bluuebunny/crossref_metadata_2025 huggingface dataset:
Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components
Authors: Fountain D , Ralston M , Higgins N , Gorlin JB , Uhl L , Wheeler C , Antin JH , Churchill WH , Benjamin RJ
Published: March 2003

BACKGROUND: The recent report of hepatitis B transmission between hematopoietic progenitor and putative stem cell (HPC) components stored in liquid nitrogen led to the questioning of whether evidence existed for similar contamination by bacterial or fungal elements. STUDY DESIGN AND METHODS: Microbial contamination rates were reviewed for 704 HPC components from 255 patients over an 18‐month period. Five liquid nitrogen freezers were surveyed for microbial contamination. The literature was reviewed to ascertain the published experience of other laboratories with HPC component contamination first documented on thawing. RESULTS: Seven (1.2%) of 583 thawed components were found to be contaminated with a variety of environmental or waterborne organisms, despite a meticulous protocol to prevent contamination during thawing. All of these components had been sterile on cryopreservation. Literature review revealed a similar incidence of post‐thaw contamination from other centers. Microbial survey of liquid nitrogen freezers revealed low‐level contamination in four of five. The organisms represented were similar to those cultured from thawed HPC components. One freezer was heavily contaminated by Aspergillus species. CONCLUSION: Liquid nitrogen freezers are not sterile, and both the liquid and vapor phases are potential sources of microbial contamination of HPC components. While low‐level contamination by environmental organisms may be common, the occurrence of heavy contamination by potential pathogens such as Aspergillus species suggests that monitoring of liquid nitrogen sterility may be indicated. Strategies to assess and prevent microbial transmission from liquid nitrogen to HPC components need further development.
Anaphylatoxins in fresh‐frozen plasma
Authors: Sonntag Josef , Stiller Brigitte , Walka Matthias M. , Maier Rolf F.
Published: March 2003

BACKGROUND: Fresh‐frozen plasma (FFP) is widely used in patients with coagulation disorders and simultaneous complement activation. Complement activation in FFP itself is poorly investigated. STUDY DESIGN AND METHODS: The concentration of anaphylatoxins C3a and C5a, the complement precursors C1q and factor B, and complement function were measured in 40 consecutively administered FFP units in two pediatric neonatal intensive care units. In 12 samples, the measurements were also performed after incubation with inulin. RESULTS: In 15 of 40 FFP units, both anaphylatoxin concentrations were below the upper cutoff levels reported for healthy humans (C3a, 500 microg/L; C5a, 5 microg/L). Anaphylatoxin levels were higher in FFP units produced by apheresis than in those from blood donation. Complement activation of FFP by inulin increased anaphylatoxin concentration, whereas C1q and factor B levels, and complement function remained unchanged. CONCLUSION: Elevated concentrations of anaphylatoxin are frequently found in FFP units produced by apheresis. Studies are necessary to investigate the reasons for complement activation and the possibilities of prevention during apheresis. As the concentrations of complement precursors and complement function did not change with activation in FFP, these studies should include measurement of the anaphylatoxins C3a and C5a.
Differential induction of P‐selectin expression on platelets by two cell separators during plateletpheresis and the effect of gender on the release of soluble P‐selectin
Authors: P. Stohlawetz MD  , N. Hergovich MD  , G. Stiegler MD  , H.G. Eichler MD Professor  , P. Hocker MD Professor  , S. Kapiotis MD Senior Research Specialist6  , B. Jilma MD
Published: March 2003

BACKGROUND: Though a number of studies have elegantly characterized platelet activation during storage, less attention has been paid to the initial activation caused by different collection procedures. STUDY DESIGN AND METHODS: The effects of two blood cell separators on the initial activation of platelets were measured by flow cytometric analysis of P‐selectin expression in 13 male donors on one cell separator (CS 3000 Plus) and 11 men and 9 women on the other (MCS 3P). In addition, the storage and release of soluble P‐selectin (circulating P‐selectin [cP‐selectin]) by platelets were quantified, to determine whether the change in cP‐selectin is a more sensitive marker for initial platelet activation, and the influence of gender on measured endpoints was evaluated. RESULTS: The CS 3000 Plus increased the percentage of P‐selectin‐positive platelets from a median of 3.4 percent before apheresis to 7.6 percent (p = 0.006) in platelet concentrates (PCs), whereas the MCS 3P did not (p = 0.002 between the two cell separators). When preapheresis cP‐selectin levels were compared to those in apheresis PCs, cP‐selectin increased from 51 to 101 ng per mL in plasma of CS 3000 Plus PCs, whereas cP‐selectin levels increased from 53 to 78 ng per mL in MCS 3P PCs (men) and from 48 to 99 ng per mL in MSC 3P PCs (women) (p<0.005 for all). The relative increase in cP‐selectin was higher in women than in men in MCS 3P PCs (p = 0.013). Concomitantly, the amount of P‐selectin stored in platelets before apheresis decreased (p<0.025 for all). When donors undergoing apheresis on the MCS 3P were compared, the amount of P‐ selectin stored in the platelets of PCs was higher in men than women (p = 0.026). CONCLUSION: This trial shows 1) that initial activation of platelets obtained with the MCS 3P is less than that of platelets obtained with the CS 3000 Plus; 2) that the increase in cP‐selectin is a more sensitive marker for initial platelet activation than the expression of P‐selectin on the surface; and 3) that the relative amount of cP‐selectin is higher in women than in men given the same stimulus. Differences in platelet activation by various cell separators and the sex of the donor may contribute to variability of PC quality.
Cryopreservation of single‐donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second‐messenger effectors: enhanced retention of in vitro functional activity
Authors: Currie L.M. , Livesey S.A. , Harper J.R. , Connor J.
Published: March 2003

