bluuebunny/crossref_metadata_2025 huggingface dataset:
Intra‐apheresis recruitment of blood progenitor cells in children
Authors: Abe T. , Makimoto A. , Kawano Y. , Okamoto Y. , Kajiume T. , Nakagawa R. , Watanabe H. , Sato J. , Watanabe T. , Kuroda Y. , Takaue Y.
Published: March 2003

BACKGROUND: Determination of the optimal duration of apheresis requires a careful examination of blood progenitor cell (BPC) kinetics during apheresis. Intra‐apheresis recruitment of BPCs should be evaluated.

STUDY DESIGN AND METHODS: Twenty‐six apheresis procedures were performed in 13 children with various malignant disorders (ages, 10 months to 17 years; median, 7 years) to collect BPCs for autologous transplant, using a blood cell separator with 2 to 5.2 blood volumes processed. The subjects were divided into three groups according to age: below 1 year (n = 4), 2 to 10 years (n = 5), and 11 to 20 years (n = 4). BPCs were mobilized by a combination of chemotherapy and granulocyte‐colony‐stimulating factor (G‐CSF; 2–7.5 micrograms/kg/day intravenous drip). The levels of circulating CD34+ cells and colony‐ forming units‐granulocyte‐macrophage (CFU‐GM) were monitored to examine intra‐apheresis recruitment. For every 50 mL per kg or 2 L of processed blood, 5‐mL blood samples were collected via a central line.

RESULTS: In the first apheresis procedure, more CD34+ cells were mobilized by the procedure itself in the infant group than in the older groups, and the number of cells decreased with the subject's age. When the same analysis was made during the second apheresis procedure, performed 1 day later, the levels of both CD34+ cells and CFU‐GM had decreased to below the preapheresis values in all of the populations. Cell yields in the second apheresis procedure were significantly lower than those in the first.

CONCLUSION: Although several factors prevent a reliable analysis, the data suggest that the intra‐apheresis recruitment of BPCs may be age‐specific; the continuous and prolonged supply of cells from the bone marrow to peripheral blood that occurs during apheresis is more predominant in infants, which leads to the collection of proportionately more BPCs in younger children than in their older counterparts.
Rapid typing of the human Fc gamma receptor IIA polymorphism by polymerase chain reaction amplification with allele‐specific primers
Authors: Flesch B.K. , Bauer F. , Neppert J.
Published: March 2003

BACKGROUND: The human Fc gamma receptor IIa (Fc gamma RIIa) is expressed in two polymorphic forms, Fc gamma RIIa‐H131 and Fc gamma RIIa‐R131, that differ by the replacement of histidine by arginine at position 131. This replacement is caused by a single‐nucleotide exchange of A–>G. The resulting receptor forms differ in their binding to human IgG2 and mouse IgG1, which may lead to a different immunologic defense to bacterial polysaccharides and encapsulated bacteria. STUDY DESIGN AND METHODS: A rapid and easy polymerase chain reaction(PCR) method of genotyping the Fc gamma RIIa was developed. Allele‐specific primers discriminate between the Fc gamma RIIa‐H131 and the Fc gamma RIIa‐R131 forms of the receptor. The results were compared with those obtained by another DNA‐based genotyping method, in which PCR‐amplified DNA was hybridized with allele‐specific oligonucleotides, and with a functional phagocytosis assay using mouse IgG1‐coated red cells as target antigens. RESULTS: The genotypes deduced from the PCR with allele‐specific primers were in complete accordance with those obtained by the data from the hybridization of PCR‐amplified DNA with allele‐ specific oligonucleotides. Furthermore, the Fc gamma RIIa genotypes of 28 individuals in all cases corresponded to the functional phenotypes. CONCLUSION: The use of PCR with allele‐specific primers provides a rapid and easily performed method for the determination the Fc gamma RIIa polymorphism.
A combination of low‐dose cyclophosphamide and colony‐stimulating factors is more cost‐effective than granulocyte‐colony‐stimulating factors alone in mobilizing peripheral blood stem and progenitor cells
Authors: Meisenberg B. , Brehm T. , Schmeckel A. , Miller W. , McMillan R.
Published: March 2003

BACKGROUND: The use of peripheral blood progenitor cells (PBPCs) instead of autologous bone marrow leads to more rapid engraftment following high‐dose chemotherapy. Mobilization regimens differ with respect to toxicity, efficiency, and cost. STUDY DESIGN AND METHODS: Two cohorts of patients with breast cancer received one of two mobilization regimens: granulocyte‐colony‐stimulating factor (G‐CSF) at 10 micrograms per kg was given subcutaneously for 5 days, with leukapheresis begun on Day 6, or low‐dose cyclophosphamide followed by sequential granulocyte‐macrophage‐CSF (GM‐CSF) at 5 micrograms per kg for 5 days and by G‐CSF at 10 micrograms per kg, with leukapheresis begun on Day 11. Results of CD34+ cell collection, engraftment, and costs of mobilization were determined. RESULTS: The combination chemotherapy and growth factor regimen was more efficient in mobilizing CD34+ cells. Sixty‐six percent of patients reached a target 4 × 10(6) CD34+ cells per kg in a single leukapheresis session with the combination regimen, compared to 14 percent who received G‐CSF alone (p < 0.01). The mean number of leukapheresis sessions required to reach a target of 4 × 10(6) CD34+ cells per kg was 1.3 for the combination regimen and 2.7 for the regimen of G‐CSF alone (p < 0.01). One patient in the chemotherapy and growth factor group developed febrile neutropenia. Engraftment was similar in both cohorts of patients. The cost of mobilization, including all supplies and cryopreservation, was $7381 for the G‐CSF regimen and $5508 for the chemotherapy regimen (p < 0.05). This reduction was attributed to the lower number of leukapheresis and cryopreservation sessions, which outweighed the slight increase in expense for chemotherapy and growth factor in the combination regimen. CONCLUSION: This combination mobilization regimen allowed the predictable and efficient collection of CD34+ cells from the peripheral blood in a limited number of leukapheresis sessions, which reduced the cost of mobilization by approximately 25 percent.
Viremia, genetic heterogeneity, and immunity to hepatitis G/GB‐C virus in multiply transfused patients with thalassemia
Authors: Zemel R. , Dickman R. , Tamary H. , Bukh J. , Zaizov R. , Tur‐Kaspa R.
Published: March 2003

