Optimized Solid-Phase Mesh-Enhanced Sorption from Headspace (SPMESH) for Rapid Sub-ng/kg Measurements of 3-Isobutyl-2-methoxypyrazine (IBMP) in Grapes.

Parallel extraction of headspace volatiles from multiwell plates using sorbent sheets (HS-SPMESH) followed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) can be used as a rapid alternative to solid-phase micro-extraction (SPME) gas-chromatography mass-spectrometry (GC-MS) for trace level volatile analyses. However, an earlier validation study of SPMESH-DART-MS using 3-isobutyl-2-methoxypyrazine (IBMP) in grape juice showed poor correlation between SPMESH-DART-MS and a gold standard SPME-GC-MS around the compound's odor detection threshold (<10 ng/kg) in grape juice, and lacked sufficient sensitivity to detect IBMP at this concentration in grape homogenate. In this work, we report on the development and validation of an improved SPMESH extraction approach that lowers the limit of detection (LOD < 0.5 ng/kg), and regulates crosstalk between wells (<0.5%) over a calibration range of 0.5−100 ng/kg. The optimized SPMESH-DART-MS method was validated using Cabernet Sauvignon and Merlot grape samples harvested from commercial vineyards in the central valley of California (n = 302) and achieved good correlation and agreement with SPME-GC-MS (R2 = 0.84) over the native range of IBMP (<0.5−20 ng/kg). Coupling of SPMESH to a lower resolution triple quadrupole (QqQ)-MS via a new JumpShot-HTS DART source also achieved low ng/kg detection limits, and throughput was improved through positioning stage optimizations which reduced time spent on intra-well SPMESH areas.

Product inhibition in nucleophilic aromatic substitution through DPPPent-supported π-arene catalysis.

Nucleophilic aromatic substitution (SNAr) of fluorobenzene by morpholine at a bis(diphenylphosphino)pentane-supported ruthenim complex is investigated as a model system for π-arene catalysis through the synthesis and full characterization of proposed intermediates. The SNAr step proceeds quickly at room temperature, however the product N-phenylmorpholine binds tightly to the ruthenium ion. In the case examined, the thermodynamics of arene binding favor product N-phenylmorpholine over fluorobenzene binding by a factor of 2000, corresponding to significant product inhibition. Observations of the catalyst resting state support this hypothesis and demonstrate an additive-controlled role for a previously-proposed ligand cyclometalation.

Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Patient outcomes and amotosalen/UVA-treated platelet utilization in massively transfused patients.

BACKGROUND: Amotosalen/UVA-treated platelet concentrates (PCs) have demonstrated efficacy for treating and preventing bleeding in clinical trials and in routine use; however, most studies were performed in haematology/oncology patients. We investigated efficacy during massive transfusion (MT) in general hospitalized patients. METHODS: Universal amotosalen/UVA treatment (INTERCEPT Blood System) of platelets was introduced at a large Austrian medical centre. We performed a retrospective cohort analysis comparing component use, in-hospital mortality and length of stay after MT that included platelet transfusion, for two periods (21 months each) before and after implementation. RESULTS: A total of 306 patients had MT. Patients were mostly male (74%) and ≥18 years old (99%), including 93 liver transplant, 97 cardiac or vascular surgery and 51 trauma patients. There were no differences in demographics between the periods. Component use on the day and within 7 days of the MT event was unchanged post-IBS implementation, except trauma patients received fewer RBCs on the day. The mean ratio of RBC:platelets:plasma on the day of the MT was close to 1:1:1 in both periods, except for liver transplants with MT who received more plasma components. Overall, in-hospital mortality (preimplementation = 27·6% vs. postimplementation = 24·0%; P = 0·51) and median time to discharge (preimplementation = 27 vs. postimplementation = 23 days; P = 0·37) did not change, except for cardiac and vascular surgery patients who were discharged earlier. CONCLUSION: The introduction of amotosalen/UVA-treated, pathogen-reduced PC did not adversely affect clinical outcomes in massively transfused patients in terms of blood product usage, in-hospital mortality and length of stay for a range of clinical indications for platelet transfusion support.

