bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Post‐Transfusion Graft‐versus‐Host Disease with Nafmostat Mesilate, a Serine Protease Inhibitor
Authors: Ryo Ryukichi , Saigo Katsuyasu , Hashimoto Makoto , Kohsaki Masatoshi , Yasufuku Masao , Watanabe Norihisa , Okada Masayoshi , Tadokoro Kenji , Juji Takeo
Published: December 2004

Background:
Cytotoxic T lymphocytes from donors are thought to injure the target organs in post‐transfusion graft‐versus‐host disease (PT‐GVHD) through perforingranzyme‐ and Fas‐dependent cell killings. The protease involved is a serine protease, and nafmostat mesilate (NM), a serine protease inhibitor, has been found to inhibit the in vitro allocytotoxicity of the T cell clone established from a patient with PT‐GVHD, thus suggesting the usefulness of NM for treatment of PT‐GVHD. Case Report: A 47‐year‐old male with esophageal cancer, who received 3 units of packed red cells and 20 units of platelet concentrates from 5 unrelated donors, was diagnosed as having PT‐GVHD on the basis of typical clinical features, HLA typing of the patient and the responsible donor, and a mixed chimera of CD8+ lymphocytes on microsatellite DNA polymorphism analysis. NM was administered to inhibit the activity of the serine proteases, thought to be granzymes; a liver dysfunction and thrombocytopenia with leukocytopenia simultaneously improved. Subsequently, a high‐dose methylprednisolone pulse therapy and monoclonal anti‐CD3 were administered to reduce the donor's proliferating lymphocytes, which resulted in lymphopenia accompanied by elimination of the donor's lymphocytes and normalization of the CD4/CD8 ratio. However, recurrence of the proliferation of the responsible donor's lymphocytes developed after cessation of NM administration, probably because of excessive immunosuppression caused by steroids and the monoclonal anti‐CD3. Conclusion: This case indicates that administration of a serine protease inhibitor may improve PT‐GVHD symptoms by inhibiting cytotoxic T‐cell‐mediated killing of target cells in fatal PT‐GVHD. Steroids and monoclonal anti‐CD3 were probably responsible for the transient clinical improvements. More studies are required, however, on mechanisms to eliminate the donor's lymphocytes.
Clinical Evaluation of Repeat Apheresis Donors in Japan
Authors: Furuta Motomu , Shimizu Tetsuo , Mizuno Shinichi , Kamiya Tadashi , Ozawa Kazuo , Nakase Toshie , Tadokoro Kenji , Takenaka Michiko , Ohkawa Tsutomu , Yokoyama Shigeki , Ogawa Yoshiaki , Kiyokawa Hiroyuki , Shimizu Masaru , Sekine Naoki , Yoshimura Isao
Published: December 2004

AbstractBackground and Objectives: To ascertain the safety of repeat apheresis donation, hematological and biochemical tests were performed on 511 donors with a donation rate of over 6 times per year for a period of 12–19 months. Materials and Methods: Repeat donors who had apheresis more than 6 times in the previous year were chosen. Data for the repeat donors at the start of the experiments were compared with those at the end of the end of the study. Blood samples were taken prior to donation. Serum protein, albumin, immunoglobulin G, A, and M, serum ferritin levels were determined by biochemical tests. Results: When compared to prospective donors of 400 ml, WBC, lymphocytes, and serum ferritin levels were lower in a roughly frequency‐dependent manner in female and male donor groups at the beginning of the study. All the data for the male group remained almost constant with increasing frequency of apheresis donation. However, in the female group, ferritin levels significantly decreased with over 21 donations. Conclusions: The present data showed that the serum ferritin level of the female donors decreased the most with increasing frequency of apheresis donation. The cumulative RBC left in the collecting chamber and for the laboratory test is discussed in relation to a possible cause of iron deficiency in frequent apheresis donors.
Press Review
Authors: Hébert P.C. , Wells G. , Blajchman M.A.
Published: December 2004

A Multicenter, Randomized, Controlled Clinical Trial of Transfusion
Use of Recombinant, Activated Factor VII in the Treatment of Congenital Factor VII Deficiencies
Authors: Mariani G. , Testa M.G. , Di Paolantonio T. , Bech R. Molskov , Hedner U. ,   ad hoc Study Group
Published: December 2004

Background and Objectives:
Factor VII (FVII) deficiency is a rare coagulation disorder, historically treated with prothrombin complex concentrates or plasma‐derived FVII concentrates. We treated such patients (n=17) with a recombinant, activated FVII preparation. Materials and Methods: Twenty‐seven spontaneous bleeding episodes were treated and 7 major and 13 minor surgical interventions were carried out. The dosages employed ranged from 8.08 to 70.5 μg/kg body weight. Results: A mean dose between 22 and 26 μg/kg was sufficient to normalise the prothrombin time. Fifteen haemarthroses were treated with single doses and results were excellent in 13 cases. In 5/6 bleeding episodes of other types, the treatment gave either excellent or at least effective results. Haemostasis was secured in the 7 major and 13 minor surgical interventions. One patient developed antibodies 4–5 weeks after an extremely high dose. Otherwise, there were no side effects and no evidence of a thrombotic tendency. Conclusion: This recombinant concentrate is efficacious in FVII‐deficient patients. It is safe since any risk of transmission of blood‐borne viruses is eliminated.
Micro‐Column Affinity Test and Gel Test: Comparative Study of Two Techniques for Red Cell Antibody Screening
Authors: Eichler H. , Böhler A. , Hastka J. , Richter E. , Kerowgan M. , Goldmann S.F.
Published: December 2004