BACKGROUND: Thalassemia patients are at high risk for posttransfusion hepatitis. Hepatitis G virus (HGV) has been suspected of being responsible for acute and chronic hepatitis. STUDY DESIGN AND METHODS: The prevalence of HGV infection, its possible association to liver disease, the genetic heterogeneity among the various HGV isolates, and immunity to HGV were studied in 36 thalassemia patients with reverse transcriptase‐polymerase chain reaction assay and sequence analysis. RESULTS: HGV RNA was detected in seven patients (19.4%), only two of whom had evidence of hepatitis C virus infection as well. Sequence analysis of the NS3 gene from isolates of the five patients infected with HGV alone revealed 84.7 to 90.9 percent homology at the nucleotide level. Prolonged HGV viremia was not associated with significant liver enzyme elevation. All five patients were chronically infected with the same viral strain. E2 antibodies were detected in 57 percent of the HGV‐ nonviremic patients and in only 1 of 7 viremic patients. CONCLUSION: HGV is associated with persistent viremia but not with significant biochemical evidence of liver damage. There is some genetic heterogeneity among HGV isolates from thalassemia patients in Israel.
The effects of dilution on the outcome of pooled plasma testing with HIV type 1 (HIV‐ 1) RNA genome amplification as compared to the outcome of individual‐unit testing with other HIV‐1 markers
Authors: Kemper M. , Witt D. , Madsen T. , Kuramoto K. , Holland P.
Published: March 2003

BACKGROUND: Proposed testing of large plasma pools with genome amplification technology (GAT) for detection of transfusion‐ transmissible viruses may have unanticipated complications not associated with individual unit testing. One such potential complication, the effect of dilution resulting from pool formation, was the subject of the present study.

STUDY DESIGN AND METHODS: Specimens from three plasma donor HIV type 1 (HIV‐1) seroconversion panels were tested with a quantitative HIV‐1 RNA GAT assay (lower detection limit, 400 copies). GAT results were compared to HIV‐1 p24 antigen and anti‐ HIV‐1/2 enzyme immunoassay results. Effects of dilution on the detection of GAT‐positive panel specimens were assessed by terminal dilution with pooled volunteer‐donor EDTA plasma samples.

RESULTS: Low HIV‐1 RNA copy numbers (755 and 890 copies/0.1‐mL input) that were detectable in two individual plasma specimens before HIV‐1 p24 antigen were subsequently undetectable by GAT upon dilution with an equal volume of nonreactive plasma from a single donor. HIV‐1 RNA at higher copy numbers (15,500 copies/0.1‐mL input) in an HIV‐1 p24 antigen‐ reactive and anti‐HIV‐1/2‐nonreactive specimen was undetectable when diluted to 1‐in‐50 (1‐in‐50). Terminal dilution of seven HIV‐1 RNA‐ containing plasma panel specimens indicated a proportional loss of HIV‐ 1 RNA detectability with increasing dilution.

CONCLUSION: GAT for detection of HIV‐1 RNA in individual specimens was more sensitive than other HIV markers. For pooled plasma testing, GAT‐independent constraints, such as effects of dilution, may preclude the use of GAT detection as a replacement for individual unit testing with HIV serologic assays.
White cell subsets in apheresis and filtered platelet concentrates
Authors: Sowemimo‐Coker S.O. , Kim A. , Tribble E. , Brandwein H.J. , Wenz B.
Published: March 2003

BACKGROUND:White cell (WBC)‐reduced platelet concentrates (PCs) are defined by their absolute WBC count, a criterion which provides no information regarding the various WBC subsets contained in the PC. These heterogeneous cells are known to mediate different physiologic and pathophysiologic functions and account for distinct adverse transfusion responses. This study describes a method which allows the detection and quantification of these subsets and characterizes their presence in a variety of platelet components.

STUDY DESIGN AND METHODS:Random‐donor pooled PCs (RD PCs) and single‐donor apheresis PCs (SD PCs) were studied. RD PCs consisting of 6 units of 2‐ to 3‐day old PCs were randomly assigned to be filtered with one of four WBC‐reduction filters from three different manufacturers (n=34). The residual WBCs were pelleted by centrifugation and isolated on a density gradient. The various WBC subsets were quantified by flow cytometry in unfiltered and filtered PCs using fluorescence and two‐angle light scatter. SD PCs obtained with two manufacturer's systems and three processing protocols (n=30) were studied in like manner.

