 100 ng per mL of IL‐10 (p<0.001). IL‐10 also potentiated CFU–GM generation from selected CD34 PBMNCs (n = 9) with an expansion of 17.22 ± 7.04 times versus 45.67 ± 16.78 times using the SCF/IL‐3 and SCF/IL‐3/IL‐10 combination, respectively (p<0.05). Moreover, expansion‐promoting effects of IL‐10 were observed in liquid cultures containing MNCs from bone marrow (n = 4) and cord blood (n = 3), but did not reach statistical significance because of the small number of samples.

CONCLUSION: These results suggest IL‐10 as a useful cytokine to optimize progenitor cell‐expansion strategies for clinical application.
Determinants of transfusion‐associated bacterial contamination: results of the French Bacthem Case‐Control Study
Authors: Perez Paul , Salmi L. Rachid , Folléa Gilles , Schmit Jean‐Luc , De Barbeyrac Bertille , Sudre Philippe , Salamon Roger ,   for the Bacthem Group the French Haemovigilance Network
Published: March 2003

BACKGROUND : Transfusion‐associated bacterial contamination (TABC), probably the most frequent transfusion‐transmitted infection, may induce serious adverse events. Systematic information and documentation on determinants are lacking.

STUDY DESIGN AND METHODS : The Bacthem Study is a French matched case‐control study assessing TABC determinants. Included were cases of TABC reported in France in a 2‐year period, as determined from uniform definitions. Information on recipient‐, blood component‐, and donor‐related potential determinants was collected on site. ORs were estimated by conditional logistic regression.

RESULTS : Of the 158 cases of suspected TABC reported, 41 that involved transfusion with 25 RBCs and 16 platelet concentrates were included. Gram‐negative rods accounted for nearly half of the bacteria species involved and for all six deaths. In comparison with the risk of TABC for patients receiving RBCs for anemia, the risk was higher for patients receiving RBCs for pancytopenia (OR, 7.3; 95% CI, 1.3‐41.0) and for those receiving platelets for thrombocytopenia (OR, 5.3; 95% CI, 1.2‐24.1). Other potential determinants were platelet transfusion for pancytopenia (OR, 4.5; 95% CI, 0.5‐40.0), immunosuppressive treatment (OR, 2.8; 95% CI, 0.7‐10.6), shelf‐life of more than 1 day for platelets or 8 days for RBCs (OR, 2.6; 95% CI, 0.7‐9.6), and more than 20 previous donations by donors (OR, 1.9; 95% CI, 0.7‐5.3).

CONCLUSION : This first comparative study revealed TABC determinants that suggest approaches for prevention.
Changes in immunologic properties of group A RBCs during treatment with an A‐degrading exo‐α‐N‐acetylgalactosaminidase
Authors: Hoskins Lansing C. , Boulding Erwin T.
Published: March 2003

BACKGROUND: Blood group A RBCs theoretically can be converted to universal‐donor group O cells by incubation with α‐N‐acetylgalactosaminidase (A‐zyme). The purpose of this study was to compare the extent of A‐zyme treatment required to abolish several immunologic responses of group B or O recipients to group A RBCs.

STUDY DESIGN AND METHODS: A1 RBCs were incubated for 2 hours at 37°C in buffer alone (control) or with 0.1 to 5.0 units (U) of A‐zyme per mL of packed cells. They were then tested for 1) immune adherence (% rosetting) and activation of monocytic cells (% erythrophagocytosis, TNFα production), 2) activation of immune hemolysis, and 3) hemagglutination with Dolichos biflorus lectin and pooled human anti‐A serum.

RESULTS: A 2‐hour incubation with ≤5.0 U of A‐zyme per mL of packed cells abolished monocytic cell adherence and phagocytosis of group A1 RBCs coated with IgG3 A MoAb. Epitopes of A binding to IgG3 anti‐A A005 MoAb and BG‐2 MoAb differed in A‐zyme sensitivity. TNFα production in group B or O whole blood in response to the addition of A1 cells varied, but it essentially was abolished when group A cells were treated with 0.1 to 1.0 U of A‐zyme per mL of packed cells. A epitopes mediating immune hemolysis and hemagglutination with D. biflorus lectin were also cleaved by <5 U of A‐zyme per mL of packed cells. In contrast, hemagglutination with polyclonal anti‐A typing serum was diminished by only 1 to 2 serial titer dilutions.