Background and Objectives:
We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro‐column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions. Materials and Methods: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low‐ionic‐strength‐saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. Results: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti‐Fya and anti‐Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti‐Lua showed a significantly higher score with the gel test. Conclusion: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that lgM antibodies are better detected by the gel test.
Determination of Granulocyte‐Specific Antigens on Neutrophil Fcγ Receptor IIIb by PCR‐Preferential Homoduplex Formation Assay, and Gene Frequencies in the Japanese Population
Authors: Fujiwara Koki , Watanabe Yoshihisa , Mitsunaga Shigeki , Oka Takanori , Yamane Akio , Akaza Tatsuya , Tadokoro Kenji , Tokunaga Katsushi , Shibata Yoichi , Juji Takeo
Published: December 2004

Background and Objectives:
Granulocyte‐specific antigens play an important role in provoking immune neutropenia and transfusion reactions. We developed a new DNA‐typing method, PCR‐preferential homoduplex formation assay (PHFA), to determine granulocyte‐specific antigens on the neutrophil Fcγ receptor IIIb (FcγRIIIb, CD 16b), namely, the NA1, NA2, and SH antigens and their gene frequencies in the Japanese population. Materials and Methods: Four hundred unrelated healthy Japanese blood donors were typed using PCR‐PHFA. To confirm the accuracy of the results of FcγRIIIB genotyping using PCR‐PHFA, PCR‐sequence‐specific primer (SSP) typing and PCR‐restriction fragment length polymorphism (RFLP) typing were carried out in another 20 samples for comparison. Results: The results of PCR‐PHFA typing agreed well with other methods. The frequencies of the FcγRIIIB alleles were 62.2, 37.8, 0 and 0% for NA1, NA2, SH, and ‘NA‐null’, respectively. Conclusion: The PCR‐PHFA method can be semi‐automated easily with computer‐based assignment and is suitable for typing both small and large numbers of samples. In the Japanese population, the frequency of NA1 is about double that in Caucasians (32.5%), and the SH allele is rare.
Allogeneic Red Blood Cell Transfusion Is an Independent Risk Factor for the Development of Postoperative Bacterial Infection
Authors: Chang Hong , Hall Gregory A. , Geerts William H. , Greenwood Celia , McLeod Robin S. , Sher Graham D.
Published: December 2004

Background and Objectives:
Allogeneic red blood cell transfusions may exert immunomodulatory effects in recipients including an increased rate of postoperative bacterial infection. It is controversial whether allogeneic transfusion is an independent predictor for the development of postoperative bacterial infection. Methods: We analysed a prospectively collected database of 1,349 patients undergoing colorectal surgery in 11 centres across Canada. The primary outcome was the development of either a postoperative wound infection or intraabdominal sepsis in transfused and nontransfused patients. The effect of allogeneic transfusion on postoperative infection was evaluated with adjustment for all the confounding factors in a multiple regression analysis. Results: The 282 patients who received a total of 832 allogeneic units had a significantly higher frequency of wound infections and intra‐abdominal sepsis than the patients who were not transfused (25.9 vs. 14.2%, p = 0.001). A significant dose‐response relationship between transfusion and infectior, rate was demonstrated. Multiple regression analysis identified allogeneic transfusion as a statistically significant independent predictor for postoperative bacterial infection (OR 1.18, 95% CI 1.05–1.33, p = 0.007). Other independent predictors were anastomotic leak, repeat operation, patient age and preoperative haemoglobin level. The mortality rate was also significantly higher in the transfused group. Conclusion: These data support the hypothesis that allogeneic red cell transfusion is an independent risk factor for the development of postoperative bacterial infection in patients undergoing colorectal surgery. This association provides further reason to minimise exposure to allogeneic transfusions in the perioperative setting.
The MNS Blood Group Antigens, Vr (MNS12) and Mta (MNS14), Each Arise from an Amino Acid Substitution on Glycophorin A
Authors: Storry Jill R. , Coghlan Gail , Poole Joyce , Figueroa Dolores , Reid Marion E.
Published: December 2004

Background and Objectives:
The antigens, Vr (MNS12) and Mta (MNS14), are low‐incidence antigens of the MNS blood group system. The Vr antigen has been found only on the red blood cells (RBCs) of persons of Dutch ancestry whereas the Mta antigen has been found on the RBCs of persons from a wide geographic distribution. The objective of this study was to determine the molecular basis of Vr and Mta. Materials and Methods: Following RT‐PCR amplification of total RNA isolated from one Vr+ person (G488) and one Mt(a+) person (GH), the genes encoding glycophorin A (GYPA) and glycophorin B (GYPB) were cloned and sequenced. To confirm the point mutation observed in the cDNA from G488 (Vr+), GYPA exon 3 was cloned and sequenced from the genomic DNA of G488 and a second unrelated Vr+ person (MU). A restriction fragment length polymorphism (RFLP) assay was used to analyze genomic DNA from 11 Mt(a+) persons (10 unrelated) following PCR amplification of GYPA exon 3. Results: The coding sequence of GYPB was normal in both G488 (Vr+) and GH (Mt(a+)). Sequencing data from GYPA clones derived from G488 showed ot full length GYPA sequences: A normal GYPA M allele and a GYPA M allele with a point mutation 197C→A. Sequencing of GYPA exon 3 from G488 and MU confirmed the point mutation. Sequencing data drom GYPA clones derived from GH showed two full length GYPA sequences: a normal GYPA M allele and a GYPA N allele with a point mutation 230C→T.RFLP analysis based on the point mutation showed that DNA from 11 Mt(a+) samples were heterozygous for the point mutation. Conclusion: The Vr antigen arises from a point mutation 197C→A on GYPA which is predicted to change serine at position 47 to tyrosine. This change introduces a new α‐chymotrypsin cleavage site. The Mta antigen arises from a point mutation 230C→T which is predicted to change threonine at position 58 to isoleucine.
Studies on the Removal of Abnormal Prion Protein by Processes Used in the Manufacture of Human Plasma Products
Authors: Foster Peter R. , Welch Anne G. , McLean Carol , Griffin Brenda D. , Hardy John C. , Bartley Anthony , MacDonald Shirley , Bailey Andrew C.
Published: December 2004