Impact of platelet pathogen inactivation on blood component utilization and patient safety in a large Austrian Regional Medical Centre.

BACKGROUND: In clinical studies, pathogen inactivation (PI) of platelet concentrates (PC) with amotosalen and UVA light did not impact patient risk for haemorrhage but may affect transfusion frequency and component utilization. We evaluated the influence of platelet PI on PC, red cell concentrate (RCC) and plasma use and safety in routine practice in a large regional hospital. STUDY DESIGN AND METHODS: Comparative effectiveness of conventional vs. PI-treated PC was analysed during two 21-month periods, before and after PI implementation. RESULTS: Similar numbers of patients were transfused in the pre-PI (control, 1797) and post-PI (test, 1694) periods with comparable numbers of PC (8611 and 7705, respectively). The mean numbers of PC per patient transfused (4·8 vs. 4·5, P = 0·43) were not different but days of PC support (5·9 vs. 5·0, P < 0·01) decreased. Most patients received RCC (86·8% control vs. 84·8% test, P = 0·90) with similar mean numbers transfused (10·8 vs. 10·2 RCC, P = 0·22), and fewer patients (55·4% control vs. 44·7% test, P < 0·01) received less plasma units (mean 9·9 vs. 7·8, respectively, P < 0·01) in the test period. The frequencies of transfusion-related adverse events (AE) were comparable (1·3% vs. 1·4%, P = 0·95). Analysis of haematology-oncology (522 control, 452 test), cardiac surgery (739 control, 711 test), paediatric (157 control, 130 test) and neonate (23 control, 20 test) patients revealed no increase in PC, plasma and RCC utilization, or AE. CONCLUSION: Component utilization and patient safety were not impacted by adoption of PI for PC. RCC use per patient was comparable, suggestive of no increase in significant bleeding.

Investigation of bacterial inactivation in apheresis platelets with 24 or 30 hours between inoculation and inactivation.

BACKGROUND AND OBJECTIVES: Blood Centre logistics, staffing and donor scheduling may be optimized if pathogen inactivation (PI) of platelets can be delayed until Day 1, but bacteria may rapidly grow during this time. This study evaluates bacterial PI performed 24 and 30 h after collection. MATERIALS AND METHODS: PAS-3 platelet units were collected on the Amicus and subsequently inoculated (3-53 CFU/unit) with 1of 5 transfusion relevant bacterial species (n = 3/organism). Units were then stored for either 24 ± 0·3 or 30 ± 0·3 h at 20-24°C with agitation, subsequently treated with amotosalen and UVA, and stored for 7 days. Samples were taken before and after inactivation, on Days 2, 5 and 7 for BacT/ALERT testing, and on Days 5 and 7 for plate counts. RESULTS: All samples from units taken prior to inactivation either demonstrated positive plate culture counts, or, in untreated positive controls, were culture-positive during storage. All contaminated units treated with amotosalen and UVA 24 after inoculation were culture-negative on all days tested. With inactivation performed 30 h following inoculation, one of 15 units (1-of-3 replicates) was culture-positive with Klebsiella pneumonia (1 × 109 CFU/ml) by Day 5. CONCLUSION: Photochemical treatment did not inactivate 1 of 15 units to sterility in apheresis platelets stored in PAS with a 30-h delay between contamination and treatment, but did inactivate 15 of 15 units with a 24-h delay.

A clinical governance framework for blood services.