RESULTS:WBC counts for non‐WBC‐ reduced PCs averaged 3 × 10(8) in RD PCs and ranged from 8.6 to 9.6 × 10(6) per SD PC. Residual WBC counts in filtered PCs ranged from 2.3 × 10(4) to 2.2 × 10(5) and those in WBC‐reduced SD PCs averaged 2.2 × 10(5) per unit. The data demonstrate significant phenotypic differences among PCs produced with various procedures. All SD PCs and two of four filtered RD PCs contained five WBC populations including granulocytes and monocytes, while RD PCs filtered with the remaining manufacturer's devices contained only lymphocytes. CONCLUSION: The data confirm that distinct phenotypic differences exist among PCs prepared with different devices and/or procedures. It is suggested that as for non‐generic pharmaceuticals, the clinical benefits of these various PCs should be individually proved.
A Gly565→Als substitution in human erythroid band 3 accounts for the Wu blood group polymorphism
Authors: Zelinski T. , McManus K. , Punter F. , Moulds M. , Coghlan G.
Published: March 2003

BACKGROUND: Reports published in 1976 and 1980 described the low‐ incidence red cell antigen Wu. Distinction of Wu from all other known low‐incidence antigens and from the ABO, Rh, Lutheran, Duffy, Kidd, P, and X‐linked blood group systems allowed Wu to be placed in the International Society of Blood Transfusion's 700 series, designated as 700013. Recently, a blood donor apparently homozygous for Wu has been identified. This report documents the serologic and molecular findings of samples from this individual and the members of his family.

STUDY DESIGN AND METHODS:Blood samples from 26 members of a kindred of Dutch descent segregating for Wu were collected and analyzed. Red cells were subjected to titration and enzymatic tests, while DNA was analyzed by polyacrylamide gel electrophoresis for single‐strand conformational polymorphism (SSCP) and nucleotide differences by DNA sequencing.

RESULTS:Serologic investigations conducted on red cells of the propositus and two of his siblings consistently revealed higher titers with various sera containing anti‐Wu than did cells from their parents or children. Treatment of intact red cells with alpha‐chymotrypsin completely abolished Wu recognition. Because erythroid band 3 is cleaved by alpha‐chymotrypsin, the possible relationship between Wu and AE1 (the gene controlling erythroid band 3 expression) was investigated by molecular methods. SSCP analysis of DNA revealed that all Wu+ family members exhibited a mobility shift in exon 14 of AE1. The nature of the SSCP was defined by DNA sequencing as a G–>C mutation that resulted in a Gly565–>Ala substitution in human erythroid band 3.

CONCLUSIONS:Three members of the kindred are homozygous for the low‐incidence red cell antigen Wu. A G–>C mutation in AE1 gives rise to a Gly565–>Ala substitution in band 3, thereby accounting for the Wu red cell polymorphism. In light of these findings, the International Society of Blood Transfusion Working Party has provisionally assigned Wu to the Diego blood group system (designated 010009 or D19).
Blood component recalls in the United States
Authors: Ramsey G. , Sherman L. A.
Published: March 2003

BACKGROUND: United States blood suppliers are required to recall marketed blood components later found to be in violation of Food and Drug Administration (FDA) regulations for safety, purity, and potency. Many recalled units have already been transfused. Analysis of the frequency and nature of blood component recalls would be useful for blood suppliers, transfusion services, and physicians.

STUDY DESIGN AND METHODS: Each blood component recall in the weekly FDA Enforcement Report from 1990 through 1997 was examined for the number of units, recall reason, and hazard class. Units for manufacturing were excluded.

RESULTS: In 8 years, an estimated 241,800 blood components were recalled, or approximately 1 in 700 units available to US hospitals. Eighty‐eight percent of recalled units were in 22 large recalls of over 1000 units each. The most common reasons were incorrect testing for syphilis (57% of units) or viral markers (19%), reactive or previously reactive donor viral markers (6‐11%), and inadequate donor‐history screening (4%). Twelve units were in the FDA's highest hazard Class I, 24 percent were in Class II, and 76 percent were in Class III. Over 43,900 units had HIV‐related problems, but only 3 units involved HIV transmission. Large recalls have declined since peaking in 1995, but units in small recalls increased 116 percent in 1997 over the previous 7‐year average.

CONCLUSIONS: Although high‐risk recalls are rare, many blood component recalls pose medical concerns for physicians and patients. The recent decline in large recalls may be due to increased FDA oversight, stricter accreditation standards for quality improvement, and more centralized donor testing in large specialized laboratories. However, smaller recalls, which involve nearly all blood suppliers, were sharply higher in 1997.
The timing of granulocyte–colony‐stimulating factor administration after chemotherapy does not affect stem and progenitor cell apheresis yield: a retrospective study of 65 cases
Authors: Lefrère F. , Audat F. , Hermine O. , Cavazzana‐Calvo M. , Belanger C. , Arnulf B. , Buzyn A. , Varet B.
Published: March 2003

BACKGROUND: The optimal time for postchemotherapy granulocyte–colony stimulating factor (G–CSF) administration before peripheral blood stem and progenitor cell (PBPC) collection is not well defined. The impact of G–CSF scheduling on the number of CD34+ cells collected by leukapheresis from 65 patients with malignant disease was studied retrospectively.

STUDY DESIGN AND METHODS: Chemotherapy was performed on Days 1 and 2 and was followed by G–CSF to mobilize PBPCs. In Group 1, 30 patients received the first dose of G–CSF immediately after the end of chemotherapy, as commonly recommended. In Group 2, 35 patients received the first G–CSF dose after the end of chemotherapy (Days 7 or 8).

