bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Antibody Detection in Pediatric Immune Thrombocytopenic Purpura: Evaluation of Three Screening Methods
Authors: Biglino Paola , Perutelli Paolo , Mori Pier Giorgio
Published: December 2004

Background and objectives: Immune thrombocytopenic purpura (ITP) is a common hematologic disorder, two forms of which occur in children. The detection of circulating platelet antibodies is helpful in diagnosis. Materials and methods: We evaluated three different immunological methods for detecting platelet antibodies in the serum of children with ITP. These were: a solid‐phase red‐cell adherence test (SPRCA), an enzyme immunoassay (EIA), and an immunofluorescence test (PSIF). Results: The sensitivity of the methods in detecting IgG antibodies ranged from 28.1 (EIA) to 39.4% (SPRCA). We also looked for IgM antibodies by PSIF, thus raising the sensitivity of this test from 32.0 to 40.0%. A combination of two tests (SPRCA and EIA) allowed us to detect 61.8% positive samples. By doing all three tests, we obtained 71.3% positive samples. Finally, we reached 73.5% by adding PSIF for IgM. We found a higher frequency of circulating antibodies in both acute and chronic ITP at onset than in clinical remission. There were a few positive sera in chronic ITP, but not in the acute form in remission. Conclusion: The individual tests each have a relatively low sensitivity, but the combination of all three increases the diagnostic effectiveness. The finding of platelet antibodies during remission may predict evoluation toward a systemic autoimmune state.
Markers of Haemostatic Activation before and after Blood Donation and Plasmapheresis
Authors: Rahr Hans B. , Roman Corina , Ingerslev Jørgen , Jørgensen Jan
Published: December 2004

Background and objectives: Previous studies have reported signs of haemostatic activation after haemorrhage. The aim of this study was to measure sensitive markers for haemostatic activation after blood donation or plasmapheresis in healthy donors. Materials and methods: Blood samples were drawn before as well as 5 min and 1 h after donation in 40 male donors, of whom 29 gave 1 unit of blood and 11 underwent plasmapheresis. Standard assays were used. Results: Plasma concentrations of the activation markers prothrombin fragment 1+2, thrombin‐antithrombin complexes, and von Willebrand factor did not increase after blood or plasma donation. Conclusions: In our opinion, these procedures are safe.
CD36 Autoantibodies and Thrombotic Diathesis, Thrombocytopenia and Repeated Early Fetal Losses
Authors: Borzini P. , Riva M. , Nembri P. , Rossi E. , Pagliaro P. , Vergani P. , Greppi P. , Tantardini P.
Published: December 2004

Background and objectives: Autoantibodies to CD36, a platelet glycoprotein, have been found in patients with thrombotic thrombocytopenic purpura, and in those with lupus‐like anticoagulant with thrombotic complications. Materials and methods: Conventional hematologic and laboratory methods were used. The patient was a pregnant woman, who had had two early fetal losses separated by a normal offspring. Despite severe thrombocytopenia, she was asymptomatic. Results: Serological investigations were strongly suggestive of CD36 autoantibodies. Neither clinical nor laboratory data were typical of those usually associated with cd36 autoantibodies, namely thrombotic thrombocytopenic purpura (TTP), systemic lupus erythematosis (SLE), or prothrombotic compliance. Prophylaxis with salicylates and prednisone was started at the 8th week of gestation, and an offspring with mild thrombocytopenia was delivered by cesarean section at the 32nd week of gestation because of abruptio placentae. Conclusions: There may be a cause‐and‐effect relationship between early fetal losses and CD36 autoantibodies.
Intersecting Pools and Their Potential Application in Testing Donated Blood for Viral Genomes
Authors: Mortimer Janet
Published: December 2004

Amplification assays for viral genomes are at present too expensive and cumbersome to be applied to individual blood donations. Through constructing interesting pools of specimens, however, it may be possible to screen as a single process large numbers of units for several low prevalence viruses using relatively few tests. The preparation of the intersecting pools that would be required is explained in this paper. If test sensitivity and specificity were adequate, the testing of these pools would permit timely release of components that had the security of genetic testing added to current serological screening programmes. The method also makes screening for other viruses feasible.
Peripheral Blood Stem Cell Transplantation
Authors: Watts M. J. , Linch D. C.
Published: December 2004

High dose chemo/radiotherapy requiring autologous haemopoietic stem cell support is increasingly used in a variety of malignant disorders. Mobilised peripheral blood stem cells (PBSC) have largely replaced the use of autologous bone marrow due to more rapid haemopoietic reconstitution with less resource use including blood and platelet transfusion requirements. PBSC graft adequacy is monitored by CD34+ cell and granulocyte‐monocyte‐colony‐forming‐cell measurements, and thresholds for rapid engraftment have been determined. Studies are in progress to determine the optimal mobilisation regimens that will permit the achievement of the necessary progenitor thresholds with only one or two aphereses. This will facilitate the use of multiple cycles of high dose therapy and possibly the use of PBSC collected by venesection rather than apheresis. PBSC are also increasingly used in the allogeneic setting where specific mobilisation protocols not using cytotoxic drugs are employed. These technical advances will aid the execution of large trials to determine the efficacy of high dose therapy.
Transfusion‐Related Immunomodulation in Chinese Children with Thalassaemia
Authors: Li Karen , Li Chi Kong , Wong Raymond P. O. , Wong Annie , Shing Matthew M. K. , Chik Ki Wai , Yuen Patrick M. P.
Published: December 2004