BACKGROUND AND OBJECTIVES: The elements of clinical governance, which ensure excellence in clinical care, can be applied to blood services. In this survey, their application in a range of blood providers was gauged, with the aim of identifying best practice and producing a generalizable framework. MATERIALS AND METHODS: The Medical Directors of members of the Alliance of Blood Operators surveyed how different elements of clinical governance operated within their organizations and developed recommendations applicable in the blood service environment. RESULTS: The recommendations that emerged highlighted the importance of an organization's culture, with the delivery of optimal clinical governance being a corporate responsibility. Senior management must agree and promote a set of values to ensure that the system operates with the patient and donor at its heart. All staff should understand how their role fits into the 'journey to the patient', and a culture of openness promoted. Thus, reporting of errors and risks should be actively sought and praised, with penalties applied for concealment. Systems should exist to collect, analyse and escalate clinical outcomes, safety data, clinical risk assessments, incident reports and complaints to inform organizational learning. CONCLUSION: Clinical governance principles from general health care can be applied within blood services to complement good manufacturing practice. This requires leadership, accountability, an open culture and a drive for continuous improvement and excellence in clinical care.

Bacterial culture of apheresis platelets: a mathematical model of the residual rate of contamination based on unconfirmed positive results.

BACKGROUND: Platelet septic reactions result from low concentrations of bacteria that escape detection by quality-control BacT/ALERT™ culture testing. We estimate the contamination rate with these bacteria at the time of testing using a mathematical model. METHODS: Culture results and reported septic reactions are described for platelets collected between January 2007 and December 2011. Initial positive results with negative confirmatory cultures were reclassified assuming some of the 'unconfirmed positive results' represent collections contaminated with low-concentration, dormant bacteria. A mathematical model based on the probability of the detection of bacteria describes the upper limit of the residual rate of contamination. RESULTS: The rate of confirmed or unconfirmed positive apheresis platelet donations was 188 per million (1:5317) and 110 per million (1:9124), respectively. The rate of post-transfusion sepsis and reported fatalities per distributed component was 1:106 931 and 1:1 015 843, respectively. A linear decrease in unconfirmed positive Bacillus spp. cultures most likely reflected diminishing environmental contamination over time. The remaining unconfirmed positive results identified similar bacteria species as those associated with septic reactions. Assuming that these represent contamination of the collection with low-concentration, dormant bacteria, the model identified a residual contamination of 3524-204 per million (1:284-1:4902) for collections contaminated with 1-20 bacteria, respectively. DISCUSSION: Greater than 99·5% of collections contain no viable, aerobic bacteria in solution at the time of early culture testing. For every confirmed positive contaminated collection detected, there are at most 19 collections with low concentrations of dormant bacteria that are not readily detected by early BacT/ALERT™ culture.

Seasonal temperature variation and the rate of donor deferral for low hematocrit in the American Red Cross.

BACKGROUND: Hematocrit (Hct) values in healthy adult populations exhibit seasonal variation, with the lowest values occurring in the summer. The extent to which environmental temperature contributes to the seasonal trend in deferral rates for unacceptable Hct in the American Red Cross was further analyzed. STUDY DESIGN AND METHODS: A centralized database of donations during 2002 to 2004, constituting 24.3 million donor presentations, was further characterized. Data on mean monthly temperature in the United States were obtained for the same period from a government agency. Multivariate regression analyses were performed to determine the relationship between Hct deferral rates among blood donors and environmental temperature and donor characteristics. RESULTS: Hct deferral rates were associated with mean monthly temperature in the United States (R(2) = 0.77). The relationship between the Hct deferral rate and environmental temperature was strongest in the region of the country with the highest seasonal variation in temperature, followed by regions with intermediate and low seasonal variation in temperature, respectively. The seasonal pattern in Hct deferral rates occurred in both sexes and across all age groups, with significantly higher Hct deferral rates occurring in June through August compared to other quarters (p < 0.0007). CONCLUSION: There is a significant seasonal pattern in Hct deferral rates that is associated with environmental temperature. The relationship between Hct deferral rates and temperature is strongest in areas of the country with greater temperature variability. The effect of seasonality on Hct deferrals should be taken into account for donor counseling, recruitment, and retention efforts.

Nucleic acid testing: update and applications.