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. This creates an inventory problem, which could be overcome by the use of cryopreservation to allow long‐term storage of platelets. It has been demonstrated that the addition to platelets of a mixture of second‐ messenger effectors (platelet storage solution), allows these cells to retain significant in vitro functional activity following cold storage. Analysis is needed of the ability of this second messenger effector mixture both to protect platelets during cryopreservation and to reduce the need for a cryoprotectant. STUDY DESIGN AND METHODS: Fresh single‐ donor platelet units (n = 8) were divided into three samples and treated with 6‐percent dimethyl sulfoxide (DMSO), 2‐percent DMSO or the platelet storage solution and 2‐percent DMSO. The samples were placed directly into a −80 degrees C freezer and stored for 1 week, after which they were thawed and analyzed for in vitro functional activity. RESULTS: Platelets cryopreserved with the platelet storage solution and 2‐percent DMSO displayed statistically higher retention of functional activity and viability‐including cell number, percent of discoid cells, extent of shape change, and hypotonic shock response‐than did platelets stored by the method using 6‐percent DMSO. In addition, the treated platelets displayed statistically lower expression of p‐ selectin. The treated platelets showed no loss of cell number, > 88‐ percent retention of discoid morphology, and > 75‐percent retention of ristocetin‐induced aggregation as compared to values for these measures in fresh platelets. CONCLUSION: The use of this platelet storage solution in the cryopreservation of platelets yields a significant improvement in their postthaw in vitro recovery and allows for a reduction of the DMSO concentration from 6 to 2 percent, with superior maintenance of in vitro viability and function.
Effective removal of copper by plasma exchange in fulminant Wilson's disease
Authors: Kiss J. E. , Berman D. , Van Thiel D.
Published: March 2003

BACKGROUND: Patients who present with fulminant hepatic failure due to Wilson's disease may develop hemolytic anemia and renal insufficiency. In this entity, acute hepatocellular necrosis triggers the release of copper ions into the circulation, which leads to toxic effects on red cell metabolic pathways and hemolysis.

STUDY DESIGN AND METHODS: The utility of therapeutic plasma exchange to rapidly remove copper and reduce toxic serum copper levels was studied in two patients with fulminant Wilson's disease.

RESULTS: Intensive plasma exchange using fresh‐frozen plasma replacement removed substantial amounts of copper from the hypercupremic patients, resulting in a rapid reduction in serum copper levels and decreased hemolysis. The net copper removal was proportional to the serum level, ranging from 7,000 to 11,800 micrograms per procedure in one patient and from 3,700 to 6,800 micrograms in the other.

CONCLUSION: Plasma exchange allows a rapid reduction in elevated serum copper levels in patients with fulminant Wilson's disease. This leads to an amelioration of hemolytic anemia and provides clinical stabilization until liver transplantation can be performed.
Evaluation of an automated microbiologic blood culture device for detection of bacteria in platelet components
Authors: Wagner SJ. , Robinette D.
Published: March 2003

BACKGROUND:Automated culture methods have been used by several investigators to detect bacterial contamination of cellular blood components. We investigated several factors affecting detection by automated culture of bacteria in platelet concentrates (PCs).These factors included the initial contamination level in PCs, the PC sample volume, the PC sample time, and the white cell level in relation to bacteria levels in the PCs.

STUDY DESIGN AND METHODS:Staphylococcus epidermidis or Escherichia coli was inoculated into freshly prepared PCs or white cell‐reduced PCs to yield colony‐forming unit (CFU) levels of 10, 1, or 0.1 per mL. At the time of inoculation (t=0) and at t=6, t=24, and t=48 hours, 0.5, 1.0, and 2.0 mL samples of the contaminated PCs were transferred into culture bottles. The presence of bacteria in the culture bottles was subsequently monitored by an automated blood culturing instrument. Bacteria levels in the PC at the time of first automated culture detection were determined by quantitative plating.

RESULTS:E. coli was detected in 92 percent of experiments when 1.0‐ or 2.0‐mL samples were taken at t=6 hours. At t=24 hours, 100‐percent detection was observed with all tested inoculation volumes; however, by that time,>10(7) CFU per mL of bacteria were present in every PC. For S. epidermidis, 89 percent and 83 percent of contaminated PCs were detected with a t=24 hour sampling time and 2.0‐ or 1.0‐mL sampling volume. Seven of 36 PCs with a 2.0‐mL sampling volume and 10 of 36 PCs with a 1.0‐mL sampling volume contained>10(6) CFU per mL of S. epidermidis at the time of first detection. CONCLUSION: Data from this preliminary evaluation suggest that sampling times of 24 hours or more would be necessary to provide confidence in detection of E. coli or S. epidermidis in PCs using this culture method.
Recovery and viability of Orientia tsutsugamwhi from packed red cells and the danger of acquiring scrub typhus from blood transfusion
Authors: Casleton B.G. , Salata K. , Dasch G.A. , Strickman D. , Kelly DJ.
Published: March 2003

BACKGROUND:The purpose of this study was to determine whether infective Orientia tsutsugamushi, the etiologic agent of scrub typhus, could survive normal blood banking processing and storage procedures.