Background and Objectives:
To identify if any process steps used in plasma fractionation may have a capability of removing agents of human transmissible spongiform encephalopathy (TSE). Materials and Methods: Sixteen fractionation steps were investigated separately by adding a preparation of hamster adapted scrapie 263K to the starting material at each process step and determining the distribution into resultant fractions of protease‐K‐resistant (abnormal) prion protein by Western blot analysis. Results: A number of process operations were found to remove abnormal prion protein to the limit of detection of the assay. These were cold ethanol precipitation of fraction IV (log reduction, LR, ≥3.0) and a depth filtration (LR ≥4.9) in the albumin process; cold ethanol fraction I+III precipitation (LR ≥3.7) and a depth filtration (LR ≥2.8) in the immunoglobulin processes and adsorption with DEAE‐Toyopearl 650M ion exchanger (LR ≥3.5) in the fibrinogen process. In addition, a substantial degree of removal of abnormal prion protein was observed across DEAE‐Toyopearl 650M ion exchange (LR = 3.1) used in the preparation of factor‐VIII concentrate; DEAE‐cellulose ion exchange (LR = 3.0) and DEAE‐sepharose ion exchange (LR = 3.0) used in the preparation of factor‐IX concentrates and S‐sepharose ion exchange (LR = 2.9) used in the preparation of thrombin. Conclusions: Plasma fractionation processes used in the manufacture of albumin, immunoglobulins, factor‐VIII concentrate, factor‐IX concentrates, fibrinogen and thrombin all contain steps which may be capable of removing causative agents of human TSEs.
Pretransfusion Testing without Serologic Crossmatch: Approaches to Ensure Patient Safety
Authors: Kuriyan Mercy , Fox Ellen
Published: December 2004

Background and Objectives:
A crossmatch is usual in pretransfusion testing, but we eliminated it at our tertiary care university hospital. In place of the crossmatch, we have introduced a system where, if an antibody screen is negative, two technologists confirm the ABO of the same patient sample, and we release blood of the patient's type without a serologic or electronic crossmatch. Materials and Methods: In 65,628 samples received for pretransfusion testing, we studied the effect of common errors that occur from the time of receipt of a sample until release of the blood unit, in order to decide if elimination of the serologic crossmatch would affect patient safety. All sample labels were inspected for acceptability. Results: A total of 1,082 (1.64%) samples were rejected for mislabeling. Discordance in patient ABO typing results between two technologists was 0.43%. Such discrepancies were resolved before the release of blood units. No donor unit mislabeling or unit release errors were detected. Conclusion: Elimination of the crossmatch for red cell antibody–negative patients is safe provided a system of error detection is used. This enhances patient care through the quicker release of blood, increased laboratory efficiency and decreased costs.
Polymorphisms of RhDVa and a New RhDVa–Like Variant Found in Japanese Individuals
Authors: Hyodo Hironobu , Ishikawa Yoshihide , Kashiwase Koichi , Ogawa Atsuko , Watanabe Yoshihisa , Tsuneyama Hatsue , Toyoda Chizu , Uchikawa Makoto , Akaza Tatsuya , Fujii Tomoyuki , Kozuma Shiro , Taketani Yuji , Juji Takeo
Published: December 2004

Background and Objectives:
Red cell type RhDVa lacks epD1 and 5 and is encoded by hybrid RHD–CE(5)–D alleles. We analyzed RhDVa and RhDVa–like samples in Japanese blood donors. Materials and Methods: Ten RhDVa samples lacked epD1 and 5 and 3 RhDVa–like variants also lacked, epD2 and a part of 6/7. We identified the full–length nucleotide sequences of the complementary DNA (cDNA) synthesized from 4 samples: 3 of type DVa and the 4th a DVa–like variant. Results: Although their sequences differed from each other, all the substitutions were exclusively in exon 5. Three DVa samples had hybrid RHD–CE(5)–D alleles, but the DVa–like variant had a unique nucleotide substitution with a single amino acid change, E233K. Exon 5 of the genomic DNA from all 13 samples was analyzed by sequencing. No other sequences were identified. Conclusion: All RhDVa and RhDVa–like variants had the substitution for E233. E233 seems to be a determinant of epD1 and 5. A new category of RhD variant, DYO, was identified.
Splenic Lymphoma Presenting as Warm Autoimmune Hemolytic Anemia Associated with Pure Red Cell Aplasia
Authors: Zeidman A. , Fradin Z. , Barac Y. , Bendayan D. , Mittelman M. , Orlin J.
Published: December 2004