Nucleic acid testing (NAT) holds the promise of closing the window of infectiousness for hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) in the general blood supply. Pioneering work by the source plasma industry with NAT for hepatitis A virus (HAV), hepatitis B virus (HBV), and parvovirus B19 suggests that, in the future, the risk of other viral infections may be reduced using similar technology. The European Commission decree that, by July 1999, all source plasma for fractionation should be NAT nonreactive for HCV at a sensitivity of 100 viral IU/mL, has driven the implementation of NAT in the United States. It is estimated that more than 95% of the US blood supply is currently tested by one of two investigational tests for HCV and HIV, and many institutions restrict the release of red blood cell (RBC) and plasma products prior to the release of NAT results. NAT implementation has been hampered by a lack of fully automated, low-cost technologies; the absence of Food and Drug Administration (FDA)-approved and validated clinical tests; and lagging turnaround times. Results from US investigational trials of the Procleix Transcription Mediated Amplification (TMA) HCV/HIV (Chiron Corp, Emeryville, CA) and the COBAS AmpliScreen (Roche Diagnostics, Indianapolis, IN) polymerase chain reaction (PCR) assays have begun to substantiate their value. While NAT assays will not replace serologic tests, they lay the groundwork for further reducing the already low risk of infection transmission through transfusion of blood components and their factor derivatives.

The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia.

Chemotaxis in leukocytes is mediated through binding of soluble chemokines to transmembrane G-protein coupled receptors. The chemokine receptor CXCR3 has been previously shown to be widely expressed on activated T cells and to mediate T-cell chemotaxis on binding to various ligands, including Mig, IP-10, and ITAC. By using immunohistochemical and flow cytometric analysis, we report that CXCR3 is also expressed on a subset of peripheral blood B cells and in distinct subtypes of B-cell lymphoma. CXCR3 immunohistochemical or flow cytometric expression was seen in 37 of 39 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (diffusely positive in 33 cases), whereas mantle cell lymphoma (30 cases), follicular lymphoma (27 cases), and small noncleaved cell lymphoma (8 cases) were negative in all but 2 cases. Strong CXCR3 expression was also seen in splenic marginal zone lymphoma (14 of 14 cases) and in the monocytoid and plasmacytic cells in extranodal marginal zone lymphoma (15 of 16 cases). This differential expression of CXCR3 in B-cell tumors contrasts with that of another B-cell-associated chemokine receptor, BLR1/CXCR5, which we show here is expressed on all types of B-cell lymphoma tested. We also report that the CXCR3 ligand, Mig, is coexpressed on tumor cells in many cases of CLL/SLL (10 of 13 cases examined) with Mig expression less frequently seen in other B-cell lymphoma subtypes. Coexpression of CXCR3 and its ligand, Mig, may be an important functional interaction in B-CLL, as well as a useful diagnostic marker for the differential diagnosis of small cell lymphomas. (Blood. 2000;95:627-632)

Massive myocardial necrosis in thrombotic thrombocytopenic purpura: a case report and review of the literature.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon syndrome resulting from diffuse occlusion of small arterioles and capillaries by hyaline microthrombi. It is characterized by fever, thrombocytopenic purpura, microangiopathic hemolytic anemia, and neurologic and renal dysfunction. While cardiac pathology in TTP is commonly seen at autopsy, clinical cardiac dysfunction is rare and typically results from conduction system involvement. While 3% to 8% of patients with TTP report chest pain on admission, reports of fatal ventricular pump failure are extremely rare. We now report a case of TTP resulting in death from widespread myocardial necrosis. This patient presented with elevated cardiac enzymes and electrocardiographic disturbances that mimicked viral myocarditis, as well as a profound thrombocytopenia. Such a case may represent the extreme of a distribution of cardiac involvement in TTP or the consequence of an unidentified autoimmune process capable of precipitating severe myocardial TTP.

Plateletpheresis efficiency: a comparison of the spectra LRS and AMICUS separators.

