Effects of storage-aged red blood cell transfusions on endothelial function in hospitalized patients.

BACKGROUND: Clinical and animal studies indicate that transfusions of older stored red blood cells (RBCs) impair clinical outcomes as compared to fresh RBC transfusions. It has been suggested that this effect is due to inhibition of nitric oxide (NO)-mediated vasodilation after transfusion of older RBC units. However, to date this effect has not been identified in human transfusion recipients. STUDY DESIGN AND METHODS: Forty-three hospitalized patients with transfusion orders were randomly assigned to receive either fresh (<14 days) or older stored (>21 days) RBC units. Before transfusion, and at selected time points after the start of transfusion, endothelial function was assessed using noninvasive flow-mediated dilation assays. RESULTS: After transfusion of older RBC units, there was a significant reduction in NO-mediated vasodilation at 24 hours after transfusion (p = 0.045), while fresh RBC transfusions had no effect (p = 0.231). CONCLUSIONS: This study suggests for the first time a significant inhibitory effect of transfused RBC units stored more than 21 days on NO-mediated vasodilation in anemic hospitalized patients. This finding lends further support to the hypothesis that deranged NO signaling mediates adverse clinical effects of older RBC transfusions. Future investigations will be necessary to address possible confounding factors and confirm these results.

Riboflavin and ultraviolet light for pathogen reduction of murine cytomegalovirus in blood products.

BACKGROUND: Two studies were performed to test the effectiveness of riboflavin and ultraviolet (UV) light treatment (Mirasol PRT, Terumo BCT) against murine cytomegalovirus (MCMV). The first study utilized immune-compromised mice to measure the reduction of cell-free MCMV. A second study used a murine model to evaluate the ability of Mirasol PRT to prevent transfusion-transmitted (TT)-MCMV infection. STUDY DESIGN AND METHODS: Human plasma was inoculated with MCMV and then treated with Mirasol PRT. The viral titer was measured using an infectious dose 50% assay in nude mice. Mice were euthanized on Day 10 posttransfusion, and their spleens were tested for the presence of MCMV DNA using polymerase chain reaction (PCR). Mirasol PRT was also evaluated to determine its effectiveness in preventing TT-MCMV in platelets (PLTs) stored in PLT additive solution. PLTs were inoculated with either cell-associated MCMV or cell-free MCMV and then treated with Mirasol PRT. Mice were transfused with treated or untreated product and were euthanized 14 days posttransfusion. Blood and spleens were assayed for MCMV DNA by real-time-PCR. RESULTS: Using nude mice to titer MCMV, a modest 2.1-log reduction was observed in plasma products after Mirasol PRT treatment. TT-MCMV was not observed in the mouse transfusion model when either cell-free or cell-associated MCMV was treated with Mirasol PRT; MCMV transmission was uniformly observed in mice transfused with untreated PLTs. CONCLUSIONS: These results suggest that using riboflavin and UV light treatment may be able to reduce the occurrence of transmission of human CMV from infectious PLTs and plasma units.

Metabolomics of ADSOL (AS-1) red blood cell storage.

Population-based investigations suggest that red blood cells (RBCs) are therapeutically effective when collected, processed, and stored for up to 42 days under validated conditions before transfusion. However, some retrospective clinical studies have shown worse patient outcomes when transfused RBCs have been stored for the longest times. Furthermore, studies of RBC persistence in the circulation after transfusion have suggested that considerable donor-to-donor variability exists and may affect transfusion efficacy. To understand the limitations of current blood storage technologies and to develop approaches to improve RBC storage and transfusion efficacy, we investigated the global metabolic alterations that occur when RBCs are stored in AS-1 (AS1-RBC). Leukoreduced AS1-RBC units prepared from 9 volunteer research donors (12 total donated units) were serially sampled for metabolomics analysis over 42 days of refrigerated storage. Samples were tested by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, and specific biochemical compounds were identified by comparison to a library of purified standards. Over 3 experiments, 185 to 264 defined metabolites were quantified in stored RBC samples. Kinetic changes in these biochemicals confirmed known alterations in glycolysis and other pathways previously identified in RBCs stored in saline, adenine, glucose and mannitol solution (SAGM-RBC). Furthermore, we identified additional alterations not previously seen in SAGM-RBCs (eg, stable pentose phosphate pathway flux, progressive decreases in oxidized glutathione), and we delineated changes occurring in other metabolic pathways not previously studied (eg, S-adenosyl methionine cycle). These data are presented in the context of a detailed comparison with previous studies of SAGM-RBCs from human donors and murine AS1-RBCs. Global metabolic profiling of AS1-RBCs revealed a number of biochemical alterations in stored blood that may affect RBC viability during storage as well as therapeutic effectiveness of stored RBCs in transfusion recipients. These results provide future opportunities to more clearly pinpoint the metabolic defects during RBC storage, to identify biomarkers for donor screening and prerelease RBC testing, and to develop improved RBC storage solutions and methodologies.