Background and objectives: Multiple transfusions can induce immunomodulation. This study was carried out to investigate the immunological status of 50 transfusion‐dependent children with β‐thalassaemia, taking into account that lymphocyte characteristics are affected by sex, age and race. We paid particular attention to the influence of transfusion and serum ferritin on the lymphocyte subsets which may be affected by the exposure to foreign antigens. Materials and methods: By multicolour immunofluorescent analysis using flow cytometry, we determined lymphocyte characteristics with regard to major subsets (T lymphocytes, B lymphocytes and NK cells), activation (membrane IL‐2 receptor CD25, HLA‐D) and memory/naive T cells (CD45RO/CD45RA). Data from 51 age‐ and sex‐balanced children served as controls. Results: The normal Chinese children had higher NK levels than the β‐thalassaemia children. The levels of CD25 and HLA‐D indicated a broad‐based increase in activation status. Memory T cells were also increased when compared with their normal counterparts. We found additional and more marked alterations in the lymphocyte subsets of those who had received over 100 transfusions. While levels of NK cells were inversely correlated with the number of transfusions, CD25+ cells increased with transfusions. Conclusion: Many multitransfused β‐thalassaemia children have altered levels of lymphocyte subsets compared with normals. What remains to be investigated is the long‐term consequence of possessing low NK and non‐MHC‐restricted T cells (CD3+CD56+CD16+) and a high activation status in terms of resistance of infections and development of malignancy.
Immunochemical Characterization of the Rh Cw Antigen Using Human Monoclonal Antibodies
Authors: Thorpe Susan J. , Boult Christina E. , Thompson Keith M.
Published: December 2004

Background and objectives: Human monoclonal antibodies have been produced against various Rh antigens. We report here the production of another one against Rh C and the first such antibody against Rh Cw. Materials and methods: Two heterohybridomas secreting IgG human monoclonal antibodies against Rh Cw and Rh C (MS‐353 and MS‐242 respectively) were produced from alloimmunized donors. Their specificity was confirmed by serological testing. Results: MS‐353 reacted with all Cw‐positive phenotypes but not with any Cw‐negative phenotypes, whereas MS‐242 reacted with all Cw‐positive and C‐positive phenotypes. Using 125I‐labelled antibodies, the average number of Cw sites/red cell was 32,000 (R1wR1w), 15,200 (R1wR1), 19,800 (R1wR2), 15,300 (R1wr), 26,200 (r′wr), and the average number of C sites per red cell on equivalent Cw/C‐positive phenotypes was similar: 22,400 (R1R1), 21,800 (R1wR1), 12,300 (R1R2), 11,700 (R1wR2), 13,400 (R1r), 15,100 (R1wr), 21,100 (r′r), 18,700 (r′wr). MS‐353 and MS‐242 were mutually inhibitory. Both antibodies specifically immunoprecipitated a 33‐ to 34‐kD polypeptide from 125I‐surface‐labelled cells. Conclusion: MS‐353 is suitable for the serological detection of Cw‐positive cells, and the immunochemical data are entirely consistent with the report that the Cw antigen is associated with a point mutation in the RHCE gene [Blood, 1995;86:1196–1201].
Genotyping of the Human Platelet Antigen‐1 by ELISA Detection of Allele – Specific Amplicons
Authors: Müller Thomas H. , Döscher Andrea , Schunter Friedrich
Published: December 2004

Background and objectives: The purpose of the study was to establish a valid and efficient method for genotyping of the human platelet alloantigen‐1 (HPA‐1) in large sample numbers. Materials and methods: Digoxigenin‐ and fluorescein‐labelled allele‐specific primers and a biotinylated common primer were included in the hot‐start PCR. Amplicons were bound to avidin‐coated plates to identify the products by ELISA. Results: This approach reduced the number of PCR analyses for HPA‐1 typing by half. PCR products were detected with high sensitivity and good reproducibility (interassay – CV: <8%) in the ELISA. The typing of 100 blood donors with both PCR plus gel electrophoresis and ELISA‐PCR showed identical results. Conclusion: This method offers efficient HPA‐1 genotyping in large numbers of donors and patients.
Collaborative Study to Assess the Suitability of a Proposed Working Reagent for Human Parvovirus B19 DNA Detection in Plasma Pools by Gene Amplification Techniques
Authors: Saldanha J. , Minor P. ,   B19 Collaborative Study Group
Published: December 2004

Background and objectives: A collaborative study was done to examine the sensitivity and specificity of assays for the detection of human parvovirus B19 DNA in plasma pools by PCR techniques and to establish a working reagent for B19 DNA testing of plasma pools. Materials and methods: Duplicate samples consisting of a tenfold dilution series of a positive cryosupernatant diluted in B19 DNA‐negative cryosupernatant were sent to 17 laboratories. Results: The sensitivity of the assays varied: 2 laboratories were able to detect the 10−7 dilution while 1 laboratory failed to detect B19 DNA in any samples. In addition, 5 laboratories obtained false‐positive results. Conclusions: In general, laboratories using assays optimised for rapid detection of B19 DNA in serum samples did not perform well, indicating that such rapid methods are not adequate for examination of plasma pools. The 10−6 dilution was detected by approximately half the laboratories and could be used as the working reagent.
Detection of Irregular Red Cell Antibodies: More than 3 Years of Experience with a Gel Technique and Pooled Screening Cells
Authors: Titlestad Kjell , Georgsen Jørgen , Andersen Helle , Kristensen Tom
Published: December 2004