Background and Objective:
Warm autoimmune hemolytic anemia (AIHA) is a condition in which peripheral red blood cell (RBC) destruction is induced by the presence of an autoantibody. Pure red cell aplasia (PRCA) represents an isolated process of decreased erythropoiesis. The combination of both is quite rare, with a very poor prognosis. We describe a patient with isolated splenic lymphoma whose presentation was a combination of AIHA and PRCA. The patient was resistant to all treatment. Materials and Methods: Erythroid colony assays were performed, in order to compare the effect of the patient's serum on colonies with that of a normal control. Results: The patient's serum significantly suppressed normal erythroid colony growth. A red cell eluate revealed the presence of a warm autoantibody. Conclusions: The patient's serum contained warm autoantibody responsible for peripheral RBC destruction and a humoral factor, perhaps the warm autoantibody, which suppressed bone marrow erythropoiesis. Establishing an early diagnosis, and treatment of the underlying disease might result in a better prognosis.
Preparation of Leukocyte‐Poor Platelet Concentrates via a Short, Hard Spin of a Pool of Buffy Coats
Authors: van Delden C.J. , Faber R.D. , de Wit H.J.C. , Sibinga C. Th. Smit
Published: December 2004

Background and Objectives:
A new method for the preparation of leukocyte‐poor platelet concentrates was developed, based on a short, hard spin of a pool of 5 buffy coats (BCs) combined with automated collection of the platelets. Materials and Methods: The characteristics of platelet concentrates (PCs) were studied as a function of the total g force applied to a pool of 5 BCs. Pools of BCs were centrifuged for 1 min with a total g force ranging from about 3,300 to 5,000 gmin (n = 7–9 per applied g force). Deceleration took place without the means of a brake. The total centrifugation time was about 11 min. The platelet‐rich plasma (PRP) fraction above the cell layer was separated by an automated component preparation device. Results: A short, hard spin with a total g force of between 3,400 and 4,600 gmin resulted in PCs that contained on average more than 290×109 platelets and less than 5×106 leukocytes without the use of a leukocyte filter, provided that the transfer of PRP was electronically checked and terminated. The cell concentrations in the PCs are a function of the total g force. Both the platelet and leukocyte levels in the concentrate decreased with an increase in the total g force applied to the pool. Conclusion: The preparation of PCs via a short hard, spin of BCs, combined with automated collection of the PRP molecular may be an alternative method for the preparation of leukocyte‐poor PCs.
Calibration of HCV Working Reagents for NAT Assays against the HCV International Standard
Authors: Saldanha J. , Heath A. , Lelie N. , Pisani G. , Nübling M. , Yu M. ,   Collaborative Study Group
Published: December 2004

Background and Objectives:
Five HCV RNA reference reagents, the Paul Ehrlich Institut (PEI) reference 75, the National Institute for Biological Standards and Control (NIBSC) reagent 96/586, the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB) Pelispy HCV RNA run control S2001, the Istituto Superiore di Sanita (ISS) reagent 0498 and the CBER panel member No. 1, were calibrated against the WHO International Standard, 96/790. Materials and Methods: The reference materials were calibrated in a collaborative study organised by NIBSC. Nineteen laboratories, using a range of qualitative and quantitative assays returned results. Results: The concentrations of the reagents were: 25,000 IU/ml for the PEI material, 710 IU/ml for the NIBSC material 96/586, 1,000 IU/ml for the CLB material, 1,700 IU/ml for the ISS material 0498 and 250 IU/ml for the CBER panel member No. 1. Conclusions: The calibration of these five reference reagents for HCV RNA nucleic acid amplification technology (NAT) assays enables them to be used for standardisation and validation of assays. Such calibrants are essential for meeting the requirements of the European Medicinal Evaluation Agency (EMEA) for the testing of plasma pools and donations for HCV RNA for the release of blood products and the PEI requirements for the release testing of erythrocyte and thrombocyte concentrates.
Natural History of Haemoglobin Levels in Female Blood Donors with Haemoglobin Levels of 120–124 g/l at Presentation
Authors: Gillon J. , Wye A. , Walker B. , Phillips P.
Published: December 2004

Background and Objectives:
In order to assess the effects of proposed changes to UK guidelines, female blood donors with venous haemoglobin (Hb) levels of 120–124 g/l were followed up over a period of 18 months in order to assess frequency of attendance and ability to donate in relation to changes in Hb and red cell indices, and to determine the effects of inter‐donation interval and iron supplementation on outcomes. Materials and Methods: Venous samples were analysed using the Haemocue haemoglobinometer at each attendance, and a donation was taken if the Hb was 120 g/l or greater. Full blood counts were done on each sample using the Coulter Model T890. A postal questionnaire was used to elicit additional information, including use of iron supplements. Results: The 392 donors enrolled in the study gave 655 subsequent attendances, resulting in 468 donations (1.5 donations/donor/year). Donors who subsequently had Hb < 120 g/l had a tendency to lower initial mean corpuscular volume (MCV), but MCV could not be used as an indicator of future performance. 23% of donors reported use of iron supplements, but those donating 3 times or more were no more likely to use iron supplementation (20%). No statistical relationship was found between inter‐donation interval and ability to donate at subsequent attendances. Conclusions: Female donors with borderline Hb levels proved to be highly committed, with a donation rate of 1.5/donor/year during the period of follow‐up. MCV is not a useful predictor of ability to donate in the future. This subset of the donor population shows wide variation in Hb level over time, and this is largely independent of iron supplementation or inter‐donation interval.
Evaluation of Leukocyte‐Depleted Platelet Concentrates Obtained by In‐Line Filtration
Authors: Ferrer F. , Rivera J. , Corral J. , González‐Conejero R. , Vicente V.
Published: December 2004

