Novel functionalities of hybrid paper-polymer centrifugal devices for assay performance enhancement.

The presented work demonstrates novel functionalities of hybrid paper-polymer centrifugal devices for assay performance enhancement that leverage the advantages of both paper-based and centrifugal microfluidic platforms. The fluid flow is manipulated by balancing the capillary force of paper inserts with the centrifugal force generated by disc rotation to enhance the signal of a colorimetric lateral flow immunoassay for pathogenic E. coli. Low-cost centrifugation for pre-concentration of bacteria was demonstrated by sample sedimentation at high rotational speeds before supernatant removal by a paper insert via capillary force after deceleration. The live bacteria capture efficiency of the device was similar to a commercial centrifuge. This pre-concentrated sample when combined with gold nanoparticle immunoconjugate probes resulted in a detection limit that is 10× lower than a non-concentrated sample for a lateral flow immunoassay. Signal enhancement was also demonstrated through rotational speed variation to prevent the flow for on-device incubation and to reduce the flow rate, thus increasing the sample residence time for the improved capture of gold nanoparticle-bacteria complexes in an integrated paper microfluidic assay. Finally, multiple sequential steps including sample pre-concentration, filtration, incubation, target capture by an integrated paper microfluidic assay, silver enhancement and quenching, and index matching were completed within a single device. The detection limit was 105 colony forming units per ml, a 100× improvement over a similar paper-based lateral flow assay. The techniques utilize the advantages of paper-based microfluidic devices, while facilitating additional functionalities with a centrifugal microfluidic platform for detection performance enhancement in a low-cost, automated platform amenable to point-of-care environments.

Antibodies to the HLA-A2 antigen prime neutrophils and serve as the second event in an in vitro model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality and has been linked to the infusion of donor antibodies directed against recipient HLA class I antigens. We hypothesize that antibodies against HLA class I antigens bind to the antigens on the neutrophil (PMN) surface and induce priming and PMN cytotoxicity as the second event in a two-event in vitro model of PMN-mediated cytotoxicity. METHODS: Isolated PMNs from HLA-A2 homozygotes, heterozygotes and null donors were incubated with a monoclonal antibody to HLA-A2 and a human polyclonal IgG to HLA-A2 and priming of the oxidase was measured. The monoclonal antibodies and PMNs from these three groups were then used in a two-event model of PMN cytotoxicity. RESULTS: The antibodies to HLA-A2 both primed PMNs from HLA-A2 homozygotes but not from heterozygotes or nulls. Antibodies to HLA-A2 also served as the second event in a two-event model to induce PMN cytotoxicity of HLA-A2 homozygous PMNs. CONCLUSION: Antibodies to HLA class I antigens may directly prime/activate PMNs through the ligation of the antigen on the cell surface, and the antigen density appears to be important for these changes in PMN physiology.

Proteomic analysis of the supernatant of red blood cell units: the effects of storage and leucoreduction.

BACKGROUND: Red blood cell (RBC) transfusion is a life-saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients. STUDY DESIGN: Five RBC units were drawn and divided (half prestorage leucoreduced (LR-RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in-gel tryptic digestion and nano-liquid chromatography tandem mass spectrometry. RESULTS: In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR-RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leucoreduction removed many platelet- and leucocyte-derived structural proteins; however, a number of intracellular proteins accumulated including peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T-phosphorylation of Prdx-6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase-9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C. CONCLUSION: We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leucoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.

The pro-inflammatory effects of platelet contamination in plasma and mitigation strategies for avoidance.