RESULTS: No difference was observed between the two groups in white cell recovery and the median number of CD34+ cells harvested. The number of leukapheresis procedures necessary to obtain the minimal number of 3 × 106 CD34+ cells per kg was the same. The proportion of patients with a failure of PBPC collection was similar, and G–CSF consumption was reduced in Group 2 without increasing infectious risks.

CONCLUSION: Early administration of G–CSF after chemotherapy appears not to be a prerequisite for satisfactory PBPC collection. This approach could allow significant savings in terms of medical cost. A randomized and prospective study would be necessary, however, to assess the validity of these conclusions.
Rapid phenotyping of HPA‐1a using either diabody‐based hemagglutination or recombinant IgG1‐based assays
Authors: Watkins N.A. , Armour K.L. , Smethurst P.A. , Metcalfe P. , Scott M.L. , Hughes D.L. , Smith G.A. , Williamson L.M. , Clark M.R. , Ouwehand W.H.
Published: March 2003

BACKGROUND: The HPA‐1 system is carried on the β3 integrin. HPA‐1a (Zwa, PlA1) is immunogenic in an HPA‐1b homozygote (HPA‐1b1b). In pregnancy, 1 of 365 women forms anti‐HPA‐1a, which causes severe thrombocytopenia in 1 in 1100 neonates. Identification of women at risk of forming anti‐HPA‐1a and the screening of donors to obtain HPA‐1a‐negative platelets for therapy need reliable, low‐cost, automated assays.

STUDY DESIGN AND METHODS: A diabody with dual specificity for HPA‐1a × D and an IgG1 anti‐HPA‐1a have been constructed by the use of the genes encoding the first anti‐HPA‐1a fragment. With these reagents, two complementary HPA‐1a phenotyping assays have been developed.

RESULTS: This diabody was used in a simple hemagglutination technique to perform HPA‐1a phenotyping on soluble glycoprotein IIb/IIIa from EDTA plasma samples. Over 1000 unselected donors have been correctly HPA‐1a‐phenotyped by use of the diabody. The human recombinant IgG1 anti‐HPA‐1a was produced in a rat myeloma cell line and was fluorescein labeled for use in a whole‐blood flow cytometric HPA‐1a phenotyping assay. This IgG1 anti‐HPA‐1a shows a clear differential between HPA‐1a‐positive and HPA‐1a‐negative platelets at nM antibody concentrations.

CONCLUSIONS: The two recombinant reagents described are highly suitable for screening and confirmatory HPA‐1a phenotyping. They permit rapid determination of the HPA‐1a phenotype and are amenable to automation.
Evaluation of a panel of human monoclonal antibodies to D and exploration of the synergistic effects of blending IgG1 and IgG3 antibodies on their in vitro biologic function
Authors: Armstrong‐Fisher S.S. , McCann Carter M.C. , Downing I. , Fraser R.H. , Inglis G.E. , Allan E.K. , Mackie A. , Prowse C.V. , Templeton J.G. , Thorpe S.J. , Urbaniak S.J.
Published: March 2003

BACKGROUND: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma‐derived polyclonal anti‐D, which constitutes the current therapeutic product. Human monoclonal anti‐D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti‐D is being evaluated in several centers.

STUDY DESIGN AND METHODS: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K‐cell) antibody‐dependent cell‐mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody‐coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays.

RESULTS: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies.

CONCLUSION: The THERAD blends containing both IgG1 and IgG3 anti‐D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.
Ultraviolet and visible light spectrophotometric approach to blood typing: objective analysis by agglutination index
Authors: Narayanan S. , Orton S. , Leparc G.F. , Garcia‐Rubio L.H. , Potter R.L.
Published: March 2003

BACKGROUND: A new blood typing technology based on ultraviolet (UV) and visible light spectroscopy (UV/visible spectroscopy) has been developed. Blood groups and types are determined by quantifying reproducible changes in the UV and visible light spectra of blood in the presence of agglutinating antibodies.

STUDY DESIGN AND METHODS: Samples of red cells in the presence and absence of agglutinating antibodies were examined by UV/visible spectroscopy. Blood groups and types were determined by comparing the optical density spectra obtained between 665 and 1000 nm. These comparisons generate numbers (agglutination index) ranging from 0 to 100, with smaller numbers corresponding to lack of agglutination and larger numbers corresponding to agglutination.

RESULTS: The optical density of agglutinated blood is dramatically different from that of unagglutinated blood. The agglutination index derived from the relative slopes of the spectra is an objective indicator of agglutination strength. An agglutination index greater than 17 consistently and accurately established blood group‐ and type‐specific agglutination.

CONCLUSION: The method accurately predicted A, B, and O blood groups, and D type in over 275 samples. Scattering theory‐based calculations of relative volumes of red cells before and after agglutination show a direct correlation with the agglutination index and provide the theoretical basis of the analysis. This quantitative technique is reproducible and has the potential for automation.
Lowering the hemoglobin threshold for transfusion in coronary artery bypass procedures: effect on patient outcome
Authors: Bracey A.W. , Radovancevic R. , Riggs S.A. , Houston S. , Cozart H. , Vaughn W.K. , Radovancevic B. , McAllister H.A. , Cooley D.A.
Published: March 2003

BACKGROUND: There is controversy regarding the application of transfusion triggers in cardiac surgery. The goal of this study was to determine if lowering the hemoglobin threshold for red cell (RBC) transfusion to 8 g per dL after coronary artery bypass graft surgery would reduce blood use without adversely affecting patient outcome.