CONCLUSION: A epitopes mediating immune hemolysis and immune adherence to and activation of monocytic cells are highly sensitive to A‐zyme cleavage, as compared to those mediating hemagglutination with monoclonal and polyclonal anti‐A.
Anemia‐induced increase in the bleeding time:implications for treatment of nonsurgical blood loss
Authors: Valeri C. Robert , Cassidy George , Pivacek Linda E. , Ragno Gina , Lieberthal Wilfred , Crowley James P. , Khuri Shukri F. , Loscalzo Joseph
Published: March 2003

BACKGROUND: Preoperative bleeding time (BT) does not correlate with postoperative bleeding in patients subjected to surgical procedures. A significant positive correlation has been reported between the BT 2 hours after cardiopulmonary bypass surgery and the nonsurgical blood loss during the first 4 hours after bypass surgery. This study was done to investigate the effect of Hct and platelet count on the BT measurement in normal, healthy men and women.

STUDY DESIGN AND METHODS: To assess the relative effect of RBCs and platelets on the BT, 22 healthy male and 7 healthy female volunteers were subjected to the removal of 2 units of RBCs (360 mL), followed by the return of the platelet‐rich plasma (PRP) from both units and the infusion of 1000 mL of 0.9‐percent NaCl. Four of the men and all seven women received their RBCs 1 hour after their removal. Shed blood levels of thromboxane B2 (TXB2), 6‐keto prostaglandin F1α, and peripheral venous Hct were measured. BTs were measured in 15 men and 13 women before and after a plateletpheresis procedure to collect 3.6 × 1011 platelets per unit.

RESULTS: The 2‐unit RBC apheresis procedure produced a 60‐percent increase in the BT associated with a 15‐percent reduction in the peripheral venous Hct and a 9‐percent reduction in the platelet count. The plateletpheresis procedure produced a 32‐percent decrease in the platelet count, no change in peripheral venous Hct, and no change in the BT. After the removal of 2 units of RBCs, the shed blood TXB2 level decreased significantly. Reinfusion of 2 units of RBCs restored the BT and restored the TXB2 level to the baseline levels.

CONCLUSION: The acute reduction in Hct produced a reversible platelet dysfunction manifested by an increase in BT and a decrease in the shed blood TXB2 level at the template BT site. Return of the RBCs restored both the BT and the shed blood TXB2 level to normal. The platelet dysfunction observed with the reduction in Hct was due in part to a reduction in shed blood TXB2 and other, unknown mechanisms.
Platelet transfusion refractoriness associated withHPA‐1a (PlA1) alloantibody without coexistent HLA antibodies successfully treated with antigen‐negative platelet transfusions
Authors: Pappalardo Philip A. , Secord Ann R. , Quitevis Philippe , Haimowitz Marcia D. , Goldfinger Dennis
Published: March 2003

BACKGROUND: Alloimmune‐mediated refractoriness to platelet transfusion is most commonly due to antibody to HLA antigens in multiply transfused or multiparous patients. Published reports of poor transfusion response due to antibodies to platelet‐specific antigens are rare and often confounded by the presence of coexistent antibodies against HLA antigens.

CASE REPORT: A case is presented of a multiparous woman with acute myelogenous leukemia whose sole cause of transfusion refractoriness was antibody to platelet antigen HPA‐1a. She responded dramatically to HPA‐1a‐negative platelet transfusion.

CONCLUSION: This case provides strong serologic and clinical evidence that platelet transfusion refractoriness may result from antibodies to platelet‐specific antigens.
Prospective RBC phenotype matching in a stroke‐prevention trial in sickle cell anemia: a multicenter transfusion trial
Authors: Vichinsky Elliott P. , Luban Naomi L.C. , Wright Elizabeth , Olivieri Nancy , Driscoll Catherine , Pegelow Charles H. , Adams Robert J. ,   for the Stroke Prevention Trial in Sickle Cell Anemia
Published: March 2003

BACKGROUND: Most sickle cell anemia patients undergo transfusion therapy to prevent complications. The Stroke Prevention Trial in Sickle Cell Anemia showed that transfusion therapy is effective in the primary prevention of stroke. Despite its efficacy, transfusion therapy is limited by alloimmunization. The purpose of this study was to determine if a multicenter trial could implement a transfusion program utilizing phenotypically matched blood to reduce alloimmunization.

STUDY DESIGN AND METHODS: One hundred thirty children underwent RBC phenotyping and antibody screening with review of blood bank records. The protocol required use of WBC‐reduced RBCs, which were matched for E, C, and Kell. Monthly alloantibody testing and review of transfusion forms were performed to determine compliance and the occurrence of any adverse events.

RESULTS: Patient RBCs expressed a low frequency of Kell (2%), E (20%), and C (25%) antigens. Sixty‐one patients received 1830 units. Ninety‐seven percent of all units were WBC reduced. Only 29 units were inadvertently not matched for E, C, and Kell. Five patients (8%) developed a clinically significant alloantibody. Four developed a single antibody to E or Kell. Three patients (5%) developed a warm autoantibody. There were 11 transfusion reactions and 8 transfusion‐associated events. Transfusion reactions included 6 febrile reactions (0.33%/unit), 3 allergic (0.16%/unit), and 2 hemolytic (0.11%/unit). Associated events included 4 episodes of hypertension (0.22%/unit), 3 crises (0.16%/unit), and 1 transient ischemic attack (0.05%/unit).