STUDY DESIGN AND METHODS:Mononuclear cells isolated from whole blood by density gradient centrifugation were inoculated with O. tsutsugamushi, Karp strain. Infection of the mononuclear cells was confirmed by Giemsa stain, direct fluorescent antibody assay, and polymerase chain reaction using primers specific for the groESL operon of O. tsutsugamushi. The quantity of rickettsial particles in each preparation was determined by direct counts from the Giemsa‐stained preparations. Infected mononuclear cells were returned to their respective aliquots of packed red blood cells, which were then either stored at 4 degrees C or glycerolized and frozen at −70 degrees C.

RESULTS:Rickettsiae survived up to 10 days (but not 30 days) of refrigerated storage and 45 days of frozen storage, as determined by inoculation of mice with 0.5‐mL aliquots of the blood components. Infection of the mice was determined by illness, death, direct fluorescent antibody assay of peritoneal smears, polymerase chain reaction of blood, and enzyme‐linked immunosorbent assay detection of antibodies in plasma. CONCLUSION: Because the quantity of rickettsiae injected into the mice was comparable to the quantity reported in the literature for human blood during natural infections, scrub typhus could present a risk in blood collected from donors in endemic areas. This may especially be true, because people can be rickettsemic before illness, after successful antibiotic treatment, and chronically after resolution of disease.
Transfusion and postoperative pneumonia in coronary artery bypass graft surgery: effect of the length of storage of transfused red cells
Authors: Vamvakas E.C. , Carven J.H.
Published: March 2003

BACKGROUND: Various bioactive substances are released from white cell (WBC) granules into red cell (RBC) components in a time‐dependent manner during blood storage. Some of these substances may have immunosuppressive effects and may contribute to transfusion‐induced immunomodulation. RBCs transfused after prolonged storage may be associated with a higher incidence of postoperative infections than fresh RBCs. This hypothesis does not seem to have been investigated in a clinical study.

STUDY DESIGN AND METHODS: The records of 416 consecutive patients undergoing coronary artery bypass graft operations at the Massachusetts General Hospital were reviewed. The association between the length of storage of the transfused RBCs, as well as the number of units of non‐WBC‐reduced allogeneic RBCs and/or platelets transfused, and the occurrence of postoperative pneumonia was calculated by logistic regression analyses adjusting for the effects of confounding factors. Among these were the numbers of days of intubation, days of impaired consciousness, and units of RBCs transfused.

RESULTS: By Centers for Disease Control and Prevention criteria, pneumonia developed in 54 patients (13.0%). Among 269 patients given RBCs, the risk of pneumonia increased by 1 percent per day of increase in the mean storage time of the transfused RBCs (p<0.005). In an analysis of all patients, the risk of pneumonia increased by 5 percent per unit of non‐WBC‐reduced allogeneic RBCs and/or platelets received (p = 0.0584).

CONCLUSION: After adjustment for the effects of the risk factors for pneumonia and the number of transfused RBCs, an association was observed between the length of storage of transfused RBCs and the development of postoperative pneumonia. This association should be investigated further in future studies of the outcomes of blood transfusion.
Complement receptor 1 red cell expression is not controlled by the In(Lu) gene
Authors: Moulds J.M. , Shah C.
Published: March 2003

BACKGROUND: The In(Lu) gene reportedly suppresses several blood group antigens that are not part of the Lutheran system, including the high‐incidence antigens of the Knops blood group system. Because complement receptor 1 (CR1), which is known to carry the Knops system antigens, has a red cell (RBC) expression polymorphism, the role of In(Lu) in the expression of the Knops system antigens was reinvestigated.

STUDY DESIGN AND METHODS: Blood samples from nine donors having the Lu(a–b–) phenotype were obtained and immediately phenotyped for Lub, Kna, McCa, Sla, and Yka. The samples were also tested for Lua, P1, and AnWj. Immunoblots were performed to study both the CR1 and Lutheran glycoproteins from these donors. RBC expression of CR1 was quantified with an enzyme‐linked immunosorbent assay, and the genetic inheritance of the high‐expression (H) or low‐expression (L) allele for CR1 was determined by Southern blot.

RESULTS: Lub was demonstrable only by absorption and elution techniques on all nine samples; however, the high‐incidence Knops system antigens were readily detectable by hemagglutination. Two Lu(a–b–) donors (sibs) demonstrated weak Lutheran glycoprotein bands of 78 and 85 kDa on immunoblots, while the other seven Lu(a–b–) samples had no detectable glycoprotein. All donors had CR1*1, and one donor also had CR1*2 on immunoblot. Only one donor was homozygous for the L allele, and all had RBC copy numbers of CR1 within the normal range.

CONCLUSIONS: Nine donors with the Lu(a–b–) phenotype showed suppression of the Lutheran system antigens but normal expression of CR1 glycoprotein and the Knops system blood group antigens. This suggests that the genes that suppress Lutheran system antigens do not suppress CR1 or its related blood group antigens.
Immunoglobulin isotype identification in red cell antibodies using flow cytometry
Authors: Price W.R. , Johnson S.T. , Curtis B.R.
Published: March 2003

BACKGROUND: Identifying the isotype of an immunoglobulin (IgM vs. IgG) detected in a patient sample is especially important in anticipating the risk of hemolytic disease of the newborn. Currently, 2‐mercaptoethanol (2‐ME) treatment of a sample is used in the authors' laboratory to degrade IgM, and this is followed by retesting. This method has multiple drawbacks. The purpose of this study was to develop a flow cytometry (FC) assay that would replace the 2‐ME treatment protocol (2‐ME treatment).

