bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐Dose Intravenous Immunoglobulins in HIV‐1‐Infected Adults with AIDS‐Related Complex and Walter‐Reed 5
Authors: Schrappe‐Bächer M. , Rasokat H. , Bauer P. , Bendick Ch. , Bube F.W. , Degenhardt St. , Fätkenheuer G. , Heiniger H.J. , Heitmann K. , Imbach P. , Krickeberg H. , Mauff G. , Meller M. , Mertens Th. , Morell A. , Perret B.A. , Plum G. , Ramon A. , Salzberger B. , Schaad U.B. , Siebel E. , Späth P.J. , Stützer H. , Türk D. , Krueger G. R. F.
Published: March 2009

Abstract. The influence of high‐dose intravenous immunoglobulins (HD‐IVIG) on the clinical status and T4 cell count of adults with AIDS‐related complex (ARC) and Walter‐Reed 5 (WR5) was evaluated in a randomized double‐blind longitudinal study. Inclusion criteria were: (1) T4 cells <400/μl and (2a) oral thrush or cutaneous anergy or (2b) two clinical ARC criteria (fever, diarrhea, weight loss, fatigue, night sweats). Thirty patients [28 males, 2 females, median age 41 (24–64) years] with ARC (n = 8), WR5 (n = 12) and both (n = 10) were stratified according to their T4 cell count (≥ vs. <300/μl). Fifteen patients received 0.4g/kg body weight IVIG and 15 placebo (albumin 0.03%) every other week for 26 weeks with follow‐up for another 26 weeks. The clinical status was defined as a score consisting of fever, diarrhea, night sweats, fatigue, weight loss, oral candidiasis and mucosal or cutaneous herpes simplex. Clinical examination and routine laboratory assessments were performed before initiation of the study and before each administration, lymphocyte phenotyping every 4 weeks and cutaneous reaction, serology and lymphocyte stimulation every 12 weeks. Both groups were comparable in initial clinical symptoms and laboratory values. Seven patients developed AIDS (treatment group: 3, placebo group: 4), 1 patient died by homicide. After 26 weeks, the clinical score (particularly fatigue and fever) was significantly improved in the treatment group, while the T4 cell count and other clinical and immunological parameters remained unaltered. This limited effect was still evident at termination of the study after 52 weeks. In conclusion, HD‐IVIG can improve the clinical status of patients with advanced HIV‐1 infection without obviously correcting the underlying impaired cellular immunity. The substitution of intact antibodies in the state of functional hypogammaglobulinemia is suggested as possible therapeutic mechanism.
Virological Examinations of Patients with AIDS‐Related Complex/Walter‐Reed 5 Enrolled in a Double‐Blind Placebo‐Controlled Study with Intravenous Gammaglobulin Administration.
Authors: Mertens Th. , Ramon A. , Kruppenbacher J. P. , Heitmann K. , Pika U. , Leyssens N. , Lievens M. ,   ARC‐IVIG Study Group Cologne/Berne
Published: March 2009

Abstract. Thirty patients with AIDS‐related complex/Walter‐Reed 5 enrolled in a placebo‐controlled double‐blind study with high‐dose intravenous gammaglobulin administration were tested by quantitating HIV Western blot and other serological tests for viral antibodies. Furthermore, conventional virus isolation attempts were performed. Absence or loss of p24 antibodies during the study period was associated with progression to AIDS (p = 0.01) and thereby was an earlier prognostic parameter of a poor prognosis than T4 cell count. Neither changes in antibody patterns against other HIV polypeptides, HIV titers in the immunofluorescence test nor demonstration of HIV antigen were significantly associated with progression to AIDS. Cytomegalovirus (CMV) could be isolated from two duodenal biopsies of a patient who developed AIDS at the same time, but a concomitant serological diagnosis of CMV infection was not successful. Though signs in the serology of human herpesviruses (herpes simplex virus, CMV, Epstein‐Barr virus), possibly indicating a reactivation of latent infections, could be observed in some instances, a correlation with clinical symptoms or the clinical outcome was not feasible, perhaps also because of a poor standardization of some of the test kits used. All patients were positive for IgG antibodies against the three herpesviruses when entering the study. High prevalence of hepatitis B virus (HBV) markers was found (83% anti‐HBc positive), only 1 patient being chronically infected and highly infectious, as shown by HBV‐DNA hybridization. No significant difference between treatment and placebo group was observed with the parameters tested in this study.
Cellular Immunologic Parameters in HIV‐Positive Patients with AIDS‐Related Complex and Intravenous Immunoglobulin Therapy1
Authors: Krueger G.R.F. , Ramon A. , Degenhardt St. , Schrappe‐Bächer M. , Rasokat H. , Koch B. , Deninger J.
Published: March 2009

Abstract. In a randomized double‐blind longitudinal study with 30 HIV‐1‐positive patients with AIDS‐related complex or stage Walter‐Reed 5 disease, the effectiveness of intravenous immunoglobulin (IVIG) was tested for correcting eventual immune dysregulation. Although the IVIG‐treated patients showed an improvement of their clinical score, no significant changes were observed in lymphocyte phenotypes, activation markers, immunoglobulins and subclasses, lymphocyte turnover or in indicators of acute inflammation. Since severe bacterial infections or autoimmune processes usually leading to IVIG therapy were not prevalent in the patients of the study, such therapy should probably be reserved for later stages of the disease. HIV‐1 antigen expression in blood lymphocytes remained uninfluenced by IVIG treatment.
Lymphocyte Proliferation in AIDS‐Related Complex/Walter‐Reed 5 Patients: Response to Herpes Simplex Virus and Tuberculin Antigen and Mitogen during Intravenous Immunoglobulin Treatment
Authors: Krickeberg H. , Mauff G. , Mertens Th. , Plum G. , Heitmann K. ,   The ARC‐IVIG Study Group Cologne/Berne
Published: March 2009