Background and Objectives:
The use of leukocyte‐depleted platelet concentrates (PCs) is justified, yet questions remain regarding their properties. We have assessed the in vitro quality of WBC‐reduced PCs obtained by using a new in‐line filter. Materials and Methods: Twenty blood units were randomized for standard component preparation, or for processing including in‐line leukodepletion of platelet‐rich plasma using an ATS™ PL Pall filter. The resultant PCs were compared during storage for several in vitro platelet quality parameters, content of cytokines and anaphylatoxins, and coagulation markers. Results: In‐line filtration of platelet‐rich plasma through ATS PL was highly efficient rendering PCs with 99.93% less white blood cells (WBCs) than standard PCs. During storage for up to 9 days, leukocyte‐depleted units displayed platelet content, biochemical parameters, and platelet surface expression of glycoproteins lbα, llb/lla, CD62 and CD63, similar to those of standard units. However, in‐line leukocyte‐reduced PCs displayed no significant accumulation of IL‐6 and IL‐8 during storage in contrast to standard units. Moreover, while storage promoted complement activation with C3a and C4a liberation in both WBC‐reduced PCs and standard units, the concentration of these anaphylatoxins after the 9‐day storage period was higher in the former group. Finally, all units, standard and leukocytereduced, displayed a similar storage‐promoted rise in TGF‐β1, and a mild prolongation of activated partial thromboplastin and prothrombin times. Conclusion: Inline filtration during component preparation appears as an easy and effective procedure for obtaining prestorage leukocyte‐depleted PCs. During subsequent blood bank storage, these WBC‐reduced PCs display, in comparison with standard PCs, normal in vitro platelet properties, decreased accumulation of cytokines IL‐6 and IL‐8, and increased complement activation.
Erratum
Authors: Caspari G. , Barbara J.A.J.
Published: December 2004

Anti‐HEV in Blood Donors: What Is Test and What is Virus?
Improved Sensitivity in the Measurement of Residual Leukocytes in Platelet Products Using an Automated Leukocyte Counter
Authors: Nagahasi H. , Yabusaki K. , Matsui H. , Hirono T. , Yokokawa N. , Satake M. , Tadokoro K. , Juji T.
Published: December 2004

Background and Objectives
: Leukocytes remaining in platelet concentrates may be responsible for side effects caused by platelet transfusion. A simple method of high sensitivity for determining trace numbers of leukocytes is currently needed. Materials and Methods: An automated leukocyte counter, the LD‐1000, the principle of which is a combination of spining down on the observation field of stained nuclei derived from a 100 μl neat sample and image digitization with a charge coupled device camera, was newly developed and tested for sensitivity and reproducibility. Results: While the theoretical lower limit of detection was 0.01 cells/μl, the limit of detection of the LD‐1000 was verified to be 0.2 cells/μl. A good correlation (r = 0.86) was observed between the results obtained using the Nageotte method and the LD‐1000. Repeated measurements also confirmed satisfactory reproducibility. Conclusions: The instrument provides a new method of enumerating residual leukocytes in platelet products with better sensitivity and easier procedure compared to the Nageotte method and will be useful for quality assurance purposes at blood centers.
Hepatitis C Virus: Routes of Infection and Genotypes in a Cohort of Anti‐HCV‐Positive French Blood Donors
Authors: Elghouzzi M.H. , Bouchardeau F. , Pillonel J. , Boiret E. , Tirtaine C. , Barlet V. , Moncharmont P. , Maisonneuve P. , du Puy‐Montbrun M.C. , Lyon‐Caen D. , Couroucé A.M.
Published: December 2004

Abstract
Background:
We evaluated and analysed risk factors of HCV‐infected blood donors according to HCV genotypes in order to improve the transfusion policy and safety of blood supply. Materials and Methods: HCV‐RNA was analysed in sera from 518 anti‐HCV‐positive blood donors, who were invited to medical consultation and interview as to risk factors by means of an extensive questionnaire. HCV genotyping was done on all samples positive for HCV‐RNA. Results: Of the 518 sera, 399 (77%) were HCV‐RNA positive, and 394 of 399 HCV genotypes were identified. Major genotypes were 1b (34.3%), 3a (24%), 1a (19.5%) and 2 (11.4%). Of the donors, 289 (55.8%) were interviewed regarding their risk behaviour: 27% were former intravenous drug users (IVDUs), 26% had been transfused, 8% had a history of invasive diagnostic procedures, and 13% a history of surgery. Among the 224 interviewed donors, genotypes 1a and 3a were mainly associated with IVDU (51 and 45% respectively) and genotype 1b, with transfusion and nosocomial infections (40 and 25%, respectively). Conclusion: In this population of anti‐HCV ‐positive blood donors, nosocomial infection may be a route of HCV spread, but the main risk factor remains IVDU, particularly in young men. The transfusion policy will improve if predonation interviews of such young men are done with a specific and sensitive questionnaire.
Effects of Leucocyte Depletion on Rheologic Properties of Human CPDA‐1 Blood
Authors: Walter R. , Brand B. , Mark M. , Schnyder L. , Stifanic M. , Reinhart W. H.
Published: December 2004