STUDY DESIGN AND METHODS: A preliminary FC assay was developed, modified, and refined through the use of stock antibodies. Then, 10 samples containing antibodies were tested in parallel by the FC assay and 2‐ME treatment.

RESULTS: When a 10‐unit mean channel fluorescence change was used as an index of a positive result, the FC assay detected all isotypes identified by 2‐ME treatment. The FC assay was also able to identify mixtures of isotypes. One antibody that had not reacted in conventional agglutination testing was detected by the FC assay. The amount of fluorescence and the agglutinating strength of the antibody did not parallel each other. In one case, this discrepancy may have reflected an antibody that was primarily IgA.

CONCLUSIONS: The FC assay appears to be as accurate as 2‐ME treatment in differentiating IgG from IgM. The FC assay produces a positive endpoint for both isotypes, will identify IgA, requires less sample, and has no odor.
The cost‐effectiveness of reducing donor exposures with single‐donor versus pooled random‐donor platelets
Authors: Lopez‐Plaza I. , Weissfeld J. , Triulzi D.J.
Published: March 2003

BACKGROUND: Single‐donor platelets (SDPs) are frequently preferred over pooled random‐donor platelets (RDPs) to reduce donor exposures and the risk for virus transmission or HLA alloimmunization. Transfusion‐associated virus‐transmission risks have significantly decreased, which suggests that white cell reduction by filtration eliminates any difference in the risk of alloimmunization in transfused leukemic patients. Health care reform pressures of make it appropriate to examine the cost‐effectiveness of SDPs versus RDPs in reducing donor exposures.

STUDY DESIGN AND METHODS: A decision analysis model was developed and sensitivity analyses were used to assess the incremental cost (dollars/quality‐adjusted life‐year) associated with the use of SDPs versus RDPs for adult patients undergoing hematopoietic progenitor cell transplantation or primary coronary artery bypass grafting (CABG).

RESULTS: Among transplant patients, the incremental cost of choosing SDPs as opposed to RDPs ranged from $168,700 to $519,822 per quality‐adjusted life‐year. For patients undergoing primary CABG, the incremental cost was $192,415 (females) and $216,280 (males). Variations in the cost differential between SDPs and RDPs, the number of random‐donor platelets in the RDP, and the risk of bacterial sepsis markedly influenced cost‐effectiveness. The model was minimally affected by variations in the risks of transmission of HIV and hepatitis B and C, and human T‐lymphotropic viruses.

CONCLUSION: In comparison with other accepted medical interventions, the use of SDPs as opposed to RDPs may not be a cost‐effective method of reducing donor exposures in the adult patient populations studied. SDPs were more cost‐effective in patients undergoing primary CABG than in leukemia patients undergoing hematopoietic progenitor cell transplantation. Regardless of diagnosis, decreasing the acquisition cost differential would have the greatest impact on improving the cost‐effectiveness of SDPs, as opposed to RDPs, to decrease donor exposures.
Hemolytic transfusion reaction due to anti‐Tca
Authors: Kowalski M.A. , Pierce S.R. , Edwards R.L. , Ethirajan S. , Ryan K. , Livergood M.P.
Published: March 2003

BACKGROUND: Anti‐Tca detects a high‐incidence antigen in the Cromer blood group system. Cromer system antibodies have not usually been associated with hemolytic transfusion reactions or hemolytic disease of the newborn.

CASE REPORT: Anti‐Tca (initially identified in the patient's serum in 1982) was not detected when she was admitted to the hospital with upper gastrointestinal. bleeding. Three units of red cells were administered. The patient was discharged, but was readmitted to the hospital after her hemoglobin fell to 7.1 g per dL. Antibody detection tests remained negative and three additional units were transfused. Over the next 7 days, her hemoglobin steadily fell to 5.5 g per dL. The level of lactate dehydrogenase rose to 1257, the plasma hemoglobin rose to >16 mg per dL, and the haptoglobin decreased to <6 mg per dL. Five days after transfusion, her direct antiglobulin test was weakly reactive with complement‐specific antiglobulin reagents. Eluates were nonreactive. Anti‐Tca was detected in her serum; no other antibodies were detected. Differential typing failed to detect any circulating Tc(a+) red cells. The antibody was strongly reactive in a monocyte monolayer assay.

CONCLUSION: Although Cromer system antibodies have generally not been proven to be clinically significant in transfusion therapy, the destruction of red cells from six units of transfused Tc(a+) red cells in this patient indicates that anti‐Tca may have destructive potential in some patients.
Coexpression of tissue factor and tissue factor pathway inhibitor by human monocytes purified by leukapheresis and elutriation.Response of nonadherent cells to lipopolysaccharide
Authors: Nguyên P. , Broussas M. , Cornillet‐Lefèbvre P. , Potron G.
Published: March 2003

BACKGROUND: Counterflow centrifugal elutriation is the method of choice for obtaining a large quantity of highly purified monocytes. In spite of the fact that this technique has been used for many years to isolate monocytes for cellular immunotherapy, it is not known whether the process of elutriation can stimulate tissue factor (TF) expression and therefore trigger coagulation in patients receiving these cell preparations. The aim of the present study is thus to identify TF and TF pathway inhibitor (TFPI) in elutriated monocytes and to evaluate their ability to trigger thrombin generation.

STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis and elutriation in sterile, endotoxin‐free conditions. TF and TFPI mRNA is detected by reverse transcription‐polymerase chain reaction. TF and TFPI are measured by enzyme‐linked immunosorbent assay in cell lysates. TF antigen expression on cell surface is evidenced by direct‐flow cytometry. Two functional tests (a chronometric test and an amidolytic assay) assess the capacity of monocytes to trigger thrombin generation. The response to lipopolysaccharide (LPS) is evaluated with each technique. Monocytic cell line THP‐1 is used as a positive control.