CONCLUSION: This is the first multicenter study to show that extended RBC phenotyping can be implemented nationwide. Compared to studies, the alloimmunization rate dropped from 3 percent to 0.5 percent per unit, and hemolytic transfusion reactions dropped by 90 percent. It is recommended that all transfused sickle cell anemia patients be antigen matched for E, C, and Kell. Patients should be closely monitored during transfusions to avoid preventable risks.
DNA analysis for the Dombrock polymorphism
Authors: Rios Maria , Hue‐Roye Kim , Lee Agnes H. , Chiofolo Joseph T. , Miller Jeffrey L. , Reid Marion E.
Published: March 2003

BACKGROUND: RBC typing for Doa and Dob is notoriously difficult, and inaccurate typing can predispose patients to hemolytic transfusion reactions. The DO1/DO2 polymorphism is associated with three nucleotide changes: 378 C>T, 624 T>C and 793 A>G. While the 378 C>T‐ and 624 T>C‐containing codons are silent mutations, the 793 A>G polymorphism in codon 265 encodes asparagine for Doa and aspartic acid for Dob.

STUDY DESIGN AND METHODS: Described here are two PCR‐RFLP assays, one using the Mnl I site associated with 624C (DO2) and the other altering two nucleotides within the sense primer, which allows recognition of 793G by the Eam 1105 I.

RESULTS: The assays have been performed on over 100 samples for which the RBC typing of one or both antigens was known. Eight samples had been historically mistyped by hemagglutination.

CONCLUSION: This RFLP assay provides a practical method for typing donor blood for Dombrock alleles.
Screening for WBC antibodies by lymphocyte indirect immunofluorescence flow cytometry: superior to cytotoxicity and ELISA?
Authors: Lubenko Anatole , Rodi Katherine M. , Johnson Andrew C.
Published: March 2003

BACKGROUND: Cytotoxic WBC antibodies are found in patients who have refractoriness to platelet transfusion (RPT) or are experiencing febrile transfusion reactions (FTRs) and in sera giving so called nonspecific hemagglutination by IAT (N/S IAT). Sera from such patients were screened for WBC antibodies regardless of the ability to fix complement using a flow cytometric (FC) lymphocyte indirect immunofluorescence test (LIFT) to compare FC‐LIFT with a routine lymphocytotoxicity test (LCT) for WBC antibody detection.

STUDY DESIGN AND METHODS: Serum from 104 patients with RPT, 87 with FTR, and 147 with N/S IAT were tested in parallel by using FC‐LIFT and LCT. Sera giving discrepant results were re‐tested with an HLA class I antibody ELISA to assess whether they were HLA‐specific.

RESULTS: Of the sera tested, 175 were LIFT positive, and 146 were LCT positive. Fifty‐five had antibodies that were detectable only by LIFT; 26 were positive only by LCT. Of these 81 discrepant sera, 30 of 63 were positive in HLA ELISA.

CONCLUSION: FC‐LIFT detects more WBC antibodies than does LCT or ELISA, and it is a superior screening technique. Because some cytotoxic antibodies are detectable only by LCT, comprehensive WBC antibody screening would require the application of both techniques. However, because FC assessments of cytotoxicity have been described, LCTs may become redundant for WBC antibody screening.
K, Fya, and Jka phenotyping of donor RBCs on microplates
Authors: Diedrich Beatrice , Andersson Jan , Sallander Suzanne , Shanwell Agneta
Published: March 2003

BACKGROUND: In many cases, the search for compatible blood for patients with clinically significant RBC alloantibodies is difficult and time‐consuming. To date, it has been considered necessary only to phenotype the blood donors for ABO group and D. There has been long experience with automated routine analysis (ABO, C, c, D, E, and e typing and RBC antibody screening), using robotic dispensers and computerized interpretation of microplate results. The purpose of this study was to explore the possibilities of also phenotyping for K, Fya, and Jka, as antibodies directed against these antigens (together with Rh antigens) are the most common clinically significant alloantibodies in the Swedish population.

STUDY DESIGN AND METHODS: One thousand thirty‐one EDTA samples from blood donors were phenotyped for K, Fya, and Jka by use of an IAT with PEG on microplates. The findings were compared to those using conventional IAT in tube's and the microcolumn gel test (DiaMed‐ID, DiaMed).

RESULTS: All typing results with the microplate method were correct. All reactions for K and Fya typing could be interpreted by the computer. The results for Jka were indeterminate in 1.4 percent (14/1031) of the samples.