Abstract
Background and Objectives:
Leucocyte depletion improves the quality of stored blood units. We have studied its role on blood viscosity. Materials and Methods: Viscosity of CPDA‐1 blood units was measured in a Couette viscometer at shear rates of 94.5 and 0.1 s‐1 prior to and following filtration with the Leukotrap® A1 system on day 0 and after 21 days at +4°C. Results: On day 0, high but not low shear viscosity was significantly decreased. The red blood cell morphology was unaffected. On day 21, blood viscosity was increased similarly for unfiltered and filtered samples at both shear rates. The echinocytosis observed after storage correlated with the increase in viscosity. Conclusion: Leucocyte depletion is associated with a decrease in high shear viscosity. This effect is, however, completely lost after 21 days.
Influence of Methylene Blue Photoinactivation Treatment on Coagulation Factors from Fresh Frozen Plasma, Cryoprecipitates and Cryosupernatants
Authors: Aznar J.A. , Bonanad S. , Montoro J.M. , Hurtado C. , Cid A.R. , Soler M.A. , De Miguel A.
Published: December 2004

Abstract
Objective:
To study the influence of virus photoinactivation with methylene blue (MB) on the coagulation factors of fresh frozen plasma (FFP) and the corresponding cryoprecipitates and cryosupernatants derived from it. Materials and Methods: The photoinactivation procedure of the German Red Cross (Springe) was applied using Biomat (Grifols, Spain). Twenty isogroup pools of three plasma units were made from 60 U of FFP. The pools were split into three bags. One of them was photoinactivated, and pre‐ and postinactivation samples (MB‐plasma) were obtained. The second bag was treated in the same way, followed by the preparation of MB‐cryoprecipitate and MB‐cryosupernatant. The third bag was not photoinactivated, and was processed in the same way to obtain control cryoprecipitate and cryosupernatant. The prothrombin time and activated partial thromboplastin time were analysed, as well as fibrinogen, factors (F) II, V, VII, VIII, IX, XI and XIII, antithrombin III, von Willebrand (vW) F:RCo, vWF:Ag and the multimeric structure of vWF. Results: In plasma, the proteins most sensitive to photoinactivation were fibrinogen, FV, FVIII, FIX and FXI (24, 32, 28, 23 and 27% loss, respectively). In the MB‐cryoprecipitate, the losses were higher for FVIII (23%), moderate for fibrinogen, FXIII and vWF:RCo (18, 14 and 13%, respectively) and minimal (only 3%) for vWF:Ag. In MB‐cryosupernatants, the losses were higher for FV (26%) and moderate for fibrinogen (16%), FIX (18%) and FXI (19%), as well as for FII and FXIII (15%). The multimeric structure of vWF was not modified in MB‐plasma or in MB‐cryoprecipitates. The supernatants (both MB treated as well as controls) showed an absence of multimers of very high and high molecular weight. Conclusions: The quantitative and qualitative conservation of coagulation factors achieved in MB‐plasma‐derived products suggest that they are useful for the global replacement of coagulation factors and for deficiencies in FV and FXI. In countries lacking the economic resources to obtain virally inactivated concentrates, MB‐cryoprecipitates could be useful in von Willebrand's disease and fibrinogen and FXIII deficiencies. MB‐cryosupernatants could be employed in thrombotic thrombocytopenic purpura, in the correction of total or partial deficiencies of prothrombin complex factors and in specific deficiencies of FV and FXI.
Functional Characteristics of S‐59 Photochemically Treated Platelet Concentrates Derived from Buffy Coats
Authors: van Rhenen D.J. , Vermeij J. , Mayaudon V. , Hind C. , Lin L. , Corash L.
Published: December 2004

Background:
A photochemical treatment (PCT) process for inactivation of infectious pathogens and leukocytes has been developed and evaluated using single‐donor platelet concentrates. This study assessed the application of PCT to platelets prepared from pooled buffy coats. In this study, in vitro functional characteristics of PCT platelets were compared to control platelets prepared from pooled buffy coats using the approved platelet‐additive solution T‐Sol®. Platelets in platelet PAS III additive solution without PCT were evaluated as well. PCT also included the use of a psoralen (S‐59) reduction device (SRD). Materials and Methods: Four types of platelet concentrates were compared: (1) platelet concentrate in plasma/T‐Sol; (2) platelet concentrate in plasma/PAS III; (3) platelet concentrate in plasma/PAS III, PCT, 9 h SRD and (4) platelet concentrate in plasma/PAS III, PCT, 16 h SRD. PCT occurred on the day after whole‐blood collection. In vitro assay parameters included: pH, pO2, pCO2, HCO–3, platelet count, mean platelet volume, plasma glucose, plasma lactate, total ATP, expression of p‐selectin, hypotonic shock response and electron microscopy. Results: The results indicate that PCT is compatible with platelet concentrates prepared from pooled buffy coats for up to 7 days of storage. Conclusion: The PCT process resulted in acceptable in vitro platelet functional characteristics and is currently in clinical trials to evaluate the haemostatic efficacy of PCT platelets in thrombocytopenic patients requiring multiple platelet transfusions.
Distinctive Swann Blood Group Genotypes: Molecular Investigations
Authors: Zelinski Teresa , Rusnak Alison , McManus Kirk , Coghlan Gail
Published: December 2004