RESULTS: Elutriated monocytes coexpress TF mRNA and TFPI mRNA. The expression of TF mRNA is dramatically increased by LPS activation. This is correlated with a 100‐fold increase in the amount of TF antigen in monocyte lysates. Flow immunocytometry confirms the expression of TF antigen on cell membrane in response to LPS stimulation, whereas TFPI mRNA is slightly increased after LPS stimulation. The amount of TFPI antigen in cell lysates is small when compared to that in plasma. Elutriated monocytes have a strong potential to trigger thrombin generation in response to LPS.

CONCLUSION: In spite of the coexpression of TF mRNA and TFPI mRNA, elutriated monocytes are capable of supporting prothrombinase activity. This should be taken into account in the evaluation of the safety of adoptive cellular immunotherapy.
Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt‐Jakob disease in humans
Authors: Brown P. , Cervenáková L. , McShane L.M. , Barber P. , Rubenstein R. , Drohan W.N.
Published: March 2003

BACKGROUND: Solid evidence from experimentally infected animals and fragmentary evidence from naturally infected humans indicate that blood may contain low levels of the infectious agent of Creutzfeldt‐Jakob disease (CJD), yet blood components have never been identified as a cause of CJD in humans.

STUDY DESIGN AND METHODS: Blood components and plasma fractions were prepared from the pooled blood of mice that had earlier been infected with a mouse‐adapted strain of human transmissible spongiform encephalopathy (TSE). Infectivity bioassays were conducted in healthy mice, and the brains of all assay animals dying during the course of the experiments were examined for the presence of proteinase‐resistant protein.

RESULTS: Infectivity in the blood during the preclinical phase of disease occurred in the buffy coat at infectious unit (IU) levels between 6 and 12 per mL and was either absent or present in only trace amounts in plasma and plasma fractions. Infectivity rose sharply at the onset of clinical signs to levels of approximately 100 IU per mL of buffy coat, 20 IU per mL of plasma, 2 IU per mL of cryoprecipitate, and less than 1 IU per mL of fractions IV and V. Plasma infectivity was not eliminated by either white cell‐reduction filtration or high‐speed centrifugation. Approximately seven times more plasma and five times more buffy coat were needed to transmit disease by the intravenous route than by the intracerebral route.

CONCLUSION: Epidemiologic evidence of the absence in humans of disease transmission from plasma components can probably be explained by 1) the absence of significant plasma infectivity until the onset of symptomatic disease, and comparatively low levels of infectivity during the symptomatic stage of disease; 2) the reduction of infectivity during plasma processing; and 3) the need for at least five to seven times more infectious agent to transmit disease by the intravenous than intracerebral route. These and other factors probably also account for the absence of transmission after the administration of whole blood or blood components.
ABO incompatibility as an adverse risk factor for survival after allogeneic bone marrow transplantation
Authors: Benjamin Richard J. , McGurk Siobhan , Ralston Maria S. , Churchill W. Hallowell , Antin Joseph H.
Published: March 2003

BACKGROUND: Graft ABO incompatibility has not been thought to aflect patient survival after allogeneic bone marrow transplantation, although it may be associated with prolonged erythroid aplasia and immediate or delayed hemolysis.

STUDY DESIGN AND METHODS: A retrospective analysis of a cohort of 292 allogeneic transplant recipients measured survival in a subgroup of ABO‐incompatible bone marrow graft recipients.

RESULTS: Patients with acute myelogenous leukemia or myelodysplastic syndrome receiving non‐T‐cell‐depleted bone marrow grafts had an 85‐percent greater risk of death within 100 days of transplant (relative risk, 1.85, 95% Cl, 1.33–2.58; p = 0.003) than comparable patients receiving ABO‐compatible grafts. Both ABO major‐ and minor‐mismatched graft recipients were at risk. The increased mortality rate was not due to an increase in graft failure or acute graft‐versus‐host disease; rather, patients died of multiple‐organ failure and sepsis, which is consistent with regimen‐related toxicity. This effect was not seen in a larger group of 112 chronic myelogenous leukemia patients undergoing similar treatment.

CONCLUSION: ABO incompatibility may be a significant prognostic risk factor after allogeneic bone marrow transplantation in susceptible subgroups of recipients. Care is necessary to design hematopoietic stem and progenitor cell‐processing and ‐transfusion policies to minimize this risk.
A randomized controlled trial comparing plasma removal with white cell reduction to prevent reactions to platelets
Authors: Heddle Nancy M. , Klama Luba , Meyer Ralph , Walker Irwin , Boshkov Lynn , Roberts Robin , Chambers Shelley , Podlosky Linda , O'Hoski Pamela , Levine Mark
Published: March 2003

BACKGROUND: Recent data suggest that most reactions to platelets are caused by white cell (WBC)‐derived cytokines that accumulate in the plasma portion of the component during storage. On the basis of this theory, the effectiveness of two interventions to prevent reactions, poststorage WBC reduction and plasma depletion, were compared.

STUDY DESIGN: A multiple crossover design was used, in which platelet components for transfusion to a patient randomly were WBC reduced after storage, or the plasma supernatant was removed. Adults >17 years of age, with a hematologic disease requiring platelet transfusion support, were eligible for the study. Patients were assessed for signs and symptoms characteristic of a reaction during, immediately after, and 1 hour after transfusion. Reactions were graded as mild, moderate, or severe. Interleukin 6 levels were also measured in the transfused platelet components.