STUDY DESIGN AND METHODS: Consecutive patients (n = 428) undergoing elective primary coronary artery bypass graft surgery were randomly assigned to two groups: study patients (n = 212) received RBC transfusions in the postoperative period if the Hb level was <8 g per dL or if predetermined clinical conditions required RBC support, and control patients (n = 216) were treated according to individual physician's orders (hemoglobin levels <9 g/dL as the institutional guideline). Multiple demographic, procedure‐related, transfusion, laboratory, and outcome data were analyzed. Questionnaires were administered for patient self‐assessment of fatigue and anemia.

RESULTS: Preoperative and operative clinical characteristics, as well as the intraoperative transfusion rate, were similar for both groups. There was a significant difference between the postoperative RBC transfusion rates in study (0.9 ± 1.5 RBC units) and control (1.4 ± 1.8 RBC units) groups (p = 0.005). There was no difference in clinical outcome, including morbidity and mortality rates, in the two groups; group scores for self‐assessment of fatigue and anemia were also similar.

CONCLUSIONS: A lower Hb threshold of 8 g per dL does not adversely affect patient outcome. Moreover, RBC resources can be saved without increased risk to the patient.
Variability of prothrombin time and activated partial thromboplastin time in the diagnosis of increased surgical bleeding
Authors: Murray David J. , Pennell Beverly , Olson John D.
Published: March 2003

BACKGROUND: Prothrombin time (PT) and activated partial thromboplastin time (APTT) are used to diagnose causes of increased surgical bleeding and to guide treatment of acquired coagulation factor deficiency. This study compared the sensitivity of various commercial PT and APTT tests in patients with dilutional coagulopathy.

STUDY DESIGN AND METHODS: A prospective study was used to identify patients who experienced increased surgical bleeding during elective extensive (>10 spinal segments) spinal fusion and instrumentation. In patients with clinical signs of increased bleeding, blood was obtained to compare the sensitivity of various commercial PT and APTT tests. PT, PT ratio, the International Normalized Ratio (INR), APTT, and APTT ratio were compared for their sensitivity in the diagnosis of a dilutional coagulopathy.

RESULTS: Sixteen patients experienced increased bleeding during surgery. Mean estimated blood volume lost exceeded 1 blood volume (1.14 ± 0.28). PT and APTT test results varied markedly. In the most sensitive PT and APTT tests, the results were 1.5 times the mean reference range values in all but on of the patients. The least sensitive combination of tests had results that were 1.5 times the mean reference range values in only 2 of 16 patients. Variability among tests was not reduced by the use of the PT or the APTT ratio, by the use of INR, or by incorporation of a measure of PT or APTT test sensitivity to factor‐deficient serum.

CONCLUSION: In surgical patients with dilutional coagulopathy, diagnostic and treatment decisions could depend on which PT and APTT test was used to determine the etiology of increased bleeding. This study indicates that the relationship between increased bleeding and an increased PT and APTT may be more difficult to define than is suggested by current practice guidelines. Each laboratory must establish guidelines based on reagent and instrument sensitivity to coagulation factor dilution.
Effects of methylene blue‐treated plasma on red cells and stored platelet concentrates
Authors: Perrotta Peter L. , Baril Laurie , Tead Cleone , Chapman John , Dincecco Dorothy , Buchholz Donald H. , Snyder Edward L.
Published: March 2003

BACKGROUND: Photochemical methods can effectively inactivate extracellular viruses and bacteria found in blood components. Treatment of plasma with methylene blue (MB), a phenothiazine dye, and visible light inactivates enveloped viruses including HIV‐1. The effects of MB‐treated plasma on cellular components stored in vitro have not been well characterized.

STUDY DESIGN AND METHODS: MB‐treated plasma (83 μg MB/250 mL plasma) was added to single‐donor platelets, stored AS‐1 red cells (RBCs), irradiated RBCs, and frozen‐deglycerolized RBCs. In vitro platelet assays performed after 1 and 5 days of storage in MB‐treated plasma included pH, pO2, pCO2, HCO3, platelet number, lactate dehydrogenase, glucose, osmotic recovery, and CD62 expression. RBC components were examined at specific intervals for leakage of potassium, plasma hemoglobin level, and percentage of hemolysis. Direct antiglobulin tests, osmotic fragilities, and RBC antigen stability tests were also performed on RBCs stored in MB‐treated plasma. Components stored with autologous plasma or nontreated allogeneic plasma served as controls.

RESULTS: Similar storage‐induced changes in pH, glucose, and platelet numbers, as well as increases in lactate dehydrogenase, CD62 expression, and lactate were seen in single‐donor platelets stored with MB‐treated and control plasma. Platelet morphology scores and osmotic recoveries were not altered. Plasma hemoglobin and potassium and percentage of hemolysis increased equally in the various RBC components stored with MB‐treated or nontreated plasma. Osmotic fragility and RBC antigen stability were not appreciably altered by MB‐treated plasma.