Abstract. In a randomized, controlled double‐blind study, 15 patients with AIDS‐related complex/Walter‐Reed 5 (ARC/WR5) were compared during 6 months intravenous immunoglobulin (IVIG) treatment (0.4 g/kg body weight every 2 weeks) with 15 placebo‐treated patients. This study was aimed at the lymphocyte response to T and B cell mitogens and antigens. 3H‐thymidine uptake was determined after stimulation with the unspecific mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM), formalinized Staphylococcus aureus‐Cowan I (SAC), and with the antigens tuberculin and herpes simplex virus (HSV) at the onset, on days 85, 183, 267 and 351; IgG and IgM antibodies against HSV were measured by ELISA. In addition, 30 untreated HIV‐negative controls were tested. For the T cell mitogen PHA, T‐cell‐dependent B cell mitogen PWM and B cell mitogen SAC, no differences between the two patient groups were observed before therapy nor in the course of therapy or the 6‐month observation period thereafter. The entire patient group showed significantly impaired mitogenic response on day 1 as compared to the controls. There was no significant difference in response to tuberculin between the patients and HIV‐negative controls, nor for both patients groups before and in the course of treatment. All patients had IgG antibodies against HSV. Three of them showed blastogenic lymphocyte response to HSV on day 1. Among 19 seropositive controls, 7 individuals showed positive HSV lymphocyte response; but for both patient groups, there was no significant difference before and in the course of the treatment and observation period. We concluded that, in spite of some clinical improvement regarding fever and fatigue during IVIG treatment of ARC/WR5 patients, there is no influence on lymphocyte function, as measured by response to mitogens and antigens.
Quantification of Complement Receptor 1 on Erythrocytes: Follow‐Up of HIV‐1‐Infected Patients with AIDS‐Related Complex/Walter‐Reed 5 under Treatment with Intravenous Immunoglobulin
Authors: Spycher M. O. , Späth P.J. ,   The ARC‐IVIG Study Group Cologne/Bern
Published: March 2009

Abstract. A sensitive flow‐cytometric method was established to quantify the number of complement receptor 1 (CR1, C3b/C4b receptor, CD35) on the surface of purified erythrocytes of 12 patients infected by HIV‐1 and showing two clinical AIDS‐related complex/Walter‐Reed 5 criteria. Erythrocytes were incubated with biotinylated monoclonal anti‐CR1 antibody followed by phycoerythrin‐streptavidin before analysis on a flow cytometer. As few as 50 binding sites/cell could be detected, making this method as sensitive as a radioimmunoassay with 125I anti‐CR1. Seven of the patients studied received an imunoglobulin preparation suitable for intravenous use during the first 6 months of the study, 5 got an equal amount of a placebo preparation consisting of human serum albumin. For a year, erythrocytes were collected and purified every 3 months, frozen and stored at ‐70°C until the end of the study, when the number of CR1 was determined. No difference between the two groups of patients was found. In 8 patients, small fluctuations of the amount of CR1/ erythrocyte were seen during the period of observation, whereas in 4 of the patients a drop of the number of CR1 was observed towards the end of the study. No correlation was found between CR1 numbers on erythrocytes and circulating immune complexes, CH50, C3 and C4 concentrations or CD4‐positive lymphocytes.
Blood Group‐Active Surface Molecules of the Human Red Blood Cell
Authors: Anstee David J.
Published: March 2009

Abstract. The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately 106 copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) has AB(H) activity and there are about 5 times 105 copies/red cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1–2 times 105 copies/red cell) are polypeptides of Mr 30,000, polypeptides of Mr 45,000–100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of Mr 30,000 and those of Mr 45,000–100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group ‘N’ antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately 105 copies/red cell. The complete protein sequence of all the above‐mentioned proteins is known, except for the Mr 30,000 and Mr 45,000–100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C. Several of the minor blood group active proteins at the red cell surface (abundance <1.2 times 104/red cell) have been the subject of recent studies. The polypeptide expressing Cromer‐related blood group antigens has been identified as decay‐accelerating factor and that carrying the Ina/Inb antigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.
Chagas' Disease and Blood Transfusion: A New World Problem?
Authors: Wendel S. , Gonzaga A.L.
Published: March 2009

American trypanosomiasis (Chagas' disease) can be transmitted by blood transfusion. For almost 40 years, this transmission has been limited to Latin America, but recently, three cases have been reported in the USA and Canada. With increasing emigration to North America and Europe, Chagas' disease may be introduced to the Northern hemisphere by transfusion of blood from carriers. This review will focus on the discovery, biology and antigenic profile of Trypanosoma cruzi (the aetiological agent of Chagas' disease), including the invertebrate vectors, animal reservoirs and transmission to humans, with special reference to blood transfusion. Finally, diagnostic tests and prophylactic measures for the prevention of Chagas' disease will be discussed.
Hepatitis A Virus Infection among the Hemophilia Population at the Bonn Hemophilia Center
Authors: Brackmann H.‐H. , Oldenburg J. , Hammerstein U. , Hanfland P. , Eis‐Hübinger A.‐M. , Gerritzen A.
Published: March 2009