CONCLUSION: The PEG‐IAT microplate method gave reliable results that were suitable for routine phenotyping, thus making available a stock of phenotyped blood at reasonable cost, ready for delivery when required.
Effects of different concentrations of anticoagulant on the in vitro characteristics of autologous whole blood
Authors: Cober Nancy , Lacasse Margaret , Bart Bevan , Rock Gail
Published: March 2003

BACKGROUND: Routinely, 450 mL of blood is collected into 63 mL of CPDA‐1, for a final anticoagulant:blood ratio of approximately 1:7 in a whole‐blood autologous unit. If less than 300 mL of blood is to be collected, the AABB standards suggest that there should be a proportionate decrease in anticoagulant. Data from an autologous blood program showed a range in volume from 92 mL to 667 mL per bag, which reflects an anticoagulant:blood ratio of 2:1 to 1:10.

STUDY DESIGN AND METHODS: To determine the effects of these ratios on the in vitro function of RBCs at various anticoagulant ratios, blood was collected into different amounts of anticoagulant, and various measurements were made during storage.

RESULTS: The number of RBCs and the MCV remained constant over time, regardless of the anticoagulant dilution used. Plasma free Hb increased with time with all dilutions. At a 1:2 ratio, it rose from 734 mg per L on Day 1 to 1805 mg per L on Day 35, and at 1:8, it was 355 mg per L for Day 1 and 854 mg per L on Day 35. Plasma sodium decreased and the potassium increased over time with all dilutions. From Day 1 to Day 35, there was a nine‐fold increase in potassium at both the 1:2 and 1:8 dilutions (2.4 to 22.9 mmol/L, 3.2 to 29.6 mmol/L, respectively). The LDH increased over time and the pH decreased in all of the dilutions. Osmotic fragility remained constant at the 1:8 dilution but decreased at all of the other dilutions with storage, with 44‐percent fragility on Day 35 at the 1:2 ratio. The WBC and platelet counts decreased consistently over time. Overall, 1 percent of the autologous units were below the cutoff volume of 300 mL at which an adjustment of the anticoagulant volume is required.

CONCLUSION: Plasma Hb and plasma potassium concentrations are considerably higher in low‐volume units, which indicates that deviation from standard collection procedures is deleterious to RBCs.
Process for the preparation of pathogen‐inactivated RBC concentrates by using PEN110 chemistry: preclinical studies
Authors: Purmal Andrei , Valeri C. Robert , Dzik Walter , Pivacek Linda , Ragno Gina , Lazo Aris , Chapman John
Published: March 2003

BACKGROUND:A pathogen‐inactivation process for RBC concentrates is being developed by using PEN110 chemistry (INACTINE, V.I. Technologies). The objective of this study was to characterize the quality of RBCs prepared by using the PEN110 process and to measure the virucidal effect achieved against two viruses.

STUDY DESIGN AND METHODS:Virology and RBC studies were conducted with standard RBC units treated with 0.1‐percent (vol/vol) PEN110 at 22°C for 6 hours. The quality of PEN110‐treated human RBCs was assessed with biochemical and phenotypic variables. The in vivo viability of PEN110‐treated RBCs in baboons was studied with the double‐label51Cr/125I method.

RESULTS:Decreases in infectious titer by inactivation of greater than a 5 log 50‐percent tissue culture infectious doses per mL of bovine viral diarrhea virus (an enveloped RNA virus) and porcine parvovirus (a nonenveloped DNA virus) was observed. RBC hemolysis was less than 1 percent after 42 days of storage, and no changes in RBC antigens were observed. The in vivo viability of PEN110‐treated baboon RBCs was unchanged from control.

CONCLUSION:The preparation of RBCs by using the PEN110 process achieved a significant viral reduction of two diverse viruses without causing adverse effects to the RBCs. The process appears to be a promising approach, thus justifying further study.
Response to vWF‐coated beads and soluble p‐selectin to characterize apheresis platelets: a comparison of three cell separators
Authors: Stiegler Gabriele , Eichelberger Beate , Leitner Gerda , Jilma Bernd , Hoecker Paul , Panzer Simon
Published: March 2003

BACKGROUND: Plateletpheresis technologies differ among various cell separators. Differences in centrifugation force, centrifugation time, and platelet concentration in the platelet concentrate may affect platelet activation and function.

STUDY DESIGN AND METHODS: In a three‐way crossover design, 12 donors were randomly assigned to three types of cell separators, two continuous flow systems (Amicus DN, Fenwall Division, Baxter and Spectra LRS, Gambro BCT) and one intermittent flow system (MCS+, Haemonetics). The response to vWF‐coated beads was determined in the peripheral blood and fresh platelet concentrates to obtain information about the initiating step of platelet aggregation, that is, platelet adhesion. Levels of soluble p‐selectin were measured as a marker of platelet activation.