Background and objectives: The purpose of this study was to evaluate more than 3 years of experience with a gel technique in combination with pooled screening cells for the detection of irregular red cell antibodies. Materials and methods: Conventional serologic methods were used for blood typing, antibody screening and cross‐matching until the end of 1992. We introduced the gel technique as a routine assay for antibody detection and identification in 1993. Results: After the tube technique had been abandoned, the number of false‐positive antibody screening tests was reduced by 71%, positive antibody screening tests by 33%, enzyme agglutination by 100% and rouleaux reactions and cold‐reacting antibodies by more than 50%. There was a 40% increase in first‐time detection of clinically relevant antibodies. We saw no increase in delayed haemolytic transfusion reactions. Conclusions: For the detection of irregular red cell antibodies, pooled screening cells in combination with a gel technique are at least as efficient and safe as a conventional tube technique with unpooled test cells.
Leukocyte Filtration Does Not Affect Lymphocyte Subpopulations and NK Cell Function in Recipients of Blood Transfusions
Authors: Mathiesen O. , Lund L. , Brodthagen U. , Gandrup P. , Grunnet N. , Balslev I. , Jersild C.
Published: December 2004

Background and objectives: The possible immunosuppressive action of blood transfusion has aroused great interest recently, particularly with respect to its effects on tumor growth and recurrence rate of malignant disease. Materials and methods: The effect of blood transfusion on lymphocyte subpopulations and NK cell function preoperatively and 6 months postoperatively was studied in 129 patients treated with elective surgery for colorectal malignancy. Forty‐two patients (33%) received blood transfusions, 21 of them randomly allocated to receive leukocyte‐depleted blood products. Investigation was by means of conventional laboratory methods. Results: In 21 patients receiving a median of 3 units of non‐leukocyte‐depleted blood products (NLD), a significant reduction in CD4+ lymphocytes (44% vs. 40%, p < 0.01) occurred. In contrast, no significant changes in CD4+ lymphocytes were observed in the 21 patients transfused with leukocyte‐depleted blood products (LD). However, with respect to lymphocyte subpopulations and NK cell function, differences between the NLD and LD groups were not significant. There was a marginal decrease in HLA‐DR+ lymphocytes in the NLD patients without a history of previous transfusion. Conclusions: There seems to be no major change in lymphocyte subpopulations and NK cell function 6 months after blood transfusion. Thus we cannot confirm our previous findings of a reduced number of CD20+ cells after blood transfusion.
Evaluation of the Microcolumn Technology for Pretransfusion Testing in Multiple Myeloma Patients
Authors: Fabijańska‐Mitek J. , Żupańska B. , Pogłód R. , Gorajek M. , Łopieńska H. , Kraj M.
Published: December 2004

Objective: Microcolumn tests are useful for serological investigations, although because of their high sensitivity, false‐positive results might be expected, e.g. in hypergammaglobulinemia. The aim of this study was to evaluate these tests in multiple myeloma. Methods: Pretransfusion testing was done in 80 patients with multiple myeloma using microcolumn and traditional tube tests. Results: All sera were negative in microcolumn indirect antiglobulin test and enzyme test, positive in 58% of samples in the enzyme tube test. The microcolumn direct antiglobulin test was positive in about 40% of samples but never in the tube direct antiglobulin test. This was not due to the presence of autoantibodies but to nonspecific binding of immunoglobulins related to their concentration in sera. Conclusion: Microcolumn tests appeared to be useful for pretransfusion testing in multiple myeloma in spite of positive autocontrols.
Photometric Evaluation of the Solid‐Phase Antiglobulin Test Using Length Measurement of the Absorption Curve
Authors: Spindler J.H. , Kerowgan M. , Eichler H. , Goldmann S. F.
Published: December 2004

Background and objectives: The very sensitive solid‐phase antiglobulin test is used widely in blood group serology. However, a disadvantage of the test is the sedimentation variability of indicator cells during centrifugation, which causes errors in the photometric evaluation. Materials and methods: To solve this problem a technique was developed which depends on the ‘length measurement of the absorption curve’ of the sedimented indicator cells. Results: 9,075 sera were tested for the presence of alloantibodies on the basis of the visual type of measurement and the newly developed technique. The agreement between the two evaluation systems was 99.94%. No false‐negative results were observed. Conclusion: The new method allows the complete automation of the solid‐phase antiglobulin test, enabling the analysis and evaluation of the solid‐phase microtiter plate assay to be more discriminating and safe.
Hepatitis G Virus Infection in Screened Chinese Blood Donors
Authors: Ren Fu‐Rong , Wang Yu , Li Hui , Chen Hong‐Song , Zhao Hai‐Yan
Published: December 2004

Background and Objectives: To determine the prevalence of the recently identified hepatitis G virus (HGV)/GBV‐C in screened Chinese paid blood donors. Materials and Methods: Two hundred and seventy‐nine plasma samples were tested for HGV RNA by RT‐PCR with nested primers from the 5'‐noncoding region of GBV‐C. All samples were obtained from plasma or blood bags that had been screened twice by routine selection tests (ALT, HBsAg, Anti‐HCV, anti‐HIV, and syphilis) and were available for clinical use. Results: HGV RNA was detected in 2 (4%) of 50 paid plasma donors from the Beijing Red Cross Blood Center, 1 (2%) of 50 paid blood donors from Taiyuan, and 9 (5%) of 179 paid blood donors from Hebei, a total HGV detection rate of 4.3% (12/279). Conclusions: Our data suggest that HGV infection is relatively frequent even in screened donors, at least in paid screened donors, although larger‐scale studies are required.
Benefit of Dynamic over Static Incubation in the Detection of a Low‐Level HBsAg (Chronic Carrier) Bone Donor
Authors: Raafat A. , Yates P. , Sellers F. , Munro H. , Dow B.
Published: December 2004