RESULTS: There were 380 analyzable platelet transfusions to 30 patients. The frequency of reactions was 25.8 percent (48/186) in the transfusions of poststorage WBC‐reduced platelets and 17.0 percent (33/194) in the transfusions of plasma‐depleted platelets (p<0.008).The severity of the reaction was graded by the patient. Severe reactions occurred more frequently in connection with poststorage WBC‐reduced platelets than with plasma‐depleted platelets: 33.4 percent (16/48) versus 18.2 percent (6/33), respectively (p = 0.048). Regression analysis identified interleukin 6 as the most significant of the evaluated factors in its correlation with the risk of reaction.

CONCLUSION: Plasma removal is more effective than poststorage WBC reduction in preventing reactions, especially severe reactions to platelets.
Genomic typing of human red cell Miltenberger glycophorins in a Taiwanese population
Authors: Shih M.C. , Yang L.H. , Wang N. M. , Chang J.G.
Published: March 2003

BACKGROUND: Antigens in the human red cell Miltenberger series are glycophorin variants of the MN (MNS) blood group system that are due to the rearrangement of glycophorin A (GPA) and glycophorin B (GPB) genes.

STUDY DESIGN AND METHODS: Taking advantage of the differences between the GPA and GPB genes, a polymerase chain reaction‐based method was developed to detect all the Miltenberger glycophorin variants and Sta subtype. GPA‐ and GPB‐specific primers were used to amplify the GPA or GPB gene, and the amplified products were used to recognize the different hybrid genes after restriction enzyme digestions.

RESULTS: Among 264 Taiwanese subjects studied, Mi.III and Sta are the most common types of Miltenberger variants found. Mi.III was present in 13 (4.92%) of 264, and Sta was found in 8 (3.03%) of 264; 1 case (0.4%) of Mi.V was also identified from the study group.

CONCLUSION: This is the first polymerase chain reaction‐based method of detecting most of the Miltenberger variants and Sta. The genomic typing results were confirmed by control DNA of identified Miltenberger phenotypes. The prevalence rates of Mi.III and Sta in this study were also consistent with other previous reports using different methods.
Nuclear matrix protein is released from apoptotic white cells during cold (1‐6°C) storage of concentrated red cell units and might induce antibody response in multiply transfused patients
Authors: Martelli A.M. , Tazzari P.L. , Bortul R. , Riccio M. , Tabellini G. , Santi S. , Frabetti F. , Musiani D. , Bareggi R. , Conte R.
Published: March 2003

BACKGROUND: A previous study showed that white cells in blood units undergo apoptosis during storage.

STUDY DESIGN AND METHODS: The present study attempts to show the release of nuclear matrix protein (NMP) in the supernatants of red cell units and to determine whether antibodies against nuclear components may be present in multiply transfused patients; the methods employed were enzyme‐linked immunosorbent assay, flow cytometry, microscopy, immunoblotting, immunofluorescence, and confocal laser‐scanning microscopy.

RESULTS: NMP is released from white cells in the supernatant of packed red cell units upon cold storage (1‐6°C). The concentration of NMP correlates well with the degree of apoptosis, as analyzed by flow cytometry, nuclear dye staining, and DNA gel electrophoresis. Immunofluorescence also shows that white cells undergoing apoptosis (pre‐G1 peak, as seen by propidium iodide staining and flow cytometry) have an NMP content lower than control cells, which confirms an actual release of NMP. Moreover, immunoblotting analysis and immunofluorescent staining showed that, in 4 of 38 multiply transfused patients, autoantibodies against NMPs were present without any clinical or laboratory sign of autoimmune disease. One of the sera, recognizing a 64‐kDa NMP, immunostained nuclear dots that were identified as coiled bodies because of their colocalization with p 80 coilin.

CONCLUSION: NMP is released in the supernatant of red cell units. The results obtained from patients suggest that nuclear proteins released during apoptosis, once transfused, may induce an immune response in multiply transfused patients.
Primary anti‐D immunization by weak D type 2 RBCs
Authors: Flegel W.A. , Khull S.R. , Wagner F.F.
Published: March 2003

BACKGROUND: D is the most immunogenic blood group antigen. In about 0.4 percent of whites, D is expressed on RBCs in a weak form. Recently, it was found that the weak D phenotypes are caused by a large number of distinct RHD alleles generally encoding altered D proteins. No particular molecular weak D type has yet been shown to induce anti‐D. The threshold of D antigen density required for anti‐D immunization is not known.

CASE REPORT: A 72‐year‐old D– white man received apparently D– RBCs. Nineteen days later, he developed a positive DAT, and anti‐D was found in his serum and an eluate from his RBCs. One donor was found to be D+ with a weak D type. The weak D type was determined by RHD exon 9‐specific nucleotide sequencing from genomic DNA. The transfusion recipient showed alloanti‐D. Ten months later, anti‐D but no other antibody was detectable; the DAT was negative and the eluate was nonreactive. The donor of the incriminated unit was D+ (ccDEe) with weak D due to the weak D type 2 allele, expressing about 450 D antigens per RBC.

CONCLUSION: This case provides formal proof that RBCs of weak D type 2 phenotype may cause alloanti‐D immunization. Among the more prevalent weak D types in whites, weak D type 2 has the lowest D antigen density. Thus, units of blood from donors of the weak D type 2 phenotype should be labeled D+; the weak D type 2 phenotype may be useful for quality assurance.
Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening
Authors: Drosten C. , Weber M. , Seifried E. , Roth W.K.
Published: March 2003

BACKGROUND: High‐throughput nucleic acid testing for transfusion‐relevant viruses by PCR requires contamination‐proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry‐over contamination by eliminating post‐PCR processing.