CONCLUSION: Plasma treated by MB photoinactivation can be used for in vitro resuspension and storage of platelets or RBCs, because of the lack of influence of MB‐treated plasma on a variety of in vitro platelet and RBC assays.
Quality assessment of platelet concentrates supplemented with second‐messenger effectors
Authors: Rivera José , Lozano Maria L. , de la Calle Javier Corral , Connor Jerome , Gónzález‐Conejero Rocío , Ferrer Francisca , Currie Laura , Garcia Vicente Vicente
Published: March 2003

BACKGROUND: While reducing the potential for bacterial contamination, the storage of platelet concentrates (PCs) at refrigerated temperatures is not routine, because of the induction of the so‐called platelet storage lesion. As the modulation of second‐messenger levels might help to overcome this drawback, a quality assessment of PCs treated with a mixture of second‐messengers effectors known as ThromboSol was performed.

STUDY DESIGN AND METHODS: The PCs were supplemented with ThromboSol or phosphate‐buffered saline, and stored in parallel at 22°C with continuous agitation or at 4°C. At 1, 5, and 9 days, an in vitro quality assessment of the PCs was performed, including measurement of cell number, metabolic and integrity markers, platelet surface expression of glycoproteins, platelet response to ristocetin and thrombin, and levels of cyclic adenosine 3′, 5′ monophosphate (cAMP) and thromboxane B2 (TxB2).

RESULTS: Control PCs stored at 4°C underwent aggregation and displayed a significant decrease in the platelet number (40% on Day 5). By contrast, the ThromboSol‐treated PCs maintained 80 percent of their initial platelet concentration after 9 days of storage at 4°C. Compared to PCs stored at 22°C, refrigerated PCs exhibited minor changes in metabolic values throughout storage, but the addition of ThromboSol induced a rise in metabolic rate during storage at 22°C. Platelet responsiveness to both ristocetin and thrombin was maximally preserved in the ThromboSol‐treated PCs stored at 4°C. These units also maintained high levels of cAMP and low concentrations of TxB2 during storage.

CONCLUSION: The pharmacologic supplementation of PCs with ThromboSol significantly favors the maintenance of in vitro integrity and responsiveness of platelets during extended storage at refrigerated temperature. This protective effect seems to be a consequence of the ability of ThromboSol's components to sustain high levels of cAMP and to inhibit TxB2 production during the entire extended‐storage period.
GB virus type C infection in patients treated for childhood acute lymphoblastic leukemia
Authors: Aricò Maurizio , Bissolati Morena , Bossi Grazia , Asti Margherita , Cerino Antonella , Caselli Désirée , Ricci Antonio , Klersy Catherine , Silini Enrico , Mondelli Mario U.
Published: March 2003

BACKGROUND: The purpose of this study was to determine the prevalence of GB virus type C (GBV‐C) infection in subjects treated for childhood acute lymphoblastic leukemia (ALL) or non‐Hodgkin's lymphoma.

STUDY DESIGN AND METHODS: One hundred forty patients (82 males) aged 4 to 27 years (median, 11) diagnosed with ALL between 1976 and 1993, were prospectively followed for a median of 5 years (range, 0.1–17) after completion of therapy. Stored sera were tested for antibody to hepatitis C virus (HCV), HCV RNA, antibody to GBV‐C E2 (anti‐E2), and GBV‐C RNA.

RESULTS: Thirty‐eight patients (27%) were exposed to GBV‐C: 30 were positive for GBV‐C RNA (mostly type 2) and 8 were positive for anti‐E2. Anti‐E2 and GBV‐C RNA were mutually exclusive: 61 patients (43%) were positive for HCV RNA, 16 (11%) were coinfected with GBV‐C and HCV. Alanine aminotransferase (ALT) levels were increased (>35 mU/mL) in 32 (23%) of 137:3 of 20 who were positive for GBV‐C and negative for HCV, 7 of 15 who were positive for GBV‐C and HCV, 15 of 44 who were negative for GBV‐C and positive for HCV, and 7 of 58 who were negative for GBV‐C and HCV (p<0.001). Median ALT values were significantly higher in patients positive for GBV‐C and HCV than in those who were positive for GBV‐C and negative for HCV (35 vs. 13 mU/mL, p = 0.003). Thirty‐one of 38 patients with GBV‐C markers were re‐tested: GBV‐C RNA was lost in 16 of 30 tested, but 7 were still GBV‐C RNA positive up to 50 months later, 3 tested positive for anti‐E2 up to 27 months later, and 1 was positive for GBV‐C RNA and anti‐E2 26 months later, while 20 tested negative for both.

CONCLUSION: GBV‐C did not behave as a liver pathogen, because ALT alterations were unrelated to GBV‐C status, but, rather, were related to HCV infection or coinfection. GBV‐C RNA was frequently lost over a relatively short period, though in some cases, it was retained for a longer time. Anti‐E2 rarely coexisted with GBV‐C RNA and might be short‐term.
Effect of recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol on the platelet storage lesion
Authors: Snyder Edward L. , Perrotta Peter , Rinder Harvey , Baril Laurene , Nichol Janet , Gilligan Diana
Published: March 2003

BACKGROUND: Platelet production is regulated by a thrombopoietic growth factor (Mpl ligand).The receptor for this platelet growth factor (Mpl) is expressed on the platelet surface membrane. A recombinant thrombopoietic cytokine, recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol (PEG‐rHuMGDF), was added to apheresis platelets in vitro to determine whether Mpl ligand–receptor binding produced any beneficial or adverse effect on the development of the platelet storage lesion during 5 days of storage.