RESULTS: Platelet yield and concentration of platelets were higher in platelet concentrates obtained with the Amicus DN than with the Spectra LRS or the MCS+. Maximal aggregation was significantly higher in platelets from the Amicus DN than in platelets from the Spectra LRS or the MCS+. Higher concentrations of soluble p‐selectin were seen in platelet concentrates obtained with the Amicus DN than in concentrates from the Spectra LRS or the MCS+, but they did not differ after correction for the number of platelets per component.

CONCLUSIONS: Different plateletpheresis procedures induced distinct changes of platelet function. Platelets collected using the Amicus DN retained the strongest adhesion capacity irrespective of their activation.
A newly developed gel centrifugation test for quantification of RBC‐bound IgG antibodies and their subclasses IgG1 and IgG3: comparison with flow cytometry
Authors: Lynen Rainer , Krone Olaf , Legler Tobias J. , Köhler Michael , Mayr Wolfgang R.
Published: March 2003

BACKGROUND: Novel gel centrifugation test (GCT) cards were evaluated with respect to their ability to estimate the quantity of IgG on RBCs and the determination of the IgG subclasses IgG1 and IgG3.

STUDY DESIGN AND METHODS: In 65 patients with a positive DAT, the amount of IgG‐γ‐, IgG1, and IgG3 on RBCs was examined by use of GCT cards and flow cytometry (FC) in parallel. The results were correlated with the presence or absence of hemolysis. In addition, D+ RBCs were studied after sensitization with anti‐D sera from 22 alloimmunized pregnant women.

RESULTS: The amount of IgG on the RBCs as determined by GCT dilution cards correlated with FC (r = 0.70, p < 0.0001). IgG subclass results as determined by GCT IgG subclass cards were confirmed by FC in 14 cases with an anti‐IgG‐γ‐chain titer ≥300, whereas IgG subclass cards were not suitable in cases with anti‐IgG‐γ‐chain titers less than 300. In 44 patients with 2+ or 3+ DAT in the GCT and anti‐IgG‐γ‐chain titer ≤30, no hemolysis was observed, whereas hemolysis occurred in 13 of 14 patients with an anti‐IgG‐γ‐chain titer ≥300. GCT data obtained by IATs with anti‐D sera were concordant with FC results.

CONCLUSION: There is a correlation between the amount of RBC‐bound IgG and immune hemolysis. The GCT cards that detect the anti‐IgG‐γ‐chain may be useful to predict hemolysis in patients with a 2+ or 3+ DAT in the GCT. The diagnostic value of GCT cards for IgG subclass testing should be investigated further.
RHC and RHc genotyping in different ethnic groups
Authors: Tax Martine G.H.M. , Van der Schoot C. Ellen , Van Doorn René , Douglas‐Berger Lotte , Van Rhenen Dick J. , Wijk Petra A. Maaskant‐van
Published: March 2003

BACKGROUND: RH genotyping assays are mainly based on research in whites. These assays may not be reliable in a multiracial society because of the genetic variation in RH among ethnic groups.

STUDY DESIGN AND METHODS: Five groups from different ethnic backgrounds were serologically typed for C and c and were genotyped on nucleotide C48 and intron 2 for RHC and RHc on nucleotides C178 and C307.

RESULTS: RHc genotyping with both methods proved to be reliable. RHC genotyping on C48 is not reliable because of a 48G>C mutation in the RHce allele (false‐positive prediction of C). This mutation was found in every ethnic group and does not affect c or e expression. RHC genotyping on intron 2 is unreliable because of r′s (Cdes) alleles (a false‐negative prediction of C). This allele was found in whites and blacks from Curaçao and South Africa. Reactions of r′s cells with anti‐C are weaker, but no negative reactions with various MoAbs were found. A new method (RHC/c/hex3‐intron 4/exon 7 multiplex PCRs) was developed based on intron 2 and r′s hybrid exon 3 characteristics (RHC) and C307 (RHc).

CONCLUSIONS: Reliable RHC and RHc genotyping is possible in different ethnic groups with the RHC/c/hex3‐intron 4/exon 7 multiplex PCR approach.
Mathematic modeling of the risk of HBV, HCV, and HIV transmission by window‐phase donations not detected by NAT
Authors: Weusten Jos J.A.M. , Van Drimmelen Harry A.J. , Lelie P. Nico
Published: March 2003

BACKGROUND: Blood transfusion centers around the world have introduced minipool NAT to reduce the risk of HBV, HCV, and HIV transmission by blood donations drawn in the infectious window phase. What would be the reduction in the residual risk when minipool NAT would be replaced by single‐donation NAT?