Background: Hepatitis B infection can be transmitted by organ or tissue transplantation. Case Report: A 40‐year‐old man received a bone graft from a donor who tested negative for HbsAg with a static‐mode assay. The same donor, on a subsequent donation, was found to be HbsAg‐positive with a dynamic mode assay. The archival sample from the initial donation also tested HbsAg‐positive with the dynamic‐mode assay. The recipient of the bone, who had received HBV vaccine in the past, did not develop clinical or laboratory evidence of HBV infection. Conclusions: This case highlights the importance of sensitive microbiological test protocols in the context of tissue banking.
Historical Note: Past, Present and Future of the Antiglobulin Test
Authors: Coombs Robin R. A.
Published: December 2004

This is a review of two invited talks on the antiglobulin reaction on the occasion of the 50th Anniversaries of the International Blood Group Reference Laboratory, now at Bristol, and the National Blood Service, London, and coincidentally the 51st anniversary of the antiglobulin test! The first talk (as specially requested) is a very personal reminiscence of the discovery, with Rob Race and Arthur Mourant, of the antiglobulin test in blood group serology. The second talk traces developments in antiglobulin testing in general immunology using, of course, isotype‐specific antiglobulin reagents as and when they became available. Special emphasis was given to: (1) testing for poorly agglutinating bacterial antibodies; (2) special procedures for measuring reaginic (IgE) antibodies to various allergens; (3) the incorporation of an antiglobulin step or stage in many of the routine automated immunoassay procedures; (4) the special experimental procedures in the form of mixed antiglobulin reactions to reveal antibodies to the surface antigens on tissue cells, and, finally, (5) by chemically coupling antiglobulin to red blood cells, a distinctive integrated immunoassay system was developed, enabling reverse passive haemagglutination as an assay for immunoglobulins, rosetting reactions to reveal native IgG receptors on lymphocytes or antibody immunoglobulin reacting with CD marker antigens; this same reagent, namely red‐cell‐labelled antiglobulin, can be used to detect antibodies reacting with either bacteria or antigens adsorbed on the walls of wells of microtitre plates. The need for improved stabilization and preservation of the antiglobulin‐linked red cells to prolong their ‘shelf‐life’ is stressed.
Mixed‐Type Autoimmune Hemolytic Anemia following Fludarabine Treatment in a Patient with Chronic Lymphocytic Leukemia/Small Cell Lymphoma
Authors: Vick Dan J. , Byrd John C. , Beal Connie L. , Chaffin Donald J.
Published: December 2004

Background and objectives: Mixed‐type autoimmune hemolytic anemia (AIHA) is a rare complication of chronic lympocytic leukemia (CLL). We report a patient with small lymphocytic lymphoma (phenotypic CLL) who developed symptomatic anemia 3 weeks after her fifth cycle of fludarabine, a T cell immunosuppressant. Materials and methods: An antibody screen and panel, direct antiglobulin test, rapid acid eluate, rabbit erythrocyte stroma (RESt) adsorption, and autoadsorption were performed. Results: Warm and cold autoantibodies were detected. Prompt treatment with corticosteroids and minimal blood transfusions led to marked improvement. Conslusion: Normally, T cells suppress polyclonal lymphocytes that produce autoantibodies. Suppression of T cells in this patient, in addition to the underlying disease process, may explain this mixed‐type AIHA, the first reported case to occur following fludarabine treatment.
High Prevalence of Parvovirus B19 lgG Antibodies among Dutch Hemophilia Patients
Authors: Mauser‐Bunschoten E.P. , Zaaijer H. L. , van Drimmelen A. A. J. , de Vries S. , Roosendaal G. , van den Berg H. M. , Lelie P. N.
Published: December 2004

Background and Objectives: Human parvovirus B19 is a potential risk to hemophiliac patients receiving blood products. Materials and Methods: To determine the prevalence of the corresponding antibody in patients with hemophilia A or B or von Willebrand's disease, we tested 326 hemophilia patients for anti‐B19 lgG. The results were compared with those of 203 age‐matched controls (male blood donors and children). Results: The overall prevalence of B19 lgG in the hemophilia patients was 302/326, and in the controls 123/203. Below the age of 10, hemophilia patients had a higher prevalence of B19 lgG (76%, 42/55) than the controls (23%, 11/48; p<0.00001). In those below the age of 5 who had been treated exclusively with monoclonally purified concentrate, it made no difference whether the product was pasteurized or solvent‐detergent treated. There was a significantly lower incidence in patients who were rarely treated. Conclusion: Parvovirus B19 is frequently transmitted in blood products. Existing virus‐inactivating methods do not prevent transmission.
DNA Analysis of Duffy Genes in American Blacks
Authors: Moulds J.M. , Hayes S. , Wells T.D.
Published: December 2004

Background: Because limited studies have shown that the Duffy genotype is not accurately reflected by the erythrocyte phenotype, we used PCR‐RFLP to determine the true FY1 and FY2 gene frequencies in blacks. Methods: Genomic DNA was extracted from 81 randomly selected black individuals, amplified using PCR, digested with BanI and electrophoresed in 1.8% gels. Direct DNA sequencing of the FY regulatory region was done on a sample which had weak Fyb antigens. Standard serological techniques were used to type the erythrocytes for Fya and Fyb. Results: The most common genotype (83%) in blacks was Fy(a−b+) but almost all of the samples serologically typed as Fy(a−b−). The new gene frequencies are 0.08 for FY1 and 0.92 for FY2. Two donors whose erythrocytes typed weakly positive for Fyb and appeared to be Fyx were homozygous for FY2 by DNA testing. DNA sequencing of the regulatory region for Duffy found a mutation in an Sp1 binding site in the Fyx donor. Conclusions: We conclude that (1) the FY2 gene occurs in a much higher frequency in blacks than previously reported which may explain the lack of anti‐Fyb in this ethnic group and (2) Fyx appears to be a the product of a mutant FY2 gene giving a quantitative variation in expression.
Effects of Blood Donation on the Physical Fitness and Hemorheology of Healthy Elderly Donors
Authors: Janetzko Karin , Böcher Regina , Klotz Karl F. , Kirchner Holger , Klüter Harald
Published: December 2004