STUDY DESIGN AND METHODS: This study was designed as a dose‐response protocol to determine the effects of adding increasing concentrations of PEG‐rHuMGDF (0.0 [control], 2.5, 25, and 250 ng/mL) to apheresis platelets stored in two types of plastic storage containers. The increasing concentrations of PEG‐rHuMGDF used simulated the theoretical peak plasma level attained in vivo, with an intravenous dose of 0, 0.1, 1.0 and 10 μg per kg of PEG‐rHuMGDF. The platelets were stored with agitation at 20 to 24°C for 5 days. A battery of in vitro assays was performed on storage Days 1 and 5, including pH, blood gases, platelet count, lactate dehydrogenase, mean platelet volume, glucose, lactate, osmotic recovery, morphology score, CD62P, and one‐dimensional polyacrylamide gel electrophoresis analyses.

RESULTS: Analysis of results on both Day 1 and Day 5 showed no significant differences among any of the three PEG‐rHuMGDF doses and the control group, for any in vitro assay. One‐dimensional polyacrylamide gel electrophoresis showed no changes among the platelet protein patterns for the three PEG‐rHuMGDF doses studied or the control. Storage‐induced changes, however, did occur equally in all four groups of platelets over the 5 days of storage.

CONCLUSION: The addition to stored apheresis platelets of up to 10 μg per kg of PEG‐rHuMGDF (250 ng/mL), followed by 5 days of storage at standard conditions, does not appear to promote or retard development of the platelet storage lesion.
Anti‐D quantification by flow cytometry:a comparison of five methods
Authors: Austin E.B. , McIntosh Y.
Published: March 2003

BACKGROUND Three flow cytometric methods for anti‐D quantification have been published. All use different cell sensitization and antibody detection conditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely.

STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in‐house modifications were compared. Ten serum samples containing anti‐D at levels between 11.6 and 915 IU per mL were selected for analysis, and each was tested a minimum of three times. Anti‐D bound to cells was detected with fluorescence‐labeled anti‐human IgG reagents.

RESULTS The interassay CV of the standard curves for each of the five methods was less than 10 percent. The intra‐assay CV was consistently <10 percent with four out of the five methods, but, by the fifth method, it was >20 percent in more than one‐third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the mean anti‐D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods.

CONCLUSION: Anti‐D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.
Comparison of flow cytometric assays with isotopic assays of 51chromium‐labeled cells for estimation of red cell clearance or survival in vivo
Authors: Kumpel B.M. , Austin E.B. , Lee D. , Jackson D.J. , Judson P.A. , Chapman G.E.
Published: March 2003

BACKGROUND: A comparison was made between flow cytometric and conventional radioisotopic assays in the determination of the clearance or survival of small volumes of 51chromium‐labeled D+ red cells after injection into volunteers.

STUDY DESIGN AND METHODS: Four clearance studies were performed using 4 mL of autologous D+ cells coated with anti‐D at two concentrations (5 or 10 μg anti‐D/mL red cells) transfused to two subjects at separate times. Five survival studies were carried out using 5 mL of frozen‐thawed D+ cells transfused to five D– subjects with no detectable anti‐D. Sequential blood samples were taken for gamma counting and flow cytometry. Several methods were used to stain the transfused red cells, and the data were analyzed by using three flow cytometers.

RESULTS: The determination of red cell clearance or survival by radioactivity measurements gave results consistent with published data. However, none of the flow cytometric assays exhibited the necessary sensitivity or accuracy in quantitation of the rare events to provide reliable data for the calculation of the initial clearance rate, the red cell half‐life, or the mean cell lifespan, although rough estimates of red cell clearance were obtained in some subjects. This inability to accurately enumerate rare fluorescence‐labeled cells was due mainly to the presence of “background” events, which were a considerable problem in some samples, when the coating level of anti‐D was less than 3000 molecules of IgG per cell.

CONCLUSION: Flow cytometry may enable the crude estimation of the percentage of small volumes (<5 mL) of transfused D+ red cells, but in this study it was found that this method was not sufficiently accurate to determine the initial clearance rate, red cell half‐life, or mean cell lifespan. If the proportion of transfused cells in the recipient is about 0.2 percent or less, the use of radioisotopes for labeling cells for quantitative in vivo red cell clearance or survival data should remain the method of choice.
DNA analysis in a paternity case involving a triploid fetus
Authors: Allen R.W. , Traver M. , Pritchard J.K.
Published: March 2003

BACKGROUND: A parentage testing laboratory was asked to perform testing in a case of sexual assault that resulted in the conception of a child. Samples submitted to the laboratory included blood from the mother, the alleged father, and the fetus.

CASE REPORT: DNA typing was used to determine if the suspect in this sexual assault was the father of the expected child. DNA extracted from these samples was subjected to both restriction fragment length polymorphism and polymerase chain reaction/short‐tandem repeat analysis at a total of 13 genetic loci. Examination of DNA profiles for selected markers suggested that the fetus was triploid. Triploidy was confirmed through the use of fluorescent in situ hybridization of chromosomes, employing three chromosome‐specific alpha satellite probes and fetal trophoblast nuclei. Statistical interpretation of the test results required identifying a method for calculation that would consider two transmitted paternal genes. Attempts to modify the standard method of calculating a paternity index were unsuccessful, because it was not possible to distinguish between dispermy and diandry as the mechanism of conception. Therefore, the likelihood ratio was calculated as the reciprocal of the random men not excluded value or the proportion of the population that possesses all of the paternal markers observed in the triploid fetus.