BACKGROUND: Optimizing the yields of plateletpheresis by increasing collection efficiencies may provide several benefits: to patients, by increasing the dose in single-donor platelet (SDP) units; to the collection center, by increasing the percentage of components that may be split into double units; and/or to the donor, by reducing the duration of donation. STUDY DESIGN AND METHODS: A prospective, randomized study was undertaken to compare the efficiency of platelet collection in paired donations by 21 donors on two cell separators (Spectra LRS, version [v] 5.1; and AMICUS, v2.37). The order of donation was randomly assigned; donations were performed at least 2 weeks apart. A fixed blood volume (4000 mL) was processed by utilizing standard protocols. Findings were confirmed in a retrospective, matched study that compared the two separators by using fixed collection times (90 min). RESULTS: The AMICUS and Spectra LRS separators consistently produced white cell-reduced (<1 x 10(6) white cells) components. The AMICUS harvested 32 percent more platelets on average (median, 4.9 x 10(11); range, 1.5-8.7 x 10(11)) than the Spectra LRS (median, 3.7 x 10(11); range, 2.1-7.7 x 10(11)) (p = 0.03), with mean times of 71.5 and 66.0 minutes (p = 0.03), respectively, to process 4000 mL. Mild donor reactions tended to be more common on the AMICUS separator, which used significantly more ACD (median 482 mL vs. 389 mL; p<0.0001) than on the Spectra LRS. CONCLUSIONS: The AMICUS separator harvested more platelets per unit of blood volume processed than the Spectra LRS. Possible benefits include increased dose in each single-donor unit, increased double-unit harvests, and/or shorter donation time.

The pathology of transfusion-related acute lung injury.

Transfusion-related acute lung injury is an uncommon condition characterized by the rapid onset of respiratory distress soon after transfusion. Our understanding of its pathophysiology is based on animal models of complement (C5a) and antibody-induced lung injury and a limited number of autopsies. These models suggest that transfusion-related acute lung injury is induced by granulocytes that aggregate in the pulmonary microvasculature after activation by transfusion-derived antibodies or biologically active lipids. The published autopsy reports provide little support for this model, as they are invariably confounded by underlying pulmonary infection, preexisting disease, and resuscitation injury. We report the case of a previously well 58-year-old man who died of transfusion-related acute lung injury within 2 hours of the onset of pulmonary distress; autopsy showed evidence of massive pulmonary edema with granulocyte aggregation within the pulmonary microvasculature and extravasation into alveoli. Electron microscopy revealed capillary endothelial damage with activated granulocytes in contact with the alveolar basement membranes. These findings provide direct support for the proposed model of transfusion-related acute lung injury pathogenesis.

Transfusion with blood from a donor with chronic myelogenous leukemia: persistence of the bcr/abl translocation in the recipient.

BACKGROUND: Transfusion of cells harvested from patients with chronic myelogenous leukemia (CML) as a therapeutic measure for patients with granulocytopenia was popular in the 1970s, when studies examining the persistence of transfused donor cells were limited by a lack of molecular techniques. Blood samples from a patient who recently received an inadvertent transfusion of CML cells were evaluated for the presence of the bcr/abl translocation characteristic of CML. CASE REPORT: The patient, a 67-year-old man with a history of congestive heart failure, myocardial infarct, hypertension, diabetes mellitus, and chronic renal failure, was transfused for bleeding from colonic angiodysplasia. A volunteer blood donor reported that he had been diagnosed with CML 10 days after his donation. Three days after the donation, blood components from the donor with CML had been administered to the patient as nonirradiated red cells and platelets. Evaluation of donor blood by a reverse-transcriptase polymerase chain reaction showed the b3a2 transcript, indicating a bcr/abl translocation. Periodic testing of the patient's peripheral blood by the same technique demonstrated the presence of the b3a2 transcript on Days 74 and 75 after transfusion. The patient died of congestive heart failure 8 months after the transfusion. CONCLUSION: In this rare case of accidental transfusion of neoplastic cells, the findings document the persistence of the donor's neoplastic clone in the recipient for 75 days.

ABO incompatibility as an adverse risk factor for survival after allogeneic bone marrow transplantation.

BACKGROUND: Graft ABO incompatibility has not been thought to affect 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% CI, 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.