Background: International regulations for blood donation recommend a maximum donor age of 65 years. As the average population age is steadily rising in western societies, a considerable group of volunteers is lost to the donor base. Study Design and Methods: In a prospective study we investigated the effect of a 450‐ml whole blood donation on the physical fitness and hemorheology of regular elderly allogeneic blood donors (n = 24, aged 63–69 years, mean = 65). Results were compared with a younger group of regular donors (n = 23, aged 55–62 years, mean = 58) and a group of elderly subjects (n = 7, aged 63–66 years, mean = 65), who did not donate blood for this study. Assessing the physical fitness, we determined the submaximal physical working capacity at a heart rate of 130 min−1 (PWC 130) and the maximal working capacity (MWC) by treadmill exercise testing the day before (day −1) and after donation (day + 1). The impact of the blood loss on hemorheology was examined by analyzing the plasma viscosity before, during and after donation. Results: We found an increase of mean values of PWC 130 and MWC on day +1 in all study groups, but increases were only significant in the younger group (PWC 130 p = 0.03; MWC p = 0.04). Values did not differ significantly between the three groups. Plasma viscosity decreased significantly directly after donation in both groups of donors. Conclusion: A single blood donation did not alter the physical fitness of otherwise healthy elderly people. The older blood donors and the younger controls showed a similar compensation mechanism to blood loss. We found no general reason for disqualifying blood donors aged 65 years from donating.
Impact of Storage at 22°C and Citrate Anticoagulation on the Cytokine Secretion of Mononuclear Leukocytes
Authors: Müller‐Steinhardt Michael , Kirchner Holger , Klüter Harald
Published: December 2004

Background and Objectives: Peripheral blood mononuclear cells (PBMC) and cytokines accumulating in platelet concentrates (PC) during storage have been implicated in causing non‐hemolytic transfusion reactions, in particular after prolonged PC storage. We investigated the impact of major storage parameters, such as storage time, anticoagulation and temperature, on the capability of PBMC to secrete cytokines. Materials and Methods: In a first study, PBMC from whole blood donations (n = 16) were exposed to standard PC storage conditions: 22°C, citrate phosphate dextrose (CPD) anticoagulation. Secretion of the cytokines interleukin‐1β (IL‐1β), IL‐2, IL‐6 and interferon‐γ (IFN‐γ) on mitogenic stimulation was measured directly after blood donation and after 1, 3 and 5 days of storage. In a second study, paired whole blood samples (n = 24) were investigated for the mitogenic induction of IL‐2, IL‐6, IFN‐γ and IFN‐α. Cytokine values were compared with respect to incubation temperature (22 vs. 37°C) and anticoagulant (CPD vs. lithium‐heparin). Results: PBMC stored at 22°C with CPD anticoagulation showed an altered cytokine secretion pattern in comparison to freshly donated cells. After storage for 3 days, IL‐2 release was decreased (p < 0.05) and after 5 days secretion of all cytokines was significantly decreased, though still detectable (p < 0.01). In the second study, CPD anticoagulation resulted in a significantly impaired cytokine secretion compared with lithium‐heparin. Cytokine secretion at 22°C were significantly lower (p < 0.001) compared with 37°C for both anticoagulants. Conclusion: Storage of PBMC at 22°C with CPD leads to an impaired cytokine response, but PBMC are still capable of secreting cytokines after 5 days of storage. PBMC remain a potential source of cytokines even after 5 days of storage in CPD at 22°C.
Lactated Ringer's Solution versus Hydroxyethyl Starch for Volume Replacement in Autologous Blood Donors with Cardiovascular Disease: A Controlled, Randomized Trial
Authors: Kasper Stefan‐Mario , Dahlmann Heinz , Mellinghoff Hermann , Ellering Jürgen , Baumann Marc , Buzello Walter
Published: December 2004

Background and Objectives: The study was designed to evaluate whether volume replacement following blood donation can prevent arterial hypotension in autologous blood donors with cardiovascular disease. Materials and Methods: One hundred nineteen autologous blood donors with known cardiovascular disease were randomly allocated to receive, following withdrawal of 500 ml of blood, either no infusion (control group) or a 25 ml/min intravenous infusion of either 1,500 ml of lactated Ringer's solution (LRS) or 500 ml of 6% hydroxyethyl starch (HES). Starting before phlebotomy, arterial blood pressure was measured oscillometrically every 5 min until 90 min after donation. Results: Group means showed little difference between the groups in blood pressure throughout the monitoring period. The proportion of patients who at least once had a ≥20% decrease from baseline in systolic blood pressure was 3–5 times greater in the control group than in the LRS and the HES group (50 vs. 10 and 15%, respectively; p < 0.001 on χ2 analysis for a 2 × 3 table). Systolic hypertensive episodes (≥ 20% increase over baseline) were observed more frequently in the LRS group than in the control and the HES group (41 vs. 10 and 18%, respectively; p=0.003). Conclusion: Both LRS and HES, administered at a volume ratio to blood loss of 3:1 and 1:1, respectively, significantly reduced the incidence of systolic hypotensive episodes in autologous blood donors with cardiovascular disease. LRS at a 3:1 volume ratio to blood loss was associated with a high rate of systolic hypertension.
Decrease in the Concentration of Vitamin E in Blood and Tissues Caused by Di(2‐Ethylhexyl) Phthalate, a Commonly Used Plasticizer in Blood Storage Bags and Medical Tubing
Authors: Manojkumar V. , Nair K.G. Padmakumaran , Santhosh A. , Deepadevi K.V. , Arun P. , Lakshmi L.R. , Kurup P.A.
Published: December 2004