Background and Objectives:
Phenotypically, Sw(a+) erythrocytes have been classified as either 700:4,41 or 700:4,–41. Since anti‐700.4, in particular, and sometimes anti‐700.41 are contained in reagents defining other low‐incidence antigens that are members of the Diego blood group system, we undertook the current investigation in an attempt to establish whether or not Swann antigens are also Diego system members. Materials and Methods: DNA from the members of three unrelated kindreds whose red cells type as Sw(a+) was isolated and analyzed for variation in SLC4A1 (solute carrier family, anion exchanger member 1 gene) by single‐strand conformational polymorphism (SSCP) and DNA sequence analyses. Results: Polymerase chain reaction‐amplified exon 16 SLC4A1 products from the DNA of all Sw(a+) individuals displayed a mobility shift by SSCP. A similar mobility shift was not observed in the DNA from Sw(a–) family members or in the amplified DNA from control individuals. DNA sequencing revealed different mutations, CGG→CAG and CGG→TGG, that result in Arg646Gln and Arg646Trp substitutions in erythroid protein band 3, respectively. Conclusion: Through genotypic analyses, we have characterized two point mutations related to the Swann blood group. The possible relationship between the resultant amino acid substitutions and the expression of Swann antigens has been discussed.
White Blood Cell Subsets in Buffy Coat‐Derived Platelet Concentrates: The Effect of Pre‐ and Poststorage Filtration
Authors: Ledent Elisabeth , Semple John W. , Berlin Gösta
Published: December 2004

Background and Objectives:
Our objective was to study the effect of storage time on the filtration of platelet concentrates (PCs). We compared the total number of white blood cells (WBC), as well as the distribution of WBC subsets, in units filtered before and after storage. Materials and Methods: Buffy coat‐derived PCs were filtered either fresh or after 5 days of storage, and total WBC were enumerated by flow cytometry. WBC subsets were analyzed by flow cytometry with three‐color fluorescence. Results: The total number of white cells before filtration was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. Although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in both fresh and stored units; the percentage of T cells was decreased, whereas the percentage of B cells and monocytes was increased after filtration. Conclusion: Our results suggest that prestorage WBC filtration of platelet concentrates is superior in reducing the absolute numbers of WBC. However, both pre‐ and poststorage WBC filtration significantly affect the proportions of WBC in the final product, decreasing the number of T cells while apparently increasing the proportion of MHC class II‐positive cell populations.
Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes
Authors: Moldenhauer A. , Nociari M. M. , Dias S. , Lalezari P. , Moore M. A. S.
Published: December 2004

Background and Objectives Dendritic cells (DCs) are promising adjuvants for clinical immunotherapy, but they are scantily distributed. Therefore, numerous in vitro methods have been developed to expand these cells while maintaining their normal functions. Current culture systems generally require the use of fetal bovine serum (FBS)‐supplemented media in order to attain DCs with high immunostimulatory activity. However, the presence of exogenous animal proteins sets limits for their use in clinical trials. The purpose of this study was to establish a simple, efficient and FBS‐free method for the generation of human DCs for clinical application.

Materials and Methods We compared monocyte‐derived DCs generated in a standard FBS‐supplemented medium vs. DCs generated in an autologous plasma (AutoPl)‐supplemented medium, with regard to their yield, function and longevity. Peripheral blood monocytes were isolated from buffy coats by two consecutive 2‐h adherence steps in tissue culture flasks. The adherent cells were differentiated into DCs within 2 weeks by adding granulocyte–macrophage colony‐stimulating factor (GM–CSF), interleukin‐4 (IL‐4), c‐kit ligand and tumour necrosis factor‐α (TNF‐α). Every 2–3 days, the cells in suspension were analysed for their immunophenotype and apoptosis rate by flow cytometry. Their function was demonstrated by their allostimulatory and migratory capacity, as well as by their proteolytic activity.

Results We show that more than 30 × 106 DCs can be achieved per unit of buffy coat using either AutoPl‐ or FBS‐supplemented media. The purity of the DCs was 53·4% and 65% (P > 0·05) in AutoPl‐ and FBS‐based medium, respectively. DCs grown in AutoPl media showed a CD80high CD83+ CD86high CD14neg HLA‐DR+ CD1aneg phenotype, while FBS‐generated DCs exhibited a CD80high CD83+ CD86high CD14neg HLA‐DR+ CD1ahigh phenotype. The apoptosis rate in both culture conditions increased from 10% to 25% over 1 week. AutoPl‐generated DCs were shown to be equally strong stimulators for proliferation of allogeneic T lymphocytes as FBS‐generated DCs. In addition, the capacity to migrate in response to macrophage inflammatory protein‐1α (MIP‐1α) and stromal‐cell‐derived factor 1α (SDF‐1α) was similar in both groups, whereas the response to MIP‐3β was reduced in AutoPl‐derived cells. Zymography analysis of supernatants from 5‐day‐old cultures demonstrated that AutoPl‐generated DCs produced higher amounts of matrix metalloproteinases, suggesting that they have an enhanced capability to traffic through peripheral tissues.