BACKGROUND AND OBJECTIVES: Plasma and platelet concentrates are disproportionately implicated in transfusion-related acute lung injury (TRALI). Platelet-derived pro-inflammatory mediators, including soluble CD40 ligand (sCD40L), accumulate during storage. We hypothesized that platelet contamination induces sCD40L generation that causes neutrophil [polymorphonuclear leucocyte (PMN)] priming and PMN-mediated cytotoxicity. MATERIALS AND METHODS: Plasma was untreated, centrifuged (12,500 g) or separated from leucoreduced whole blood (WBLR) prior to freezing. Platelet counts and sCD40L concentrations were measured 1-5 days post-thaw. The plasma was assayed for PMN priming activity and was used in a two-event in vitro model of PMN-mediated human pulmonary microvascular endothelial cell (HMVEC) cytotoxicity. RESULTS: Untreated plasma contained 42±4·2×10(3)/µl platelets, which generated sCD40L accumulation (1·6-eight-fold vs. controls). Priming activity and HMVEC cytotoxicity were directly proportional to sCD40L concentration. WBLR and centrifugation reduced platelet and sCD40L contamination, abrogating the pro-inflammatory potential. CONCLUSION: Platelet contamination causes sCD40L accumulation in stored plasma that may contribute to TRALI. Platelet reduction is potentially the first TRALI mitigation effort in plasma manufacturing.

Glial tumors in the MNU rat model: induction of pure and mixed gliomas that do not require typical missense mutations of p53.

Gliomas were induced in adult male Sprague-Dawley rats by continuous exposure to 100 ppm of N-nitrosmethylurea (MNU) in drinking water. Latency periods for such tumors were 20 and 50 weeks following completion of exposure intervals of 20, 15, and 10 weeks, respectively. Based on histomorphology and the pattern of GFAP immunoreactivity, a large percentage of MNU-induced tumors (>40%) were anaplastic mixed gliomas, having both neoplastic astrocytic and oligodendroglial components. Typical oligodendrogliomas and astrocytomas also occurred less frequently. Unlike the majority of tumors induced by ethylnitrosourea (ENU), MNU yielded glial tumors that did not express synaptophysin. Anaplastic mixed gliomas and glioblastoma multiforme (GBMs) had no missense p53 mutations in the commonly mutated exons 4 through 8 and did not overexpress wild-type p53, suggesting that MNU-induced oncogenesis in rat brain tumors may not require inactivation/alteration of the p53 tumor suppressor gene. The K-ras gene was also analyzed and found to have no activating mutations in brain tumors. This model is suitable for studying genetic events leading to the majority of gliomas that apparently express functional p53.

Leukemias, myeloma, and other lymphoreticular neoplasms.

BACKGROUND: The purpose of this study was to assess the occurrence of various morphologic types of leukemia and myeloma within patient demographic groups and to correlate findings with data-reporting periods and other variables, such as 5-year relative survival. METHODS: Data from 31,850 cases of multiple subgroups of acute and chronic leukemia, 12,237 cases of myeloma, and 321 cases of "other" lymphoreticular neoplasms were collected by the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program. The data were examined by age, sex, race, age-specific and age-adjusted incidence rate, and patient 5-year relative survival during three reporting periods: 1973-1977, 1978-1982, and 1983-1987. RESULTS: The age-adjusted incidence rate for all categories of leukemia combined has been constant, but there has been an increase in the relative frequency (percentage) of acute lymphoid leukemia (ALL) in the general population and a rising incidence rate of myeloid leukemia in the black population. The increase of ALL is offset by a decline of acute myeloid leukemias (AMLs) and acute leukemia, not otherwise specified. The age-adjusted rate of ALL in whites, 1.5 per 100,000 per year, is twice that of blacks, 0.8. The rates for each of the major categories of leukemia are considerably higher in males than in females. Five-year survival rates changed very little for leukemias over the 15 years of the study except for ALL, in which there was a marked improvement between the first (1973-1977) (39.1%) and second (1978-1982) (51.3%) reporting period. The SEER data confirm that multiple myeloma is predominantly a disease of late adulthood and occurs more frequently in blacks and males. The incidence rate of multiple myeloma has not changed during the 15 years surveyed. The 5-year relative survival rate has remained nearly constant for multiple myeloma. There is a marked difference in 5-year relative survival rates for patients with plasmacytoma of bone marrow (45.7%), multiple myeloma (25.9%), and plasma cell leukemia (13.0%). CONCLUSIONS: Shifts in the relative frequencies of leukemia types may have been affected by changes in classification criteria, changes in the use of histologic terms over time, and the expanded use of immunophenotyping and other technology to characterize acute leukemias. Incidence rates and 5-year relative survival rates for myeloma have remained stable.