Following a publication on the sudden outbreak of hepatitis A (HAV) infections in Italian hemophiliacs after treatment with a solvent detergent (S/D) virus‐inactivated factor VIII concentrate, we retrospectively examined our patients for HAV seroconversions and clinical symptoms of an acute HAV infection at our center. We found that between 1988 and 1992, 17 hemophilia A patients displayed HAV IgG seroconversions. Of these patients, 13 also had an HAV IgM seroconversion and 10 exhibited clinical symptoms of an acute HAV infection. A feature common to all was the fact that in the months prior to seroconversion they had been treated exclusively with the S/D‐virus‐inactivated product Octavi manufactured by Octapharma in Düsseldorf (FRG). None of the hemophilia A patients who had been treated with other products and who were susceptible to hepatitis A displayed seroconversion in the above‐mentioned time span.
Hepatitis A in French Hemophiliacs
Authors: Goudemand J. , Parquet A. , D'Oiron R. , Stieltjes N. , Guérois C. , Fonlupt J. , Alcalay M. , Négrier C. , Cazenave J.P. , Briquel M.E. , Fressinaud E.
Published: March 2009

The prevalence of serum antibodies to hepatitis A virus (HAV) in 793 hemophilia A (HA) and 89 hemophilia B (HB) patients coming from 10 French Hemophilia Centers and treated since 1986–1987 with solvent/detergent (SD)‐treated products is reported. The results indicated seropositivity to HAV of 29.9% in HA and 40.4% in HB patients. There was no difference among the patients according to severity of the disease, HIV serology or administration of factor VIII during the last 12 months. Seropositivity increased with age from 5.2% in HA children to 42.4% in adults (in HB the respective prevalences in the same groups were 7.7% and 56.1%). When compared to normal controls (n = 585), the prevalence of HAV seropositivity was not excessive in HA patients (n = 206). 19/20 children exclusively treated with a very‐high‐purity SD‐factor VIII concentrate (Centre régional de transfusion sanguine, Lille) remained HAV seronegative. Six cases of HAV contamination were reported in patients with severe HA, probably reflecting the level of HAV endemy in the normal population in France. No special risk of HAV transmission linked to the SD products used in France since 1986 had thus been identified.
Is the Apparent Outbreak of Hepatitis A in Belgian Hemophiliacs due to a Loss of Previous Passive Immunity?
Authors: Peerlinck K. , Vermylen J. , Goubau P. , Coppens G. , Desmyter J.
Published: March 2009

Several reports describing outbreaks of hepatitis A in hemophilia A patients transfused with solvent/detergent‐treated factor VIII concentrates have raised concern about possible transmission of hepatitis A by these concentrates. We recently witnessed such an outbreak of hepatitis A in 6 hemophilia A patients; review of the clinical data did not disclose any increased risk factor for community‐acquired hepatitis A. A case‐control study comparing the prevalence of anti‐hepatitis A IgG antibodies in hemophiliacs and age‐matched controls showed a lower seroprevalence in hemophiliacs. This might be due to passive protection acquired through transfusion of the previously used immunoglobulin‐containing cryoprecipitate. The outbreak of hepatitis A could be explained as a catch‐up phenomenon linked to the loss of passive protection with the use of purer factor VIII concentrates.
The Outbreak of Hepatitis A in Italian Patients with Hemophilia: Facts and Fancies
Authors: Mannucci P.M. , Santagostino E. , Di Bona E. , Gentili G. , Ghirardini A. , Mele A. , Schiavoni M.
Published: March 2009

To determine whether an outbreak of hepatitis A that occured in 52 Italian hemophiliacs was acquired through the infusion of contaminated factor VIII or through environmental enteric transmission, a case‐control study of the first 29 infected patients was carried out. Case patients were neither more nor less likely than controls (hemophiliacs without HAV infection) to have traveled to high‐risk countries, consumed raw shellfish or had contact with persons with jaundice. The case patients, however, were more likely than controls to have received a factor VIII concentrate treated with solvent‐detergent and to have had larger infusions of the concentrate during the presumed HAV incubation period. In the PCR analysis of HAV sequences from implicated lots of factor VIII, HAV sequences were found in 5 of 12 lots of factor VIII implicated in the outbreak. Three different strains of HAV were recovered, suggesting that lots were not contaminated from the same plasmapheresis donor. To obtain molecular evidence that the HAV detected in the factor VIII preparations was responsible for transmission of HAV, serial serum samples from two patients were tested for HAV gene sequences and compared with those of the implicated lots. The genomic sequences of HAV obtained for two matched sets of factor VIII and recipient serum samples were identical within each set, but different for the two sets.
Inactivation of Hepatitis A Virus by Pasteurization and Elimination of Picornaviruses during Manufacture of Factor VIII Concentrate
Authors: Hilfenhaus J. , Nowak T.
Published: March 2009

Hepatitis A virus (HAV) infections have been reported among hemophiliacs who received factor VIII concentrates which had been purified by ion‐exchange chromatography and treated by the solvent detergent (SD) method. Since the virus inactivation procedure of our manufacturing process is heat treatment of the stabilized, aqueous protein solution at 60°C for 10 h (pasteurization), we investigated whether this method inactivated picornaviruses such as HAV and poliovirus type 1, which we routinely use as a test virus for non‐enveloped viruses. HAV was substantially inactivated by pasteurization but the stabilizers used in the manufacturing process of the commercial products considerably delayed HAV inactivation. Residual infectious HAV was found even after 10 h heat treatment of the stabilized preparation. Thus HAV is more stable in the presence of stabilizers than poliovirus type 1. Furthermore, we studied stage by stage the elimination of poliovirus type 1 by the manufacturing procedure of these pasteurized factor VIII concentrates. Three other stages of the manufacturing process apart from pasteurization eliminated poliovirus by approximately three orders of magnitude each. Taking into account this efficient elimination of the picornavirus poliovirus and the substantial inactivation of HAV by pasteurization, we conclude that a high margin of safety exists for pasteurized factor VIII concentrates regarding HAV. This conclusion is supported by the fact that no HAV infection has been reported in hemophilia patients treated with pasteurized factor VIII concentrates. Furthermore, in a retrospective study, none of 95 patients subjected to a long‐term treatment with pasteurized factor VIII concentrates had developed anti‐HAV seroconversion as a result of this treatment.
The Natural History of Hepatitis A: The Potential for Transmission by Transfusion of Blood or Blood Products
Authors: Lemon Stanley M.
Published: March 2009