STUDY DESIGN AND METHODS: A mathematic model was developed to estimate the probability of virus transmission by blood transfusion when NAT screening methods are used for virologic safety testing. The major assumptions used are threefold: 1) The viral nucleic acid concentrations in the early window phase of infection double in 2.8 (HBV), 0.74 (HCV), and 0.90 (HIV) days. 2) The detectability of low copy numbers of viral DNA or RNA by the screening assay can be described with a probit model. 3) The probability of infection depends linearly on the logarithm of the administered dose, with 50‐percent infectivity rates at 10 (HBV and HCV) or 1000 (HIV) viral nucleic acid copies per transfusion unit (estimates based on NAT studies with samples of known infectivity in chimpanzees).

RESULTS: A reasonably simple equation was obtained that allows studying the effect of the sensitivity of the NAT assay and of the pool size used for screening on the residual risk of transfusion‐transmitted infection. The computations are illustrated by using observed sensitivity estimates of various NAT methods. By using epidemiologic data among European donors over 1997 as baseline, the calculations predict that the incidence of virus transmission per 10‐million RBC transfusions reduces with the following numbers when lowering the test pool size from 96 to 1 (single‐donation testing): HBV from 11 to 13 to 3.3 to 5.1, HCV from 1.7 to 2.0 to 0.5 to 0.8, and HIV from 0.47 to 0.62 to 0.010 to 0.045 (ranges for the different NAT screening methods).

CONCLUSION: A proper mathematic model for the calculation of residual infection risk by blood transfusion helps understand the impact of introducing new NAT methods for blood safety testing.
Detection of antibodies in acid eluates with the gel microcolumn assay
Authors: Greco Victoria A. , Byrne Karen M. , Procter Jo L. , Stroncek David F.
Published: March 2003

BACKGROUND : Gel microcolumns can be used to detect unexpected serum antibodies and to determine ABO blood group and Rh phenotype. DATs can also be performed with this system. The purpose of this study was to compare the gel microcolumn to the tube IAT using anti‐IgG for the detection of antibodies eluted from RBCs.

STUDY DESIGN AND METHODS
: Acid eluates were prepared from 30 peripheral blood and 41 umbilical cord blood samples. Twelve of the 71 eluates made were from control samples (known DAT negative). Specificities of eluted antibodies were determined by both tube and gel assays with a three‐cell screen plus A
1 and B cells, as determined by blood type.

RESULTS
: Ten of 30 peripheral blood eluates were reactive in both assays. Eighteen were nonreactive in both assays, and two from patients with autoimmune hemolytic anemia were reactive by gel assays and nonreactive by tube assays. Thirty three of the 41 cord blood eluates were reactive in both assays. Eluates from 2 of the 35 DAT‐positive samples reacted with A
1
and B cells by the tube method but were nonreactive by the gel method. Of the 33 cord blood eluates that were reactive by both assays, antibody specificity differed for two samples. When tested by tube assay, these eluates reacted with both A
1
and B cells, whereas the same eluates tested by gel assay showed one reacting with only A
1 cells and the other with only B cells.

CONCLUSION S: Results of testing eluates in gel assays were similar to those obtained in tube assays. The gel assays may be better at detecting antibodies eluted from RBCs from patients with autoimmune hemolytic anemia, and tube assays may be better at detecting isohemagglutinins eluted from umbilical cord blood.
Ultrastructural changes and activation differences in platelet concentrates stored in plasma and additive solution
Authors: Wagner Thomas , Vetter Angelika , Dimovic Natascha , Guber S.E. , Helmberg Wolfgang , Kröll Wolfgang , Lanzer Gerhard , Mayr W.R. , Neumüller Josef
Published: March 2003

BACKGROUND : The aim of this study was to demonstrate how ultrastructural morphology of platelets stored in different media correlate with the appearance of particular activation markers on their cell surface.

STUDY DESIGN AND METHODS : Concentrates of buffy coat‐derived platelets were stored in plasma or a glucose‐free citrate‐acetate‐NaCl platelet additive solution (PAS2, Baxter Healthcare Corp.). Activation markers on platelets were measured by flow cytometry and compared with changes in the platelet cell surface as demonstrated by electron microscopy. Levels of the vasoactive cytokines vascular endothelial growth factor (VEGF) and RANTES (regulated upon activation, normal T‐cell expressed and secreted) were determined in the storage medium of the platelet concentrate.