Conclusions Our findings indicate that plastic‐adherent peripheral blood cells, when cultured with GM–CSF, IL‐4, c‐kit‐ligand and TNF‐α in autologous human plasma‐supplemented media, are a potent source of functional DCS that may be of value for human therapy.
Red cell transfusion practice following the transfusion requirements in critical care (TRICC) study: prospective observational cohort study in a large UK intensive care unit
Authors: Chohan S. S. , McArdle F. , McClelland D. B. L. , Mackenzie S. J. , Walsh T. S.
Published: December 2004

Background and Objectives The Transfusion Requirements In Critical Care (TRICC) study found that critically ill patients tolerate a restrictive haemoglobin transfusion threshold. We investigated red‐cell transfusion practice since publication of the TRICC study in a large Scottish teaching hospital intensive care unit (ICU).

Materials and Methods We prospectively collected daily data for a 6‐month period on haemoglobin concentrations, red‐cell transfusions and indications for transfusions, throughout ICU stay for all patients who stayed for longer than 24 h in the ICU.

Results A total of 176 patients were studied, who utilized 1237 ICU days. Of these 176 patients, 52% received red‐cell transfusions. A haemoglobin concentration of ≤ 9 g/dl was measured in 55% of patients; this occurred by day 1 and day 2 in 52% and 77% of these cases, respectively. Overall the haemoglobin concentration was ≤ 9 g/dl for 45% of all patient days. Total red‐cell use was 3·1 units per admission (0·47 units per patient day). Only 18% of transfusion episodes were required as a result of haemorrhage. For ‘non‐haemorrhage’ transfusion episodes, the median pretransfusion haemoglobin concentration was 7·8 g/dl (interquartile range: 7·4–8·4 g/dl), and 64% of transfusion episodes were for 2 units.

Conclusions Clinicians in our centre were conservative, in keeping with recent transfusion guidelines, but deviated from the TRICC protocol by transfusing at haemoglobin concentrations of between 7 and 9 g/dl, rather than below 7 g/dl, and by prescribing 2 unit transfusions. Significant numbers of red‐cell units are still used in the critically ill.
Properties of a new intravenous immunoglobulin (IGIV‐C, 10%) produced by virus inactivation with caprylate and column chromatography
Authors: Lebing W. , Remington K. M. , Schreiner C. , Paul H.‐I.
Published: December 2004

Background and Objectives Current manufacture of intravenous immunoglobulin (Gamimune®N) uses four cold‐ethanol precipitation steps and solvent–detergent treatment. Our objective was to design a new manufacturing process to maximize immunoglobulin G (IgG) purity, achieve robust viral safety, preserve all the biological activities of antibody and avoid unnecessary protein loss.

Materials and Methods The new process combines multiple functions in single steps. Caprylate is added to precipitate non‐IgG proteins and to inactivate enveloped viruses. Two successive anion‐exchange columns are used to purify IgG and remove caprylate. The new product, IGIV‐C (Gamunex™, 10%) is formulated with glycine at 100 mg/ml IgG, pH 4·25. Vials are incubated for 21 days at 23–27 °C in a final virus‐inactivation step.

Results Compared with the process for production of Gamimune®N, that for IGIV‐C requires a shorter production time, achieves more robust virus inactivation, increases IGIV yield from plasma, improves physiological IgG subclass distribution (resulting in higher levels of IgG4), and improves purity, with lower levels of IgA (40 µg/ml), IgM (< 2 µg/ml) and albumin (< 20 µg/ml). Antibody binding, opsonization and protective activities are similar.

Conclusions Compared with the current commercial process, the new IGIV‐C manufacturing process produces a more highly purified preparation that contains slightly higher levels of IgG4 and retains antibody activities required for clinical efficacy.
Flow cytometry characterization of white cell‐reduced blood: apoptosis markers and morphology of postfiltration elements
Authors: Tazzari P. L. , Bontadini A. , Fruet F. , Tassi C. , Ricci F. , Manfroi S. , Conte R.
Published: December 2004

Background and Objectives Apoptosis affects white blood cells (WBCs) contained in packed red blood cell (RBC) units. This phenomenon was recently described also in residual WBCs after filtration. The aim of this study was to better characterize the residual WBCs postfiltration by using apoptosis markers and morphology.

Materials and Methods Immunofluorescence, flow cytometry and cell‐sorting techniques were utilized.

Results Residual leucocytes of leucodepleted packed RBC units showed increasing values of apoptotic elements in a time‐course experiment. We also demonstrated that these elements are positive for APO 2·7 monoclonal antibody (mAb), poly ADP‐ribose polymerase (PARP) cleavage and fluorescein isothiocyanate (FITC)‐conjugated N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (Z‐VAD‐FMK), all of which indicate that programmed death is a feature of this population of cells. Phenotypic analysis with CD45 side‐scatter gating demonstrated also that CD15 and CD16 granulocyte‐associated antigens are present on a subset of postfiltration leucocytes. Moreover, the expression of human leucocyte antigen (HLA) class I antigens is maintained. Sorting of CD45‐positive cells and morphological analysis of these samples confirmed that leucocytes in postfiltration units have morphological characteristics of dying cells.

Conclusions Our study extends previous observations regarding the morphology and function of apoptotic cells in leucodepleted blood units, which suggested the presence of apoptotic cells in postfiltration leucocytes. Cleaved PARP, APO 2·7 mAb and positivity for the FITC‐conjugated Z‐VAD‐analogous reagent strongly suggest the activation of programmed death pathways. In addition, the maintained granulocyte‐associated and HLA class I antigens might recall an immune response in multitransfused patients.