A study of the natural history and risk factors for hepatitis A can shed light on the potential for contamination of plasma concentrate with hepatitis A virus (HAV). According to the long‐term Sentinel Counties Study conducted by Alter and colleagues at the Centers for Disease Control and Prevention in Atlanta, the most frequently reported risk factors for HAV infection are living with a patient who has hepatitis, homosexual activity, and close contact with young children. International travel to hepatitis A endemic areas and illicit parenteral drug use were less frequently documented risk factors, although illicit injectable drug use has been considered more significant in other hepatitis A studies. Approximately 40% of patients with hepatitis A reported no apparent risk factors. Hepatitis A occurs most often today in the 5‐ to 30‐year‐old age group. Young adults, who are also eligible donors, are thus at risk of infection. The natural history of hepatitis A was studied in New World owl monkeys. Fecal shedding of infectious virus was detected by 4 days after intravenous injection of infectious material and peaked at almost 10 million infectious particles per gram of feces just prior to onset of chemical evidence of liver disease. Viremia of substantial magnitude occurred throughout most of the 4‐week incubation period and was maximal during the prodromal stage, prior to the development of clinical, chemical, or serologic manifestations of infection. Although the magnitude of hepatitis A viremia has not been well documented in humans, it is likely to reach levels of 104–106 infectious particles per milliliter of blood. Thus, although almost all HAV infections are acquired by the fecal‐oral route, blood‐borne transmission of hepatitis A from asymptomatic individuals who are incubating the disease and who are also plasma donors is a possibility. A review of the solvent‐detergent anion‐exchange chromatography method of producing factor VIII concentrates suggested that this process may not be adequate for the elimination of all infectious HAV from plasma pools.
CRYOPRESERVATION OF RED BLOOD CELLS
Authors: ROWE ARTHUR W.
Published: March 2009

“There is practically no mention in the literature of attempts to keep red blood cells alive in vitro, yet methods for their preservation might have much practical importance, and certainly would possess theoretical interest. Kept cells could be utilized for serum reactions or for transfusion. This last possibility is the more worth considering…” (P. Rous and J.R. Turner, J. Exp. Med. 1916)
RECENT ADVANCES IN THE PREPARATION AND STORAGE OF RED CELLS
Authors: HÖGMAN CLAES F.
Published: March 2009

The policy of blood collection for transfusion and the supply of fractionated plasma products differ between countries which has a strong impact on the choice of methods for RBC preparation and storage. Separation into components and storage of red cells in ASs are commonly used. New methods for improved preparation have been developed, allowing better standardization of the blood components. Several new compositions of AS have been described which improve quality with respect to oxygen affinity of the RBC haemoglobin and prolong shelf live. Multiple component systems using apheresis technique have been developed, particularly designed for autologous use.
Molecular Genetics of the ABO Histo‐Blood Group System
Authors: Yamamoto Fumi‐ichiro
Published: March 2009

Molecular genetic study of the histo‐blood group ABO system has elucidated the allelic basis of this genetic locus. Comparison of the nucleotide sequence has identified in the coding region differences which change amino acid sequences of the glycosyltransferases coded by these genes. Effects of the differences (mutations) on the specificity and activity of the glycosyltransferases have been examined.
Performance Evaluation of a Particle Agglutination Test for Antibody to Human Immunodeficiency Virus 1: Comparison with Enzyme Immunoassay
Authors: Elavia A.J. , Thomas A. , Nandi J. , Coyaji G.D. , Bhavalkar‐Potdar V.
Published: March 2009

A performance evaluation of a particle agglutination test (PAT), manufactured by Fujirebio Inc., Japan (Serodia‐HIV), for antibody to human immunodeficiency virus type 1 (anti‐HIV‐1) was carried out and compared with a currently available enzyme immunoassay (EIA), manufactured by Genetic Systems Corp., USA, (HIV‐1/HIV‐2 EIA). Testing 2,878 Indian donor and patient samples, both tests showed 100% sensitivity and comparable specificity (PAT: 99.8%; EIA: 99.7% among donor samples). We conclude that PAT is a specific and sensitive test for anti‐HIV‐1; it is simple to perform and does not require sophisticated equipment. Hence it is suitable for mass screening of blood donors in a developing country like India, especially in rural areas where presently no HIV‐testing facilities are available.
In vitro Cytokine Treatment of B Cell Defects in HIV‐Infected Hemophilia Patients
Authors: Weimer Rolf , Zipperle Silvia , Daniel Volker , Opelz Gerhard
Published: March 2009