CONCLUSION: Calculation of a likelihood ratio employing the exclusionary power of a collection of DNA markers appears to be the only method suitable for assigning weight to the significance of DNA matches between an alleged father and a child who is triploid.
Evaluation of the putative role of C‐C chemokines as protective factors of HIV‐1 infection in seronegative hemophiliacs exposed to contaminated hemoderivatives
Authors: Barretina J. , Blanco J. , Gutiérrez A. , Puig L. , Altisent C. , Espanol T. , Caragol I. , Clotet B. , Esté J.A.
Published: March 2003

BACKGROUND: Overproduction of β‐chemokines and genetic variations in chemokine receptors have been correlated with protection against infection by HIV‐1 or slow progression to AIDS in infected individuals.

STUDY DESIGN AND METHODS: The protective role of chemokines and their receptors was evaluated in a group of seven uninfected (seronegative) hemophiliacs transfused with hemoderivatives presumably contaminated with HIV‐1. This group was compared to a group of seven infected (seropositive) hemophiliacs and a group of healthy donors (controls). The CD4+ cell count, intracellular cytokine levels, β‐chemokine levels in plasma, β‐chemokine production by PBMNCs, and expression of chemokine receptors CCR5 and CXCR4 in CD4+ cells were evaluated. The occurrence of protective genotypes in CCR5, CCR2b, and SDF‐1 (stromal cell‐derived factor 1) genes and susceptibility to infection by HIV‐1 were also studied.

RESULTS: Significant differences in the production and plasma levels of β‐chemokines among the three groups were not detected. Lower IL‐2 and IFN‐γ production was observed in the uninfected exposed hemophiliacs than in the controls. Genetic analysis of CCR5, CCR2b, and SDF‐1 showed several polymorphisms associated with resistance in some HIV‐exposed uninfected hemophiliacs. However, these genetic features cannot explain the protection of all exposed hemophiliacs. In fact, only one patient, carrying two copies of CCR5 from which 32 bp was deleted, showed low CCR5 expression and low susceptibility to infection by a CCR5‐using HIV‐1 strain. In contrast, PBMNCs from all other individuals supported infection in vitro by both CCR5‐ and CXCR4‐using HIV‐1 strains.

CONCLUSION: It is not possible to assign to β‐chemo‐kines and polymorphisms in chemokine receptors a central role in preventing HIV‐1 infection. Natural protection against HIV‐1 infection is likely to be due to a multiplicity of factors.
Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield
Authors: Hildebrandt M. , Serke S. , Meyer O. , Ebell W. , Salama A.
Published: March 2003

BACKGROUND: In immunomagnetic selection of CD34+ cells from HPC transplants, not all factors that affect yield and purity of CD34+ cells are known.

METHODS: Forty‐three consecutive procedures of immunomagnetic selection of CD34+ cells from peripheral blood HPCs and bone marrow harvests (autologous harvests, n = 27; allogeneic harvests; n=16) were performed by use of a cell selection system (Isolex 300i, Baxter Immunotherapy). The composition of the starting component and the subsets of CD34+ cells were analyzed for correlation with the yield and purity of the final component.

RESULTS: The mean purity of the final components was 84.3 percent (range, 27‐99%), and the mean yield was 51.4 percent (range, 9.4‐80.4%). Partial regression analysis showed that, among the factors correlating with purity and/or yield, the RBC volume in the starting fraction had the highest predictive impact on the purity and yield of CD34+ cells, even after the exclusion of procedures using bone marrow harvests as an HPC source (beta coefficient, –0.704; p = 0.001).

CONCLUSION: The use of the Isolex 300i system allows efficient recovery of CD34+ cells in routine selection procedures. The volume of RBCs in the starting component should be minimized to ensure a high yield and purity of the final component.
Severe HDN due to anti‐Ce that required exchange tranfusion
Authors: Wagner T. , Resch B. , Legler T.J. , Mossier C. , Helmberg W. , Köhler M. , Lanzer G.
Published: March 2003

BACKGROUND: Rh system antibodies are commonly encountered in blood bank practice as well as during pregnancy. Nevertheless, no examples of anti‐Ce (RH7) have been reported as a cause of HDN that requires exchange transfusion.

CASE REPORT: A 38‐year‐old woman in her fourth pregnancy was typed as blood group O D+, C–, c+, E+, e–. Anti‐C and anti‐e were detected in her serum during a routine prenatal work‐up. Further evaluation, including flow cytometric analysis, revealed the presence of a strong anti‐Ce and a weak anti‐e. Her partner was typed as group A D+, C+, c–, E–, e+. A seemingly healthy male infant was delivered at 40 weeks of gestation. The infant's RBCs were typed as group O D–, C+, c+, E+, e+ with a positive DAT (titer 128). Twenty‐five hours after birth, the baby had to be transferred to the neonatal intensive care unit because of rapidly rising total serum bilirubin. Despite intensive treatment, including double phototherapy, albumin infusion, and the administration of furosemide and IVIG, the total serum bilirubin level increased during the following day and exchange transfusion with 2 units of type O D–, C–, c+, E+, e– had to be performed; this resulted in a prompt decrease in total serum bilirubin without relapse.

CONCLUSION: Anti‐Ce caused severe HDN requiring exchange transfusion. This highlights the need for a close follow‐up throughout pregnancy if unexpected RBC antibodies are present, to permit the provision of compatible blood in case of a rare antibody.