Red blood cells stored for increasing periods produce progressive impairments in nitric oxide-mediated vasodilation.

BACKGROUND: Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)-mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow. STUDY DESIGN AND METHODS: AS-1 RBC units stored up to 42 days were sampled at selected storage times. Samples were added to aortic rings ex vivo, a system where NO-mediated vasodilation could be experimentally controlled. RESULTS: RBC units showed storage-dependent changes in plasma hemoglobin (Hb), RBC 2,3-diphosphoglycerate acid, and RBC adenosine triphosphate conforming to expected profiles. When freshly collected (Day 0) blood was added to rat aortic rings, methacholine (MCh) stimulated substantial NO-mediated vasodilation. In contrast, MCh produced no vasodilation in the presence of blood stored for 42 days. Surprisingly, the vasoinhibitory effects of stored RBCs were almost totally mediated by RBCs themselves: removal of the supernatant did not attenuate the inhibitory effects, while addition of supernatant alone to the aortic rings only minimally inhibited MCh-stimulated relaxation. Stored RBCs did not inhibit vasodilation by a direct NO donor, demonstrating that the RBC-mediated vasoinhibitory mechanism did not work by NO scavenging. CONCLUSIONS: These studies have revealed a previously unrecognized vasoinhibitory activity of stored RBCs, which is more potent than the described effects of free Hb and works through a different mechanism that does not involve NO scavenging but may function by reducing endothelial NO production. Through this novel mechanism, transfusion of small volumes of stored blood may be able to disrupt physiologic vasodilatory responses and thereby possibly cause adverse clinical outcomes.

Transfusion-transmitted cytomegalovirus (CMV) infections in a murine model: characterization of CMV-infected donor mice.

BACKGROUND: Donor and recipient mechanisms that modulate the incidence and severity of transfusion-transmitted cytomegalovirus (TT-CMV) are unclear. The kinetics of murine CMV (MCMV) infection in the peripheral blood of donor mice were investigated to determine the utility of this model for studying TT-CMV. STUDY DESIGN AND METHODS: BALB/cByJ mice, experimentally infected with Smith strain MCMV, were killed at serial time points up to 28 days after infection. Peritoneal exudate cells (PECs), peripheral blood white blood cells (WBCs), plasma, and marrow were tested for MCMV DNA with quantitative polymerase chain reaction (PCR), replication-competent virus with quantitative culture, and transcription of viral genes with reverse transcription (RT)-PCR targeted at the immediate-early 1 (ie1) gene. RESULTS: PECs, macrophages infected by MCMV shortly after intraperitoneal inoculation, demonstrated high mean levels of MCMV DNA (10(5)-10(7) genome equivalents [geqs]/10(5) PECs), virus production (10(1)-10(4) infectious virions/10(5) PECs), and ie1 gene transcription, demonstrating productive infection. In contrast, while MCMV loads averaged 10(4) to 10(6) geqs per 10(5) peripheral WBCs, all WBC samples were uniformly negative for MCMV ie1 expression by RT-PCR and for culturable virus, consistent with latent MCMV infection. Plasma and marrow showed lower viral loads than WBCs and PECs and were all negative by culture and RT-PCR analysis. CONCLUSIONS: Following experimental MCMV infection, murine peripheral blood WBCs appear to be latently infected with virus (MCMV DNA-positive; MCMV RNA-negative; MCMV culture-negative), similar to the latently infected human monocytes in peripheral blood of CMV-seropositive donors. These donor kinetics suggest that the experimental MCMV system can be used to effectively model the mechanisms of TT-CMV infections in humans.

Comparison of cytomegalovirus polymerase chain reaction and serology for screening umbilical cord blood components.