HIV‐infected patients exhibit defects in B cell differentiation and in the IL‐6 response of B cells, in association with autoantibody formation against T cells. These autoantibodies have been implicated as important factors in the development of immunodeficiency disease. As the restoration of defective B cell responses might prevent autoantibody formation and the resulting immunosuppression, we studied whether in vitro treatment with recombinant IL‐2 (rIL‐2), recombinant IL‐4 (rIL‐4) or recombinant IL‐6 (rIL‐6) might restore the response of B cells of HIV‐infected patients. B cells of 6 HIV‐negative hemophilia patients, 4 HIV‐positive patients at CDC stage II, III, 4 HIV‐positive patients at CDC stage IV, and 6 healthy controls were tested in Staphylococcus aureus Cowan I (SAC‐I)‐stimulated B cell cultures and Pokeweed mitogen (PWM)‐stimulated allogeneic B and T cell cocultures. B cell differentiation was assessed in a reverse hemolytic plaque assay and by ELISA determination of IgM, IgG and IL‐6 in culture supernatants. In vitro application of rIL‐6 resulted in suppression of both elevated unstimulated and mitogen‐stimulated B cell responses in a dose‐dependent manner which was in part due to feedback inhibition. PWM‐ and SAC‐I‐stimulated IgG and IgM responses, respectively, could be restored after addition of 10 U/ml rIL‐2 in HIV‐negative patients, but not in HIV‐positive patients. Addition of rIL‐4 to cultures resulted in suppression of both unstimulated and mitogen‐stimulated IL‐6 secretion and B cell responses. Severely depressed B cell responses in CDC IV patients were not significantly affected by cytokine application. These results indicate that defective Ig responses in HIV‐negative patients may be restored by rIL‐2 treatment whereas HIV‐induced B cell defects are not corrected by supply of T cell help or cytokines promoting B cell growth and differentiation.
Cytokines in Platelet Concentrates Prepared from Pooled Buffy Coats
Authors: Klüter Harald , Müller‐Steinhardt Michael , Danzer Susanne , Wilhelm Dorothea , Kirchner Holger
Published: March 2009

Platelet concentrates (PC) prepared from pooled buffy coat (BC‐PC) contain a variable number of leukocytes from different donors. We questioned whether storage of BC‐PC can lead to a lymphocyte activation in the sense of a mixed lymphocyte reaction. BC‐PC were prepared from four ABO‐identical buffy coats and we undertook leukocyte analyses and measurement of different cytokines on days 1, 3 and 5 of PC storage (n = 72). Cytokine content was also determined in freshly prepared plasma (n = 48) and PC prepared by thrombapheresis (SD‐PC) (n = 12). As control, we studied lymphoproliferation of pooled peripheral blood mononuclear cells from four individuals in 10 mixed lymphocyte cultures (MLCs) under optimal conditions. In the BC‐PC, whole blood count and lymphocyte analysis showed a mean leukocyte contamination of 64±28 times 106 per unit with a proportion of lymphocytes of 66.7±13%. In the MLC, levels of interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) were increased on day 3 and 5 of storage (p<0.001). In a proportion of BC‐PC, tumor necrosis factor‐α (72.2%) and IL‐2 (43.1%) were detectable immediately after preparation, whereas IFN‐γ (4.2%), interleukin‐1β (4.2%) and interleukin‐8 (11.1%) were only found in some BC‐PC. In all cases, initial values of cytokines did not increase during storage. Cytokine measurement in FFP and SD‐PC showed similar results. The study demonstrates that cytokines are detectable in a variety of blood products immediately after preparation. Levels of cytokines did not increase in the preparations. BC‐PC can be stored for up to 5 days without any signs of lymphocyte activation.
Relative Frequencies of the Two O Alleles of the Histo‐Blood ABH System in Different Racial Groups
Authors: Franco Rendrik F. , Simões Belinda P. , Zago Marco A.
Published: March 2009

O blood group results from the absence of glycosyltransferase activity, which is due most commonly to a single nucleotide deletion in the glycosyltransferase gene. A second type of O allele resulting from three nucleotide substitutions in the glycosyltransferase gene had its frequency recently determined in a Danish population. However, its frequency among different human populations is not known. The frequencies of the two types of O alleles were determined by DNA analysis of the glycosyltransferase gene of group O individuals of three racial groups (Caucasians, blacks and Amerindians). The mean frequency of carriers of the three‐base mutation group O gene among blacks and Caucasians is 4.7%; the mutation was not observed among 100 Amerindian chromosomes. The data reveal the relative frequencies of O alleles in different racial groups, and demonstrate that the origin of this variant predates racial divergence, since it is found equally among blacks and whites.
A Combination of Two Distinct in vitro Amplification Procedures for DNA Typing of HLA‐DRB and ‐DQB 1 Alleles
Authors: Fischer Gottfried F. , Faé Ingrid , Petrasek Monika , Moser Sabine
Published: March 2009

The differential hybridisation of oligonucleotide probes to polymerase chain reaction (PCR)‐amplified DNA has become a standard procedure for tissue typing. We describe a typing method in which differential ligation replaces differential hybridisation, which is a significant simplification of this strategy. After amplification by the PCR two labelled, sequence‐specific oligonucleotides hybridise, in the fluid phase, to one strand of heat‐denatured amplification product in juxtaposition. In the case of perfectly complementary sequences surrounding the gap, a thermostable ligase catalyses the ligation of the two oligonucleotides, otherwise they stay separated. The use of heat‐resistant ligase enables easy repetition of the denaturation‐annealing‐ligation cycle in a thermocycler. The ligation products are detected by an enzyme linked immunosorbent assay. We tested this typing approach in a model system, the characterisation of three functional alleles of HLA‐DRB 3 using three probe pairs. No discrepancies were observed in typing 100 individuals of known genotypes. A total of 33 probe pairs combined with generic and group‐specific amplification allowed the typing of alleles of HLA‐DRB and ‐DQB1 loci at low resolution. We confirmed ligation‐based typing results of 259 individuals with sequence‐based HLA‐DRB1 typing and HLA‐DQB1 typing using PCR with sequence‐specific primers (SSPs). In addition, more than 1,500 ligation‐based HLA‐DRB1 typings were concordant with SSP typing. Excellent signal‐to‐noise ratios in the enzyme‐linked immunosorbent assay make ligation‐based typing remarkably robust. The time requirement of 2.5 h post‐PCR enables practicable typing of putative organ donors. The whole procedure is more easily amenable to automation than methods based on differential hybridisation requiring additional incubators and extra handling for hybridisation and washing.
Neonatal Alloimmune Thrombocytopenia in Taiwan Due to an Antibody against a Labile Component of HPA‐3a (Bak)
Authors: Lin Marie , Shieh Shiow‐Hua , Liang Der‐Cherng , Yang Ting‐Fan , Shibata Yoichi
Published: March 2009