Background and Objectives: Significant amounts of di(2‐ethylhexyl) phthlate (DEHP) leach into blood stored in DEHP‐plasticized PVC bags. The aim of this study was to find out whether DEHP at these low levels has any effect on the concentration of vitimin E, an antioxidant which affords protection against free radical damage. Materials and Methods: DEHP was administered in low doses (150–750μg/100g body weight) to rats intraperitoneally and the concentration of vitamin E in the liver and testes was measured. Concentration of vitamin E was also measured in blood stored in glass bottles in the presence and absence of DEHP and in blood stored in DEHP‐plasticized PVC bags. Results: A decrease in the concentration of vitamin E was observed in all cases. Administration of vitamin E to rats and incorporation of vitamin E in the additive solution in the case of blood prevented this decrease. Conclusion: DEHP even at very low doses caused a decrease in the concentration of vitamin E in liver and tests of rats given this substance. Blood stored in DEHP‐plasticized bags also showed a decrease in the concentration of vitamin E.
Rapid Detection of Genetic Mutations Associated with Haemochromatosis
Authors: Guttridge M. G. , Thompson J. , Worwood M. , Darke C.
Published: December 2004

Background and Objectives: The purpose of this study was to establish a rapid method suitable for large‐scale population screening, including blood donors, for the detection of two genetic mutations at codons 63 and 282 on the HFE gene that are associated with haemochromatosis. Materials and Methods: A method using the polymerase chain reaction with sequence‐specific primers (PCR‐SSP) was designed and tested using a panel of 185 individuals previously typed for HFE mutations by PCR‐RFLP. Results: The PCR‐SSP method detected the two mutations showing complete agreement with the genotypes obtained by PCR‐RFLP. Three HFE alleles, termed HFE‐1, ‐2, and ‐3, were identified. Conclusions: This PCR‐SSP method allows efficient HFE genotyping for large numbers of individuals.
Detection of HIV‐1 Infection in Dried Blood Spots from a 12‐Year‐Old ABO Bedside Test Card
Authors: Strobel E. , Emminger Ch. , Mayer G. , Eberle J. , Gürtler L.
Published: December 2004

Background and Objectives: We tested dried blood from an ABO bedside test card which had been stored at room temperature for 12 years, to prove that a patient with HIV‐1 infection had been infected by blood transfusion. Materials and Methods: Immunoblots for HIV‐1 antibodies and threefold PCRs with halfnested primers for the HIV‐1 integrase gene were done with eluates from the dried blood spots. Results: HIV‐1 antibodies and HIV‐1 DNA could be detected in the sample from one unit of blood, but not from the two other units or from the recipient before transfusion. Conclusion: Further studies should be done on the validity of stored dried blood as an alternative to the storage of frozen donor serum for several years for ‘look‐back’ studies.
Screening of Blood Donors for Human Parvovirus B19 and Characterization of the Results
Authors: Wakamatsu C. , Takakura F. , Kojima E. , Kiriyama Y. , Goto N. , Matsumoto K. , Oyama M. , Sato H. , Okochi K. , Maeda Y.
Published: December 2004

Background and Objectives:
Human parvovirus B19 (B19 virus) can be transmitted through blood transfusion and plasma‐derived products. In a previous report, we utilized the simple hemagglutination method based on the interaction between the B19 virus and P antigen on human erythrocytes in order to screen the blood donors. We called this method receptor‐mediated hemagglutination (RHA) [Lancet 1995;346:1237–1238]. In this paper, we report on a large‐scale screening of the B19 virus by RHA and discuss the results. Materials and Methods: Donor sera from September 1995 to March 1997 and seroconversion panels were enrolled. Donor sera were examined by RHA for large‐scale screening. The positive sera in the first screening were then further investigated by the RHA inhibition test, countercurrent immunoelectrophoresis (CIE), and enzyme‐linked immunosorbent assay, and polymerase chain reaction (PCR). We also evaluated the infectivity and neutralizing activity of various kinds of sera by the erythroid colony forming unit (CFU‐e) assay. To examine the detection limits of the B19 virus by RHA, B19‐viremic sera were purified by sucrose gradient ultracentrifugation. Results: Among 257,710 sera specimens, 293 sera (0.11%) gave a positive reaction in the first screening using RHA. Out of these 293 sera specimens, 31 were positive for PCR, of which 28 were also RHA inhibition‐positive, and 25 of the 28 CIE‐positive. In the CFU‐e injury assay, all the RHA inhibition (+) sera showed a decrease in the number of erythroid colonies. The RHA inhibition (‐) PCR (+) B19 antibody (+) sera did not affect the erythroid colony formation and protected CFU‐e from injury by the B19 virus. By measuring the amount of purified B19 protein and its RHA titer, the detection limit of the B19 virus by RHA was calculated to the 0.37±0.03 ng/ml. Conclusion: These results suggest that the RHA (+) RHA inhibition (+) sera were infectious in vitro. The combination of RHA and the RHA inhibition test is considered to be useful for the large‐scale screening of infectious B19 virus in blood donors with high specificity.
Liquid‐Stored Red Blood Cells for Transfusion
Authors: Högman Claes F.
Published: December 2004