RESULTS
: The activation markers CD62P and CD63 and the binding of thrombospondin measured by flow cytometry were expressed to a higher extent in the PAS2 group compared with the plasma group. The difference reached significance on Day 3 (CD62P: 66.37 ± 2.44 vs. 37.83 ± 2.03, p < 0.001; CD63: 42.11 ± 3.29 vs. 34.84 ± 2.04, p < 0.05; and thrombospondin binding: 18.84 ± 3.9 vs. 13.98 ± 3.87, p < 0.001, respectively). The form factor that is related to changes of the platelet shape was determined by image analysis and correlated significantly with the cell surface expression of CD62P (p < 0.001) and with CD63 (p < 0.05) and with thrombospondin binding (p
< 0.05). The chemokines VEGF and RANTES were measured at higher levels in the PAS2 group.

CONCLUSIONS : With exception of baseline activation probably due to necessary handling procedures, platelets remain relatively unaltered and more stable in plasma in comparison to storage in PAS2.
Applying a novel statistical process control model to platelet quality monitoring
Authors: Perrotta P.L. , Ozcan C. , Whitbread J.A. , Finch S.J.
Published: March 2003

BACKGROUND: Many countries are implementing universal WBC reduction of blood components Thus, manufacturing procedures must include QC techniques to detect units that fail to meet established standards.

STUDY DESIGN AND METHODS: A statistical process control model, based on the exponentially weighted moving average of the cumulative distribution function (CDF‐EWMA), was developed to detect shifts in a mean and/or variance of a process. The model's parameters (weights) were optimized to maximize detection of an out‐of‐control process while minimizing sensitivity to autocorrelation. Validation was performed using a retrospective set of WBC‐reduction data obtained from a blood bank. The WBC‐reduction process was considered in control when there was 95‐percent confidence that more than 95 percent of platelet concentrates would contain less than 1 × 106WBCs (6.0 log WBC) as required by European standards. A sentry setting of 5.7 log WBCs was used to allow earlier detection of an out‐of‐control process.

RESULTS: Graphic output of the CDF‐EWMA model provided a continuous update of the probability that a WBC‐reduction process was in control. Using the validation data, the model showed that the process was in control until Observation 332, at which point residual WBCs per unit increased. However, the first platelet concentrate to exceed specified criteria (Observation 346) occurred after the model detected that the process was out of control, demonstrating the forecasting value of this model. This deviation corresponded to an equipment failure in a single apheresis instrument. The Shewhart and EWMA techniques were similarly able to detect when the process was out of control using the test data.

CONCLUSION: As a statistical process control model, the CDF‐EWMA provides real‐time estimation of the fraction of components meeting a regulatory limit. It is capable of detecting developing QC problems before units fail to meet regulatory requirements and is a potential alternative to other QC techniques for monitoring WBC reduction of blood components.
Development of a DNA‐based genotyping method for the Diego blood group system
Authors: Wu Guo‐Guang , Su Yu‐Qing , Yu Qiong , Jin Shi‐Zheng , Zhao Tong‐Mao
Published: March 2003

BACKGROUND
: The paucity of appropriate reagents for serologic typing of the Diego blood group has hindered the identification of the rare Di(b–) blood donors needed to transfuse a Di
b
antigen‐negative patient who presented with anti‐Di
b . Development of an alternative Di typing approach as a supplement to the current serologic typing method is an important and necessary goal.

STUDY DESIGN AND METHODS
:
DI1
and
DI2
alleles result from a single C to T substitution at nucleotide 2561 in exon 19 of the human anion exchanger gene causing a proline (
DI1
) to leucine (
DI2
) change at amino acid position 854. Allele‐specific primers were designed to specifically amplify the
DI1
and
DI2 alleles using a PCR‐based assay system.

RESULTS
: A PCR sequence‐specific primer (SSP) method for Di genotyping was developed, and the specificity and reproducibility of the method were assessed in a blind control study using serologic tests, family segregation, and DNA sequencing analyses. A total of 1766 DNA samples from unrelated blood donors were typed for
DI1
and
DI2
alleles and a single Di(b–) donor was identified. The frequency of
DI1
and
DI2 alleles among Chinese blood donors was 0.0357 and 0.9643, respectively.

CONCLUSION : A simple, accurate, and inexpensive DNA‐based PCR‐SSP method was established for Di genotyping. The typing results can be visualized on a single photograph within 3 hours, making this reliable method suitable for large‐scale typing of potential blood donors without serologic backup.
Seven‐day storage of apheresis platelets: report of an in vitro study
Authors: Dumont Larry J. , VandenBroeke Tania
Published: March 2003

BACKGROUND : The objective of this study was to determine the allowable platelet content limits for apheresis platelets stored for 7 days in a platelet storage bag (COBE ELP, Gambro BCT).

METHODS : Apheresis platelets under controlled concentration and volume per bag were stored in plasma up to 8 days at 22°C with horizontal agitation. Routinely evaluated in vitro platelet parameters were followed. Oxygen consumption was directly measured with a Clark‐type electrode. All components were cultured in aerobic medium on Day 7.