We report on two siblings who developed severe neonatal alloimmune thrombocytopenia (NAIT) due to an alloantibody against a labile component or components of the HPA‐3a (Baka) antigen. The antibody reacted only with fresh, unfixed platelets by the solid‐phase red cell adherence test, immunofluorescence test and mixed passive haemagglutination test. In the latter method, weakly fixed platelets also gave a weak positive reaction. Monoclonal‐antibody‐specific immobilization of platelet antigens and immunoblotting tests gave negative results. Our findings may possibly help to explain why in some cases of NAIT no platelet‐specific antibody is demonstrable in tests with fixed or solubilized platelets.
Post‐Transfusion Hyperhaemolysis in a Patient with Sickle Cell Disease: Use of Steroids and Intravenous Immunoglobulin to Prevent Further Red Cell Destruction
Authors: Cullis J.O. , Win Nay , Dudley J.M. , Kaye T.
Published: March 2009

Delayed haemolytic transfusion reactions (DHTRs) are seen more frequently in patients with sickle cell disease (SCD) than in other groups of patients, and are characterised by a positive direct antiglobulin test and the appearance of previously undetected red blood cell (RBC) alloantibodies in the patient's serum. Recently a syndrome of post‐transfusion hyperhaemolysis has been described in children with SCD, characterised by destruction of both autologous and transfused RBCs with negative serological findings: continuation of RBC transfusion exacerbated haemolysis further. We describe a case of life‐threatening posttransfusion hyperhaemolysis in an adult patient with SCD in whom severe anaemia necessitated further RBC transfusion, which was successfully performed in conjunction with intravenous immunoglobulin. This approach may be useful in the management of post‐transfusion hyperhaemolysis in SCD as well as in the management of severe DHTRs.
Thermal Stability of Human Immunoglobulins with Sorbitol: A Critical Evaluation
Authors: González Martin , Murature Domingo A. , Fidelio Gerardo D.
Published: March 2009

The effect of the additive sorbitol on the thermal stabilization of human IgG was investigated by differential scanning calorimetry and size exclusion chromatography. In the presence of 33% sorbitol, the temperature at which denaturation of IgG began (Ti) was increased from 52 to 65°C. Similarly, the temperature of the maximum heat capacity (Tmax) was increased from 69 to 76°C. Sorbitol also decreased dimer aggregation and the extent of oligomerization during heating compared with IgG dissolved in phosphate buffer. Sorbitol at 33% prevented massive protein denaturation but a 10–15% of oligomerization of high molecular weight aggregates with turbidity could not be avoided when heating for 10 h at 60°C. The use of sorbitol 33% to avoid heat denaturation of human IgG during viral inactivation did not prevent protein aggregation or the appearance of turbidity. Consequently, further processing will be required to achieve a product suitable for pharmaceutical use.
A Solid‐Phase Test Using Antiglobulin and Enzyme‐Enhanced Techniques for Erythrocyte Antibody Screening during Pregnancy
Authors: Sallander S. , Shanwell A. , Pegert S. , Jakobsson L. , Wiechel B.
Published: March 2009

We have modified previously described solid‐phase tests for erythrocyte antibody screening to develop a method, suitable for antiglobulin‐ and enzyme‐enhanced techniques (SPH‐IAT and SPH‐ENZ). In this study we compared the SPH tests with an autoanalyzer (AA) technique. The results were more specific with the SPH tests than with the AA. Of 4,234 unselected samples from pregnant women, screen‐positive samples were reduced from 96 (2.27%) by the AA, to 56 (1.32%) by the SPH tests. This difference was due to the reduced number of false‐positive reactions with the SPH tests, 0.47% compared to 1.44% with the AA. Antibodies detected by the AA and the SPH‐IAT and ‐ENZ were: 9 Rh prophylaxis, 2 anti‐c, 1 anti‐K and anti‐M, and 1 anti‐Jka. Antibodies detected only by the SPH tests were 1 anti‐K, 1 anti‐Lea (SPH‐IAT and ‐ENZ), 1 anti‐M (SPH‐IAT) and 4 anti‐Jka (SPH‐ENZ). One anti‐C, 2 anti‐D, 3 Rh prophylaxis and 1 anti‐E were detected by the AA and the SPH‐ENZ but failed to react by the SPH‐IAT. One anti‐Leaand 8 Rh prophylaxis antibodies were detected by AA only. All clinically important antibodies were detected by the SPH tests. We conclude that the SPH‐IAT and SPH‐ENZ are screening methods with high specificity that are readily adaptable to larger series of samples from pregnant women and suitable for automated handling throughout the screening and identification process.
K12 Is Located on the Kell Blood Group Protein in Proximity to K/k and Jsa/Jsb
Authors: Reid M.E. , Øyen R. , Redman C.M. , Gillespie G. , Jones J. , Eckrich R.
Published: March 2009