Blood transfusion in a modern sense means the transfusion of red cells, when necessary supplemented by other components. The demand for plasma and plasma fractions and for platelets for therapeutic use has had an influence on the technique for preparing red cells. Automated devices have made it possible to perform collection as well as separation under more standardized conditions. Improved techniques for storage of red cells have prolonged the shelf life somewhat but most of the available methods disregard the rapid loss of 2,3 diphosphoglycerate and the accompanying increase in oxygen affinity. Methods are available which reduce the number of contaminating leukocytes to low levels, but information is still incomplete as to the degree of depletion actually needed.
Hepatic Dysfunction in a Population of Antibody‐Deficient Patients: Prevalence, Aetiology and Outcome of PCR Screening for Hepatitis C and G Viruses
Authors: O'Leary P.F.G. , Collingham K. , Skidmore S. , King J. , Bennett C.L. , Williams P.E. , Pillay D. , Thompson R.A.
Published: December 2004

Background and Objectives:
A group of 40 antibody‐deficient patients receiving regular infusions of intravenous immunoglobulin underwent close monitoring in an attempt to identify hepatic dysfunction. The continuing risk of hepatitis virus transmission, especially hepatitis C virus via immunoglobulin products prompted this policy. We report our findings. Methods: Screening included measurement of transaminase levels at each infusion. The patients were also tested for evidence of infection with hepatitis viruses B, C and G. Results: Abnormal liver function tests were identified in 6 cases. However, a blood‐borne viral aetiology was not found in any of these cases. Additional investigation allowed an alternative aetiology to be identified in most cases. One patient found to be positive for hepatitis G virus (HGV)‐RNA by reverse transcriptase‐polymerase chain reaction has no evidence to date of clinical problems as a result. Interpretation: The results are reassuring in that definite iatrogenic hepatitis virus transmission has not been found in this cohort, despite long‐term treatment with a wide range of immunoglobulin products. The source of infection of the single patient infected with HGV remains as uncertain as the pathogenic potential of this virus. However, as long as the risk of immunoglobulin‐associated viral transmission continues, a strict monitoring programme such as ours should continue to facilitate prompt detection of cases with abnormal liver function. Liver dysfunction in this group requires full investigation and we cannot exclude infection with hitherto unidentified blood‐borne viruses.
Removal and Inactivation of Hepatitis B Virus from Contaminated Pooled Plasma in a Large‐Scale Manufacturing Process for Factor VIII and Human Serum Albumin
Authors: Murozuka T. , Aoki M. , Kimura N. , Sotoyama K. , Abe I. , Izumi H. , Emura H. , Takeda Y. , Nanakawa H. , Katsubayashi Y. , Takeuchi T. , Ito H. , Hirakawa S. , Mitsunaga S. , Tadokoro K. , Kanemitsu K. , Miyamoto M. , Yamanaka R. , Nishioka K.
Published: December 2004

Background and Objectives:
The Japanese Red Cross Society recalled one lot of monoclonal‐antibody‐purified factor VIII (F VIII) and two lots of human serum albumin (HSA) 5 months after preparation of the final products, because of a procedural error that led to contamination by a unit of plasma positve for hepatitis B surface antigen (HBsAg). We evaluated the effectiveness of virus inactivation/removal in a large‐scale process for manufacturing F VIII and HSA. Materials and Methods: HBV DNA in the retained samples in process was measured by the polymerase chain reaction (PCR). The kinetics of virus inactivation by solvent‐detergent (S/D) treatment was examined using model viruses. We also did a look‐back survey of the patients who received corresponding products. Results: Contaminated hepatitis B virus (HBV) DNA became undetectable beyond fraction S IV‐I in the albumin process, and immunoaffinity chromatography in the F VIII process, respectively. The model viruses were inactivated within 5 s by S/D treatment. There is no evidence that patients were infected by HBV after transfusion of these products. Conclusion: We conclude that virus inactivation/removal was effectively achieved in a large‐scale manufacturing process for F VIII and HSA.
Comparison of Four HTLV‐I and HTLV‐I + II ELISAs
Authors: Vrielink H. , Reesink H. W. , Habibuw M. R. , Schuller M. , van der Meer C.B.M. , Lelie P. N.
Published: December 2004

Background:
Various countries require blood donor screening using assays applying specific HTLV‐I and HTLV‐II antigens. We evaluated the sensitivity and specificity of 4 anti‐HTLV‐I + II ELISAs (Abbott, Murex, Organon Teknika and Ortho). Methods: Panel A consisted of HTLV‐I‐positive individuals (n = 41), panel B of Mixed Titer Performance Panel 204 (Boston Biomedica Inc. panels C and D of dilution series from HTLV‐I‐positive (n = 30) and HTLV‐II‐positive (n = 20) individuals and panel E of sera from first‐time blood donors (n = 1,055). Results: In HTLV‐I and ‐II‐positive samples, a sensitivity of 100% could be observed in all 4 ELISAs. In diluted HTLV‐I‐ and ‐II‐positive samples, probit analysis revealed that the Murex assay had the highest analytical sensitivity, followed by the ELISAs from Ortho, Abbott and Organon Teknika. In specimens from first‐time donors, a specificity of 100% was observed in ELISAs from Murex, Organon Teknika and Ortho, and of 99.7% in the assay from Abbott. Conclusion: The 4 anti‐HTLV‐I + II ELISAs studied were appropriate as screening tests.
Press Review
Authors: Bayer R. L. , Lichtman S. M. , Allen S. L.
Published: December 2004

Acquired factor VIII Inhibitors – Successful Treatment with on Oral Outpatient Regimen
International Reference Reagents: Antihuman Globulin
Authors: Case J. , Ford D. S. , Chung A. , Collins R. , Kochman S. , Mazda T. , Overbeeke M. , Perera R. , Sakuldamrongpanich T. , Scott M. , Voak D. , Żupa´nska B.
Published: December 2004