STUDY DESIGN AND METHODS: Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template‐derived competitive internal control sequence designed through site‐directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single‐donor and single‐patient testing and by spiking sample mini‐pools. Three seroconversion panels were tested.

RESULTS: Sensitivity is high, reaching 300 HBV genomes per mL of single‐patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors.

CONCLUSION: The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.
Differentiation of autologous ABO, RHD, RHCE, KEL, JK, and FY blood group genotypes by analysis of peripheral blood samples of patients who have recently received multiple transfusions
Authors: Rožman P. , Dovč T. , Gassner C.
Published: March 2003

BACKGROUND: After multiple transfusions, the serologic typing of autologous blood group phenotypes is difficult, because of mixed RBC populations. The genotyping of ABO, Rh, Kell, Kidd, and Duffy systems could be used to determine autologous blood group antigen status.

STUDY DESIGN AND METHODS: Blood samples from patients and donors were analyzed before and after 26 multiple‐transfusion events. An average of 6.9 non‐WBC‐reduced RBC units with an average age of 5.9 days were administered per transfusion event. The average period of blood sampling after transfusions was 5.3 days. All samples were serologically phenotyped for ABO, Rh, Kell, Kidd, and Duffy. Pretransfusion, posttransfusion, and buccal samples from patients were genotyped for the corresponding alleles by a uniform PCR sequence‐specific primer protocol that allowed their simultaneous determination within 3 hours.

RESULTS: All posttransfusion samples exhibited mixed‐cell populations of various blood group systems on serologic testing. Genotyping from peripheral blood produced results identical to the autologous blood group phenotypes, regardless of the amount of blood transfused or of the length of the sampling period after transfusion.

CONCLUSION: A fast and reliable PCR‐sequence‐specific primer DNA genotyping assay for simultaneous determination of autologous ABO, Rh, Kell, Kidd, and Duffy blood groups can be performed on peripheral blood samples, even though the patients have recently received multiple transfusions.
Successful storage of RBCs for 10 weeks in a new additive solution
Authors: Hess J.R. , Rugg N. , Knapp A.D. , Gormas J.F. , Silberstein E.B. , Greenwalt T.J.
Published: March 2003

BACKGROUND: The effect of storing packed RBCs suspended in 300 mL of an alkaline, experimental additive solution (EAS 64) was explored.

STUDY DESIGN AND METHODS: RBC units prepared from blood collected from healthy donors into CPD were WBC reduced and stored for 10 weeks under blood bank conditions after the addition of 300 mL of EAS 64 (adenine, 2 mM; dextrose, 50 mM; mannitol, 20 mM; NaCl, 75 mM; Na2HPO4, 9 mM). For comparison, non‐WBC‐reduced units from the same donors were stored in a different additive solution (AS‐1, Baxter Healthcare) for 6 weeks. Standard methods were used for the in vitro assays. The 24‐hour in vivo recoveries were measured by using 51Cr‐ and 99mTc‐labeled RBCs.

RESULTS: Mean recovery in the EAS 64 units after 10 weeks was 84 ± 8 percent, the same as in the AS‐1 units stored for 6 weeks. For EAS 64 and AS‐1 units, respectively, the ATP of the RBCs was 85 percent and 64 percent of the initial value, hemolysis was 0.43 percent and 0.63 percent, supernatant potassium was 24 mEq per L and 44 mEq per L, and the morphologic index was 98 and 71.

CONCLUSION: RBCs suspended in 300 mL of EAS 64 can be stored satisfactorily for 10 weeks. Longer RBC storage should reduce outdating, increase availability of transfusions in remote locations, and improve the efficiency of autologous donor programs.
Survival of Ehrlichia chaffeensis in refrigerated, ADSOL‐treated RBCs
Authors: McKechnie Don B. , Slater Kimetha S. , Childs James E. , Massung Robert F. , Paddock Christopher D.
Published: March 2003

BACKGROUND: The purpose of this study was to investigate the persistence of viable Ehrlichia chaffeensis in ADSOL‐treated RBCs stored at 4 to 6°C.

STUDY DESIGN AND METHODS: The continuous monocytic cell lines THP‐1 and DH82 were infected with E. chaffeensis (St. Vincent isolate). Packed RBC units were inoculated in separate experiments with E. chaffeensis‐infected cells as final concentrations of 8.02 × 104 (DH82) and 1.43 × 104 (THP‐1) infected cells per mL. Aliquots were stored at 4 to 6°C for 1 to 42 days. At selected intervals, nucleated cells from the RBC aliquots were obtained by using a ficoll‐isopaque separation procedure. Uninfected DH82 cell cultures were inoculated with the harvested nucleated cells or supernatant. The cell cultures were evaluated for infection by weekly examination of Wright's (Diff‐Quik) stained cytocentrifuged slides. PCR amplification was also used to test the harvested nucleated cells or supernatant for the presence of E. chaffeensis DNA.

RESULTS: In both types of infected cell lines, E. chaffeensis was reisolated in DH82 cells for as long as 11 days from the cellular fraction and for up to 5 days from the supernatant fraction. PCR results were positive throughout the 42‐day testing period.