K12 is a high‐prevalence antigen, with no known antithetical partner, that is associated with the Kell blood group system. We report studies on the 5th propositus (MS) with the K:‐12 phenotype and the second case to show that the K12 is inherited. The anti‐K12 in his serum did not destroy antigen‐positive incompatible red cells transfused on at least 3 occasions over 6 years. Red cell membranes from MS possessed the Kell protein that was indistinguishable from control membranes. K12 antigen was shown by immunoprecipitation to be on a protein with an apparent molecular mass of 93,000 and by monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay to be on the Kell protein in proximity to K/k and Jsa/Jsbantigens. These data remove the K:‐12 phenotype from its current Kell‐related (or para Kell) status and elevates the K12 antigen to a bona fide member of the Kell blood group system.
Evidence that the Granulocyte‐Specific Antigen NC1 Is Identical with NA2
Authors: Bux J. , Behrens G. , Leist M. , Mueller‐Eckhardt C.
Published: March 2009

The neutrophil‐specific antigen NC1 is defined by an antibody in the serum of a mother who gave birth to a child with alloimmune neonatal neutropenia. NC1 has been reported to be associated with the neutrophil‐specific antigen NA2, but the precise relation of NC1 and NA2 remained unclear. Therefore, we investigated the serum using the antigen capture assay MAIGA and the granulocyte (GIFT) and lymphocyte (LIFT) immunofluorescence tests. In GIFT, no NA association was observed. In LIFT, serum antibodies bound preferably to lymphocytes with the HLA antigens HLA‐B7 and cross‐reacting antigens. In MAIGA, an antibody specific for the NA2 variant of the granulocyte Fcγ‐receptor III was observed. The NA2 specificity was confirmed by testing granulocytes from 40 further different donors. This indicates that the NC1 and NA2 antigens are identical. A positive GIFT result but a negative one in LIFT using cells of an NA2‐negative typed individual suggest the presence of an additional, non‐NA2‐specific granulocyte antibody.
Characterization of Mutants of the Vitamin‐D‐Binding Protein/Group Specific Component: GC Aborigine (1A1) from Australian Aborigines and South African Blacks, and 2A9 from South Germany
Authors: Kofler Andrea , Braun Andreas , Jenkins Trefor , Serjeantson Sue W. , Cleve Hartwig
Published: March 2009

The structure and organization of the human vitamin‐D‐binding protein gene (DBP, group‐specific component, GC) have recently been determined. Each ex‐on may now be amplified by the PCR method using oligonucleotide primers deduced from the intron sequences near their 5′ ends and 3′ ends. In this study we examined the anodal GC variants 1A1 and 2A9. Genomic DNA of the variant 1A1 was obtained from Australian Aborigines and from South African Bantu‐speaking Blacks. Amplification and sequencing of exon 11 of 1A1 revealed a point mutation in codon 429 at the second position. It is remarkable that this mutation was found in the Australian 1A1 variant and in the African 1A1 variant, and raises the question whether the mutation in these two ethnic groups has a common origin. Genomic DNA of the 2A variant called 2A9 was obtained from South Germany and a point mutation also concerning position 429 in exon 11 was found. The nucleotide exchange in this case, however, was at the first position of the codon. The widely distributed genetic polymorphism of DBP/GC is located in exon 11 and is characterized by substitution at amino acid positions 416 and 420. Variant 1A1 is due to a second site mutation of the allele GC*1F; variant 2A9 is due to a mutation in the GC*2 allele.
A Method to Collect, Store and Issue Multiple Aliquots of Packed Red Blood Cells for Neonatal Transfusions
Authors: Strauss R. G. , Villhauer P.J. , Cordle D.G.
Published: March 2009

Premature neonates require multiple red blood cell (RBC) transfusions. Single‐donor programs have been proposed as a means to limit donor exposures, but methods must be developed to collect, store long‐term and issue multiple aliquots of RBCs from a single donor. We evaluated a method by which RBCs could be collected, leukocyte depleted, repeatedly centrifuged for issuance as multiple small aliquots of high‐hematocrit cells and then resuspended for continued storage throughout 42 days. The quality of RBCs handled by the method were compared to cells stored in standard fashion. Leakage of intracellular potassium, hemoglobin and lactic dehydrogenase into the extracellular fluid from RBCs processed by either method was comparable — indicating maintenance of RBC integrity. Multiple cultures, taken throughout the period of storage, were sterile to document that extensive handling did not introduce contamination. This new method appears promising as a means to provide RBCs for neonates.
Pooled Platelet Concentrates Prepared by the Platelet‐Rich‐Plasma Method and Filtered with Three Different Filters and Stored for 8 Days
Authors: Boomgaard M. N. , Gouwerok C. W.N. , Palfenier C.H. , Pankalla‐Blandeau I.E. , Veldman H.A. , de Korte D. , Loos J.A.
Published: March 2009

Pooled platelet concentrates (PC) prepared by the platelet‐rich plasma (PRP) method were filtered with three different filters and stored for 8 days at room temperature. The effect of filtration on leukocyte contamination, platelet concentration, and the in vitro function, morphology, metabolism and activation of platelets were studied. Eight pools of 20 PRP‐PC were used, each pool was split into 4 equal volumes; 3 were filtered over a PL50HF, a PL‐10A and a Bio P10 filter, the 4 served as a control. After filtration, leukocyte counts exceeded 3×105 in none of the pooled PC. Platelet loss induced by filtration was about 17%. During storage, no differences in pH, PCO2, and lactate and glucose concentration were found between the filtered and the unfiltered units, nor were any differences observed between filtered and unfiltered pooled PC in aggregation upon stimulation with collagen and/or ADP, adhesion capacity to collagen in flowing blood, nucleotide content of the platelets and nucleobase concentration in the plasma, expression of activation‐dependent antigens, or platelet morphology as observed by light microscopy and by the swirling effect. Selective removal of β‐thromboglobulin (22%) by the PL50HF filter was observed. Pooled PC prepared by the PRP‐method can be filtered and stored for 8 days without detrimental effect on platelet function, metabolism or activation.
Blood Group Antigen Survival on Freeze‐Dried Erythrocytes and Ghosts after Isolation
Authors: Ruspini F. , Petitpain N. , Marchand‐Arvier M. , Vigneron C. , Janot C.
Published: March 2009