An international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti‐lgG and anti‐C3 (anti‐C3c and anti‐C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2‐ml freezedried aliquots from seven assigned distribution centres.
Allele Frequencies of Human Platelet Antigen 1, 2, 3, and 5 Systems in Patients with Chronic Refractory Autoimmune Thrombocytopenia and in Normal Persons
Authors: Thude Hansjörg , Gatzka Elfriede , Anders Olaf , Barz Dagmar
Published: December 2004

Background and Objectives:
Chronic refractory autoimmune thrombocytopenic purpura (AITP) is an autoimmune disorder due to autoantibodies against platelet glycoproteins (GP). Human platelet alloantigenic (HPA) systems are distributed to different platelet GPs. We carried out genotyping of diallelic HPA‐1, ‐2, ‐3, and ‐5 systems to clarify potential associations between HPA alleles and the development of chronic refractory AITP. Patients and Methods: DNA was isolated from 33 unrelated German patients with chronic refractory AITP and from 80 randomly selected German blood donors to determine the phenotype and allele frequencies for the HPA‐1, ‐2, ‐3, and ‐5 systems. Fragments carrying the polymorphic sequences corresponding to those alleles were amplified by the polymerase chain reaction and further characterized by restriction analysis. Results: Whereas HPA‐1, ‐3, and ‐5 allele frequencies were identical in 33 patients with chronic refractory AITP and in controls, HPA‐2 allele frequencies showed a statistically significant difference (p = 0.017). In our group of patients, the HPA‐2a allele frequency was 100%, but HPA‐2b was not seen. In contrast, the allele frequency of HPA‐2a in the control group was 92% (n = 147), and in HPA‐2b it was 8% (n = 13). Conclusion: This study suggests an association between the HPA‐2a allele and chronic refractory AITP. The HPA‐2a allele may be involved in the formation of an AITP‐specific autoepitope.
Press Review
Authors: Engelfriet C.P. , Williamson L.M. , Lowe S. , Love E.M.
Published: December 2004

Serious Hazards of Transfusion (SHOT)
Clinical Tolerance of Methylene Blue Virus‐Inactivated Plasma
Authors: Simonsen Anne Catrine , Sørensen Henning
Published: December 2004

Objectives:
Baxter's methylene blue (MB) photoinactivation of fresh frozen plasma (FFP) is a method that effectively removes any intracellular potentially infectious agent, such as prions, by filtration combined with an improved photochemical virus inactivation on a single unit of FFP. The purpose of this study was to demonstrate clinical and biochemical tolerance of MB‐treated plasma in healthy human volunteers. Methods: The design was a crossover randomized trial. Twelve subjects received alternatively treated and nontreated autologous FFP. Coagulation parameters were tested as well as clinical and biochemical parameters. Results: No clinical or statistically significant difference was detected for any of the parameters. Conclusion: Transfusion of FFP treated for viral reduction by an improved MB photoinactivation method was shown to be safe and did not affect the normal function of coagulation proteins. We consider this method as a valuable alternative to other methods described as it combines the photoinactivation with a filtration step removing any intracellular infectious agents, such as prions.
Large‐Scale Screening for Human Parvovirus B19 DNA by PCR: Application to the Quality Control of Plasma for Fractionation
Authors: Aubin J.T. , Defer C. , Vidaud M. , Montreuil M. Maniez , Flan B.
Published: December 2004

Background and Objectives:
Because human parvovirus B19 (B19) has been transmitted by various plasma‐derived medicinal products, the ‘Laboratoire français du Fractionnement et des Biotechnologies’ (LFB) implemented PCR screening of plasma pool samples for B19 DNA as part of the quality control of plasma source material. Materials and Methods: Plasma pool samples (average of 46.5 donations) were tested for B19 DNA by PCR and by immunological detection of PCR products. The viral DNA content was determined by means of a TaqManTM‐based, quantitative PCR. Results: From plasma corresponding to 2‐year collections in France, and representing 4.26 million donations from approximately 1.25 million voluntary unpaid donors, the average frequency of positive donations was 1/5,950 and reached 1/1,420 during an epidemic. Levels of B19 DNA in positive pools ranged from <102 to 1011 copies/ml. Conclusion: A large‐scale PCR plasma screening increased the safety of LFB's wide range of products with respect to B19, a virus particularly resistant to physicochemical inactivation procedures.
Plasma Levels of Eicosanoids after Transfusion of Intraoperatively Salvaged Blood
Authors: Jacobi K. , Walther A. , Lörler H. , Neidhardt B.
Published: December 2004

Background:
The interest in autologous red blood cell salvage is increasing rapidly, but it is well known that platelets and white cells are activated during cell washing. Therefore, we aimed to determine whether or not eicosanoids transfused together with the salvaged blood are responsible for nonhemolytic immunological transfusion reactions. Materials and Methods: In 11 patients who underwent radical prostatectomy we studied the release of eicosanoids, prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), in association with reinfusion of intraoperatively salvaged blood. Blood samples were taken before operation, on days 1 and 3 after operation and before and after cell washing. Results: There was a twofold increase in PGE2 and a twentyfold increase in TxB2 in the unwashed salvaged blood. Washing caused a reduction in eicosanoid plasma levels of up to 95%, but a significantly lower level in relation to its corresponding preoperative value was only found for PGE2. After transfusion of an average of 420 ml RBC sediment from washed blood, the patients' plasma concentrations of PGE2 and TxB2 on the 1st and 3rd postoperative day did not significantly differ compared to baseline values. Conclusion: Our data support the notion that RBC sediment from ‘salvaged’ and washed autologous blood contains increased amounts of PGE2 and TxB2. It remains, however, questionable if these passively infused eicosanoids become biologically active. According to our study, the PGE2 and TxB2 transfused are apparently not responsible for nonhemolytic immunological transfusion reactions.