RESULTS
: Twenty‐four components were evaluated in storage configurations (median [range], 340 [110‐402] mL, 1.32 [0.99‐2.45]× 10
6
platelets/μL, and 4.8 [1.4‐5.9]× 10
11
platelets/bag). No bacterial contamination was detected. One component had a pH value at 22°C of below 6.0 before Day 5 with attendant loss of all other in vitro function measures. The pH value at 22°C was maintained above 6.2 for the remaining 23 components. A pH value of greater than 7.4 was observed at some point in storage for 13 of 23 units, although platelet function or activation was not adversely affected. Aerobic metabolic function was maintained over 7 days with O
2
consumption of 321 μmol per hour per 10
12 platelets on Day 7.

CONCLUSION
: Although a continuing decline of platelet in vitro characteristics can be observed for storage beyond 5 days, apheresis platelets in plasma stored 100 to 400 mL per bag, 1.0 × 10
6
to 2.5 × 10
6
platelets per μL, and a maximum of 5.1 × 10
11 platelets per bag maintained in vitro platelet characteristics over 7 days of storage.
TRALI: correlation of antigen‐antibody and monocyte activation in donor‐recipient pairs
Authors: Kopko Patricia M. , Paglieroni Teresa G. , Popovsky Mark A. , Muto Kathryn N. , MacKenzie Malcolm R. , Holland Paul V.
Published: March 2003

BACKGROUND : TRALI may be a severe reaction associated with transfusion of plasma‐containing blood components. TRALI has usually been associated with antibodies against granulocytes and HLA class I antigens, but more recently with antibodies against HLA class II and monocytes. TRALI cases were investigated to determine correlation between antigen and antibody. Additionally, activation of monocytes by TRALI serums was studied.

STUDY DESIGN AND METHODS : Sixteen cases of TRALI were investigated. All patients were typed for HLA antigens. Implicated donors were screened for HLA antigens and antibodies against granulocytes and monocytes. In 6 cases, recipient monocyte activation was measured in vitro after incubation with TRALI and control serums. In four cases, monocyte activation was measured after incubation of TRALI serums against a panel of monocytes of known HLA antigen type.

RESULTS : In 14 of the 16 cases (87.5%), antigen‐antibody correlation was identified. TRALI monocytes, incubated with implicated TRALI serum (n = 6), expressed significantly greater cytokine and tissue factor (p < 0.05, repeated‐measures ANOVA) than controls. Panel monocytes incubated with TRALI serum showed increased expression of cytokine and/or tissue factor when corresponding antigen was present.

CONCLUSION : In most cases of TRALI, a correlation between antigen and antibody can be identified. Activation of monocytes and their subsequent release of cytokines may play a role in the pathogenesis of TRALI.
Measurement of intracavernosal catecholamine during a prostaglandin E1 test
Authors: Nagao Koichi , Miura Kazukiyo , Ishii Nobuhisa , Shirai Masafumi
Published: March 2003

Background : Erectile dysfunction (ED) can be a cause of male infertility among young men. The prostaglandin E1 (PGE1) intracavernous injection test, an erectile function test, is known as an objective method of examining the penile vasculature system. However, some ED patients fail to sufficiently maintain an erection because of the stress load upon them during the test. Thus, we measured changes in catecholamine levels by stress loading when we performed dynamic infusion cavernosometry (DIC).

Methods
: Among the 221 ED patients undergoing a PGE1 test, 10 were selected as the subjects for the present study. These 10 patients were 25–56 years of age, with a mean of 32.8 years, whose maximal penile rigidity was normal, but in whom penile erection could not be maintained sufficiently in the PGE1 test. Stress loading via vibratory sensory measurement during the PGE1 test suppressed penile rigidity strongly. Catecholamine levels were measured by using blood samples obtained from the corpus cavernosum and the cubital vein when erection was suppressed by stress loading, pain caused by needle insertion, and also when erection recovered from the suppression. Dynamic infusion cavernosometry was performed after each blood sampling time, to check corporeal veno‐occlusive function and cavernosal arterial flow.

Results
: Penile norepinephrine levels were 0.20 ± 0.06 ng/mL during a suppressed erection and 0.15 ± 0.03 ng/mL during an erection, showing a significantly higher level (Student’s t‐test, P = 0.0309) during suppressed erection. The results of the DIC measurement indicated a normal corporeal veno‐occlusive function and cavernosal artery in all cases.

Conclusion
: The results of the present study revealed that corporeal veno‐occlusive function and cavernosal arterial flow are normal in men who have normal maximum penile rigidity, but cannot sufficiently maintain the erection in the PGE1 test, suggesting the involvement of stress‐induced penile norepinephrine in the suppression of erection maintenance. (Reprod Med Biol 2002; 1: 11–15)