To screen and identify irregular antibodies, whatever the technique used, fresh erythrocytes (RBCs) are needed to set up the panel. Solid‐phase tests using dried blood cells are available, but the technique is based on the adherence of sensitized RBCs, which have a short life span. We have checked antigen survival on membranes with a saline test and an antiglobulin test for two methods to preserve the antigen substrate: freeze‐drying of RBCs and preparation of RBC membranes. The different antigens of the ABO, Rhesus, Kell, P, Lewis, MNSs, Lutheran, Duffy, Kidd and Li systems are well recognized on the membranes after isolation and on freeze‐dried cells. Demonstration of antigen survival leads us to consider using membranes or freeze‐dried cells in new immunological tests.
Evaluation of a Combined HIV‐1/2 and HTLV‐I/II Assay for Screening Blood Donors
Authors: Flanagan P. , McAlpine L. , Ramskill S.J. , Smith A.G. , Eglin R. , Parry J.V. , Mortimer P.P.
Published: March 2009

A combined immunoassay for the simultaneous detection of antibodies to HIV‐1/2 and HTLV‐I/II (Bioelisa, Launch Diagnostics) has been evaluated to determine its suitability for routine use in blood donor screening. 84,222 donations were tested from 76,452 donors. One HIV‐ and 1 HTLV‐I‐positive donor were identified. The specificity was 99.7%, and the sensitivity for anti‐HIV‐1, anti‐HIV‐2, and anti‐HTLV‐I on 173 positive sera was 100%; 2 of 25 anti‐HTLV‐II‐positive sera were non‐reactive. Although the specificity of the assay is not as high as that of HIV‐1/2 kits currently in UK transfusion use, the information gained about donor HTLV antibody status makes the test an attractive alternative to them.
Simultaneous DNA Typing of Human Platelet Antigens 2, 3 and 4 by an Allele‐Specific PCR Method
Authors: Tanaka Shigenori , Taniue Atsuko , Nagao Nobuo , Ohnoki Shiro , Shibata Hirotoshi , Okubo Yasuto , Yamaguchi Hideo
Published: March 2009

We developed an allele‐specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy‐three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA‐2, ‐3 and ‐4 in parallel without the use of platelets, platelet‐specific alloantibodies or restriction enzymes.
The Immunoglobulin VH Gene, VH4–21, Specifically Encodes Autoanti‐Red Cell Antibodies against the I or i Antigens
Authors: Smith Graham , Spellerberg Myfanwy , Boulton Frank , Roelcke Dieter , Stevenson Freda
Published: March 2009

Most autoanti‐red cell antibodies found in patients with cold agglutinin disease are specific for the I or i carbohydrate antigenic determinants. However, antibodies specific for other antigens such as Pr or Sa can also be found, and these are identified by their pattern of reactivity with enzyme‐treated red cells. Recently, it has been shown that the vast majority of anti‐Ii antibodies react with a monoclonal anti‐idiotypic antibody (9G4); this reactivity arises from restriction of the immunoglobulin heavy chains used to encode the antibodies to a single VH4–21 gene, VH4–21. The 9G4 antibody appears specific for this gene product, and we have used it to analyse VH4–21 gene involvement in encoding a spectrum of red cell antibodies of various specificities. The results support the strong association between usage of this gene and anti‐Ii specificity and indicate that it is not generally used by other specificities. In particular, it is striking that the unsubstituted type 2 oligosaccharide antigens (I and i) induce a highly restricted autoantibody response very different from that induced by the sialylated type 2 antigens (Sia‐b,‐1 and lb). The 9G4 antibody therefore provides a simple tool for discrimination between these autoanti‐red cell antibodies, which should be of use in red cell serology.
Mild Course of Fetal Rh D Haemolytic Disease due to Maternal Alloimmunisation to Paternal HLA Class I and II Antigens
Authors: Eichler H. , Zieger W. , Neppert J. , Kerowgan M. , Melchert F. , Goldmann S.F.
Published: March 2009

The patient is a pregnant women of African origin with a prior history of spontaneous abortion and newborn dystrophy. The investigation showed an anti‐Rh D antibody (IgG isotypes 1 and 3) with an indirect antiglobulin tube test (IAT) titre of 1:512. The monocyte monolayer assay (MMA) proved clearly the interaction of Fc receptors with the maternal anti‐D, and so a clinical significance was expected. In spite of this, no signs of severe haemolysis in the Rh‐D‐positive and direct antiglobulin test‐positive fetus could be observed. Furthermore, two HLA class I and II alloantibodies (anti‐A10, anti‐DR13) directed against paternal and fetal antigens were detected in the serum of the gravida. Both antibodies showed an inhibitory effect on the in vitro phagocytosis capacity of mononuclear cells expressing at least one of the corresponding HLA antigens (immunophagocytosis inhibition (IPI) test). Thus, the mild course of haemolytic disease may be explained by an effective inhibition of the fetal mononuclear phagocyte system by maternal HLA class I and/or class II antibodies resulting in a diminished destruction of anti‐D‐coated fetal red blood cells.