BACKGROUND: Recipients of umbilical cord blood (UCB) transplants are susceptible to opportunistic infections, including cytomegalovirus (CMV). To prevent CMV transmission from UCB donors, most laboratories perform serology on corresponding maternal samples and quarantine units when the mother has immunoglobulin M (IgM) anti-CMV. STUDY DESIGN AND METHODS: UCB units and associated samples (UCB plasma and red cell pellet; maternal whole blood and serum) from two cord blood banks were tested with two validated CMV polymerase chain reaction assays (UL54 and UL93 targets). Results were compared with maternal CMV serology (IgG and IgM). RESULTS: Only 4 of 48 (8.3%) quarantined CMV IgM-positive units were also CMV nucleic acid testing (NAT)-positive (651-68,600 copies/mL). In contrast, 1 of 200 "CMV-safe" UCB units (CMV IgM-equivocal or -negative) had CMV DNA (0.5%). The corresponding maternal samples were CMV NAT-negative. Positive maternal IgM serology demonstrates only modest sensitivity (80%) and specificity (82%) and poor positive predictive value (8%), when correlated with the presence of CMV DNA in UCB units. CONCLUSION: CMV NAT may be a useful adjunct to serologic screening, potentially reducing wastage of IgM-positive and NAT-negative units while also detecting potentially infectious units that would pass serologic screening. A prospective clinical trial to further evaluate the role of CMV NAT in UCB transplantation appears warranted.

Photochemical treatment of platelet concentrates with amotosalen hydrochloride and ultraviolet A light inactivates free and latent cytomegalovirus in a murine transfusion model.

BACKGROUND: A photochemical treatment (PCT) process utilizing amotosalen hydrochloride and long wavelength UVA light has been developed to inactivate pathogens in PLTs. This study investigated the effects of amotosalen/UVA treatment on free and latent murine CMV (MCMV) in PLT preparations using a murine model of transfusion-transmitted CMV (TT-CMV). STUDY DESIGN AND METHODS: In a model of latent MCMV infection, "donor" mice received 1 x 10(6) plaque-forming units (PFUs) MCMV and were rested 14 days. Subsequently harvested, pooled, and washed WBCs were PCR positive for MCMV. Murine WBC doses of 1 x 10(4), 1 x 10(5), and 1 x 10(6) were added to human apheresis PLTs in 35 percent autologous plasma and 65 percent PLT AS (PAS). The WBC-PLT products were treated with 150 micro mol/L amotosalen and 0.6 J per cm2 UVA and transfused via tail vein injection into recipient mice. Recipients were killed on Day 14. Blood and spleens were collected and assayed for MCMV by PCR. In a parallel model of active infection with free virus, human PLT in 35 percent autologous plasma and 65 percent PAS were dosed with 1 x 10(5) and 1 x 10(6) PFUs of MCMV. All other procedures were as described above. RESULTS: In the absence of amotosalen/UVA-pretreatment, transfusion of PLT latently or actively infected with MCMV produced TT-CMV in a dose-dependent fashion. In contrast, all transfusion recipients of identical PLT preparations pretreated with amotosalen/UVA were uniformly PCR negative for MCMV (abrogation of TT-CMV; p < 0.05). CONCLUSIONS: PCT of PLT preparations with the specified doses of amotosalen hydrochloride and UVA light prevents transfusion transmission of free and latent MCMV in a murine model. These results suggest that PCT of human PLTs with amotosalen/UVA should also effectively abrogate TT-CMV in the clinical setting.

Prevalence of the fragile X syndrome in African-Americans.

Since the development of a molecular diagnosis for the fragile X syndrome in the early 1990s, several population-based studies in Caucasians of mostly northern European descent have established that the prevalence is probably between one in 6,000 to one in 4,000 males in the general population. Reports of increased or decreased prevalence of the fragile X syndrome exist for a few other world populations; however, many of these are small and not population-based. We present here the final results of a 4-year study in the metropolitan area of Atlanta, Georgia, establishing the prevalence of the fragile X syndrome and the frequency of CGG repeat variants in a large Caucasian and African-American population. Results demonstrate that one-quarter to one-third of the children identified with the fragile X syndrome attending Atlanta public schools are not diagnosed before the age of 10 years. Also, a revised prevalence for the syndrome revealed a higher point estimate for African-American males (1/2,545; 95% CI: 1/5,208-1/1,289) than reported previously, although confidence intervals include the prevalence estimated for Caucasians from this (1/3,717; 95% CI: 1/7,692-1/1,869) and other studies. Further population-based studies in diverse populations are necessary to explore the possibility that the prevalence of the fragile X syndrome differs among world populations.