CONCLUSION: Cell‐associated E. chaffeensis remains viable in ADSOL‐treated RBCs stored at 4 to 6°C for at least 11 days. These data suggest that transfusion‐acquired infection is possible. Successful reisolation was achieved from the supernatant fraction, which suggests that RBC products treated with a WBC‐reduction procedure may still present a risk for transfusion transmission. No correlation between PCR positivity and viability of bacteria was noted.
An amino acid substitution in the putative second extracellular loop of RBC band 3 accounts for the Froese blood group polymorphism
Authors: McManus Kirk , Lupe Krystine , Coghlan Gail , Zelinski Teresa
Published: March 2003

BACKGROUND: The low incidence RBC antigen Fra has been excluded from 17 of the 25 established blood group systems. Previous genetic analysis assigned the gene controlling Fra expression to the same chromosomal region as the solute carrier family 4, anion exchanger member 1 gene (SLC4A1). Because SLC4A1 encodes RBC band 3 and controls the expression of Diego blood group system antigens, the possible relationship of Fra to the Diego blood group system was investigated by molecular analysis of SLC4A1.

STUDY DESIGN AND METHODS: Blood samples were obtained from the members of two unrelated Mennonite kindreds segregating for Fra. DNA was extracted, amplified by PCR using intronic primer sets flanking exons 11‐20 of SLC4A1, and screened by single‐strand conformation polymorphism (SSCP) analysis. Those exons displaying SSCPs were subjected to DNA sequence analysis.

RESULTS: An exon 13 SSCP mobility shift was observed in the DNA from all Fr(a+) persons that was not seen in the DNA from Fr(a‐) family members or control subjects. Linkage between the exon 13 SSCP and Fra was established, with peak lods = 3.62 at θ = 0.00 for combined paternal and maternal meioses. DNA sequencing revealed a GAG → AAG mutation that underlies a Glu480Lys substitution in RBC band 3.

CONCLUSIONS: A point mutation in exon 13 of SLC4A1 accounting for a Glu480Lys substitution in band 3 controls Fra expression. On the basis of these our results, the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens has assigned Fra to the Diego blood group system, with the designation DI20.
Validation of selected donor‐screening questions: structure, content, and comprehension
Authors: Orton Sharyn L. , Virvos Victoria J. , Williams Alan E.
Published: March 2003

BACKGROUND: Donor questioning is an integral component of blood safety, designed to identify and interdict donors considered to be at elevated risk of transfusion‐transmissible infection. Questions are frequently added to this screening procedure, but they are rarely evaluated for comprehension.

STUDY DESIGN AND METHODS: Seven American Red Cross blood donor history questions were selected for evaluation. Focus groups were conducted using individuals from high school/college, church, business, and other sources, who had never donated blood. Both sexes and various age and racial groups were represented. The donation process and regulatory requirements or guidelines were explained. Participants were asked to consider 1) content, 2) clarity 3) likelihood of asking for more information, 4) suggestions for improvement, and 5) how a need for clarifying information would best be met.

RESULTS: Constructive, helpful, and consistent comments obtained might be utilized in revising questions.

CONCLUSION: Focus group discussions can be useful in providing guidance for developing screening questions that can be easily understood.
CD34+ cell mobilization for allogeneic progenitor cell transplantation: efficacy of a short course of G–CSF
Authors: De Fabritiis Paolo , Iori Anna Paola , Mengarelli Andrea , Gozzer Maria , Ferrazza Giancarlo , De Propris Maria Stefania , Romano Atelda , Arcese William
Published: March 2003

BACKGROUND: G–CSF‐mobilized PBPCs are considered the richest source of HPCs for both autologous and allogeneic transplantation, but, despite their wide use, the best dose and schedule for G–CSF administration have not been definitively established.

STUDY DESIGN AND METHODS: With a target of collecting from the peripheral blood ≥4 × 106 CD34+ cells per kg of body weight of the recipient, the short‐course administration of glycosylated G–CSF (gly‐G–CSF) in 30 healthy donors for an allogeneic transplantation was investigated. Gly‐G–CSF was given subcutaneously at a dose of 10 μg per kg per day in two divided doses over 3 days and was followed by a leukapheresis (on the 4th day) 12 hours after the last dose.

RESULTS: A median of 53.5 circulating CD34+ cells per μL (range, 19‐190) was found in the 30 donors on the day of first leukapheresis, which allowed a median CD34+ cell collection of 6.0 × 106 per kg of body weight of the donor and 6.5 × 106 per kg of body weight of the recipient. In 25 (83%) of 30 donors, a single procedure was sufficient to collect the target CD34+ cells, while in the other 5, two leukapheresis procedures were required. Hematologic reconstitution was observed in all patients at a median of 14 days (range, 10‐23) for neutrophils and 14.5 days (range, 11‐46) for platelets. With a median infusion of 3.9 × 108 CD3+ T‐lymphocytes per kg of body weight of the recipient (range, 1.3‐7.8), acute and chronic GVHD occurred in 13 (43%) of 30 and 15 (60%) of 25 evaluable patients, respectively. After a median follow‐up of 337 days from transplant, 22 (73%) of 30 patients are alive in complete remission.

CONCLUSION: A schedule consisting of 3‐day administration of gly‐G–CSF followed by a single leukapheresis can be proposed and widely accepted by healthy donors, as 84 percent of them reach the target in the estimated time with a reduced drug exposure. The cost of the procedure is reduced, in terms of both the growth factor administration and the number of leukapheresis procedures. The search for the optimum methods of donor management may improve the acceptability of this procedure and increase the number of allogeneic transplantations from PBPCs.